Discovery of NIRF theranostic probes targeting amyloid-β and cholinesterases in Alzheimer’s disease models

Domain: neuroscience
Published: 2026-01-26
Editorial outcome: major_revision

Abstract
Alzheimer’s disease (AD) remains a major unmet medical challenge, with limited tools that integrate early diagnosis and therapeutic intervention. Considering the pivotal roles of amyloid-β (Aβ) and cholinesterases (ChEs) in AD etiology, we report dual-functional theranostic NIR-I probes. The lead candidate, I-43, exhibits favorable NIR optical properties (Stokes shift ≥ 220 nm) and binds strongly to Aβ fibrils, with Kd values of 58.2 ± 9.7 nM for Aβ 1-40 and 104 ± 25 nM for Aβ 1-42. Histological staining of brain tissues from transgenic APP-PS1 mice and human autopsy samples confirms selective detection of Aβ plaques with a high signal-to-noise ratio and minimal cross-reactivity toward pathogenic tau tangles and α-synuclein. In addition, I-43 exhibits fluorescence response toward AChE, shows inhibitory activity (IC50 = 0.38 µM), and enhances memory in a scopolamine-induced amnesia in Swiss albino mice. Despite this, limited aqueous solubility and metabolic stability necessitate structural modifications and formulation strategies to broaden the scope in preclinical studies. Herein, we demonstrate that probes can be engineered to label key AD biomarkers with ChEs inhibitory activity, paving the way to an alternate theranostic approach in AD management.

Introduction
Alzheimer’s Disease (AD) is an insidious and irreversible cerebral atrophy that accounts for 70–80% of individuals diagnosed with dementia1. Clinical manifestations typically encompass amnesia, and aging may cause diverse functional disabilities like aphasia, disorientation, and ataxia2. Toxic amyloid-β (Aβ) deposition and cholinergic impairment have been consistently identified as signature neuropathological hallmarks in postmortem analyses of brains from AD patients. The onset of AD is closely linked to the accretion of amyloidogenic Aβ (mainly Aβ 40 and Aβ 42) peptides into well-ordered β-sheet oligomers and subsequent insoluble fibrils, ultimately depositing as senile plaques within cerebral tissues3,4. In turn, the existence of these pathogenic Aβ forms (oligomers, and fibrils) within biological systems serves as critical AD biomarkers, detectable via advanced imaging and cerebrospinal fluid analysis, thereby enabling early diagnosis5,6. Additionally, cholinesterases (ChEs), particularly acetylcholinesterase (AChE), regulate synaptic transmission by hydrolyzing acetylcholine and remain key therapeutic targets in AD7,8. Usually, cholinergic dysregulation in the basal forebrain promotes a cascade of events that causes synaptic failure, accelerates irreversible neurodegeneration, and drives cognitive decline in AD9–11. Its colocalization with diffuse pre-amyloid deposits and mature plaques in AD brains, along with peripheral anionic sites (PAS) mediated acceleration of Aβ oligomer and fibril formation, highlights interconnected pathological events in AD12–15. Similarly, toxic Aβ species also exacerbate cholinergic dysfunction, even in the prodromal phases16. A definitive cure remains out of reach because of elusive underlying causes of triggering events in AD onset, coupled with a multifaceted and intertwined etiology. To date, clinically approved AChE inhibitors provide only transient relief from impaired memory in AD, while amyloid-β (Aβ) targeted therapies failed to arrest or attenuate neurodegeneration in the long term. A decade-long (10–20 years) preclinical phase of AD is marked by irreversible damage to the neurons prior to the commencement of apparent clinical symptoms. It poses a formidable barrier to curative therapies17. Early detection in prodromal stages is thus critical for effective interventions but remains challenging due to diagnostic limitations. Approved radiotracers excel at visualizing Aβ plaques but rely on non-readily available tools and expensive, short-lived isotopes like [18F] or [11C], which limit their widespread use for routine screening18–20. Similarly, only a few AChE-specific radiotracers are available, such as [11C]MP4A, rarely used in diagnosis due to sensitivity issues21,22. Non-radiative NIR (Near-infrared) fluorescence chemical sensors (λem ≥ 650 nm) offer an affordable transformative alternative, enabling real-time, highly selective, deep tissue imaging of these biomarkers non-invasively23–27. Donor-π-Acceptor (D-π-A) NIR probes, such as DANIR, CRANAD, and BODIPY dyes (as illustrated in Fig. 1b), have been successfully tested for non-invasive NIR imaging of the brain in a murine model of AD28–33. Likewise, MYCN and BChE-NIRFP probes (Fig. 1c) were ChE-activated off-on DANIR probes to study the physiological roles of AChE and BChE, respectively, in AD pathophysiology23,34. Recent advances in NIR-based theranostics, which integrate diagnostics with therapy, hold immense promise for addressing the multifaceted pathology of AD. Hybrid constructs such as TBT, PiB-C, and cationic probes like quinolinium-based carbazole and DNTPH (Fig. 1d) serve as exemplary theranostic agents, capable of detecting Aβ fibrils while concurrently inhibiting their aggregation and mitigating pathology in preclinical models35–38. However, extensive ongoing research aimed at clinical translation underscores the need for innovative designs to advance theranostics for effective AD diagnosis and therapy.

Conversely, imaging modalities make recording in situ changes in ChE enzymes and Aβs activity simultaneously in the AD brain more challenging. Given the key role played by Aβs and the change in the level of ChEs during different stages of AD pathology, currently, there are no reported molecular theranostics capable of effectively probing these pathological hallmarks and simultaneously acting as inhibitors to modulate crucial AChE activity.

This study aimed to design a D-π-A-based core capable of detecting Aβs and ChEs while also serving as a ChE inhibition activity. We achieved this by adopting a hybridization approach, combining a ferulic acid-derived pharmacophore (ChE inhibitors), previously identified from our laboratory, with Aβ aggregate-specific D-π-A probes (DANIR-2c and MCCAD-3), as illustrated in Fig. 1f. We hypothesized that incorporating a donor-π-acceptor (D-π-A) architecture with an extended diene system would facilitate NIR deep tissue imaging. At the same time, the inclusion of bulky acceptor fragments would enhance lipophilicity and thereby strengthen binding to the hydrophobic domains of Aβ fibrils. Additionally, this design may enable selective modulation of cholinesterase (ChE) activity by engaging aromatic residues at the catalytic and peripheral sites of the enzymes. The presence of ester functionalities is expected to further enhance binding potency and overall efficacy.

In this work, I-43 (compound 33, in Fig. 1g) is identified as a lead theranostic fluorescent probe, exhibiting dual emission peaks at 690 and 725 nm upon binding to Aβ aggregates, with a high Stokes shift of ≥200 nm that enables high-contrast NIR imaging with minimal background interference and reduced excitation laser-mediated self-quenching. In biological screening studies, I-43 demonstrated specificity for Aβ plaques in histochemical (HC) and immunohistochemical (IHC) assays, with minimal cross-reactivity compared to other key neurodegenerative biomarkers, including tau and α-synuclein. Its favorable optical and physicochemical properties enable in vivo brain NIR imaging and Aβ quantification in preclinical models. Moreover, the emissivity at 710 nm upon interaction with AChE/BChE enables in situ monitoring of cholinesterase activity in cell-based models. I-43 also exhibits AChE inhibitory activity at low micoromolar concentrations and improves cognition in a scopolamine-induced amnesia model, demonstrating its theranostic potential. Together, these findings highlight the theranostic potential of I-43, offering a dual-targeting strategy for the diagnosis and modulation of key biochemical factors implicated in the onset of AD.

Results
Design, synthesis, and in vitro fluorescence assays
In line with the literature, an ideal in vivo NIR theragnostic for addressing the diverse neurobiology of the AD brain necessitates a small molecular probe (≤500 Da) with a substantial Stokes shift, deep-NIR emission maxima (≥650–900 nm), high signal-to-noise ratio, biocompatibility, acceptable brain permeability, favorable pharmacokinetics, non-toxicity, high binding affinity, therapeutic efficacy in the low μM range, and minimal undesirable interactions with surrounding biological components. Herein, the scaffold design was inspired by previous and ongoing research on the development of AD therapeutics39,40. A ferulic acid-tryptamine derivative (compound F24 or 7a) has been shown to possess sub-μM activity against AChE (IC50 = 5.74 ± 0.13 µM), and BChE (IC50 = 14.05 ± 0.10 µM), as seen in Fig. 1f40,41. Thus, we hypothesized that integrating ferulic acid pharmacophore with the DANIR-2c framework using a molecular hybridization approach could benefit in two ways: N,N-dimethylamine substituted donor-acceptor framework from DANIR-2c acts as recognition of misfolded protein (Aβs) with effective intramolecular charge transfer (ICT)42, and ChEs recognizing ferulic acid fragment for anticholinergic activity. The bulky groups (R) at the acceptor fragment contribute to steric management, enabling precise targeting of structural variations in ChE active site pockets. Drawing from previous research and addressing specific design challenges, this integrated approach represents a significant step forward in advancing our ability to visualize and manage biochemical factors responsible for AD onset in living organisms. It is noteworthy that Seo et al. (2016) developed an NIR probe that selectively targets tau (compound 16 or 3h, Fig. 1e), which shares partial structural similarity with our designed probes. Interestingly, our designed probes lack the methylene spacer in cyano aryl ester fragments. Further, the unique feature indicating the robustness of the designed probes is the presence of a single methoxy group at the 2nd position of N,N-dimethylaniline, and other features mentioned above25. Targeted probes were synthesized according to synthetic protocols in supplementary methods and characterized well with NMR (1H, 13C, and 19F) and high-resolution mass spectroscopy (Supplementary Figs. 1–41 and Supplementary Table 1). HPLC purity suggested that most probes were ≥95% pure (Supplementary Fig. 42 and Supplementary Table 2).

We began our screening by measuring the optical properties of the developed NIR probes in DMSO, followed by 1× PBS (containing 20% DMSO as a cosolvent) to mimic physiological conditions. UV–Vis and fluorescence analyses (Supplementary Figs. 43 and 44 and Supplementary Table 3) revealed that all probes exhibit absorption maxima at 490–495 nm, broad emission profiles with maxima in the deep NIR-I region (690–740 nm), and large Stokes shifts (~235 nm). These features support their utility in acquiring high-contrast deep tissue optical imaging, with enhanced signal-to-background ratios and minimal interference from excitation light. In addition, high molar absorptivity (ε; Supplementary Table 3) enables efficient excitation at low concentrations, thereby minimizing phototoxicity. However, the apparent reason for low quantum yields (0.0017–0.034; Supplementary Table 3) of these probes is linked to the extended polyenic π-system, which promotes nonradiative decay from the excited state and leads to fluorescence quenching. Balanced hydrophobicity and hydrophilicity are critical for ensuring target affinity, brain permeability, and aqueous solubility. Investigational studies have shown that conventional dyes are primarily bound to Aβ fibrils through hydrophobic interactions35,43,44. To leverage this interaction mode, bulky hydrophobic fragments were introduced into the acceptor region of the probes, yielding moderate cLogP values (4.82–5.95; Supplementary Table 3), which are suitable for brain targeting. We hypothesize that this hydrophobic character not only enhances selectivity towards ChEs but also strengthens binding affinity to β-sheet-rich, hydrophobic Aβ fibrils, thereby supporting dual-target engagement.

Given their favorable optical and physicochemical properties, fluorescence-based in vitro assays were conducted against Aβ pre-aggregates, β-sheet-rich Aβ fibrils, and freshly prepared bovine serum albumin (BSA) to identify candidates capable of selectively labeling pathogenic Aβ species. To assess specificity, the lead compounds were further tested against recombinant tau and α-synuclein proteins. The aggregation states of Aβ 1-40 and Aβ 1-42 (Supplementary Fig. 45a, b) were validated by ThT fluorescence assay and transmission electron microscopy (TEM), whereas pathogenic tau and α-synuclein were characterized by TEM (Supplementary Fig. 45c). As shown in Supplementary Figs. 46–47 and Fig. 2a, I-30, I-32, I-33, I-34, I-35, I-36, I-38, I-39, I-42, and I-43 exhibit distinct fluorescence enhancement in response to Aβ 1-42 aggregates, with negligible signals observed for monomeric forms. In addition, probes I-33 to I-36 showed enhanced fluorescence with BSA, indicating undesirable off-target signals and leading to their exclusion from further investigation. I-32, I-38, and I-43 demonstrated the most pronounced and selective responses toward aggregated Aβ (****p<0.0001) compared to Aβ monomers and BSA, supporting their continued investigation. These probes exhibited emission maxima at 725 nm (I-32), 720 nm (I-38), and dual peaks at 690/725 nm (I-43) upon binding to aggregated Aβ (Supplementary Fig. 47 and Fig. 2a), all falling within the deep NIR window (650–900 nm) preferred for in vivo optical imaging. This fluorescence enhancement is likely driven by sensitivity to microenvironmental polarity or restricted intramolecular rotation upon intercalation into the hydrophobic grooves of β-sheet-rich Aβ aggregates. To explore this phenomenon, solvent-dependent fluorescence studies were conducted using toluene, DCM, THF, MeOH, ACN, DMF, and DMSO (Fig. 2c, d and Supplementary Figs. 48 and 49). We found that absorbance maxima remained largely unaffected by solvent polarity changes, indicating minimal dependency on the solvent permittivity, with a 20–30 nm shift in MeOH to other aprotic solvents, possibly due to hydrogen bonding interactions. However, a bathochromic emission shift (λem) and a decrease in emissivity were observed with the increase in the solvent’s polarity (Fig. 2e and Supplementary Fig. 49). In typical TICT (twisted-ICT) fluorophores, a more polar solvent system stabilizes the excited state by promoting a stronger charge transfer character, shifting emission to red, but may increase nonradiative decay due to enhanced internal conversion from the twisted or distorted excited-state geometry. Therefore, we employed the Lippert-Mataga equation to determine the correlation between Stokes shifts (Δν; Supplementary Table 4) values of probes in different solvents based on solvent orientation polarizability [Δf(ε, n)], given in Supplementary Table 545. I-32 showed minimal sensitivity to solvent polarity, as evident from the Δν vs solvent polarizability plot (Supplementary Fig. 49a). In contrast, I-38 and I-43 displayed good linear correlations (r² values 0.870 and 0.7668, respectively; Fig. 2e and Supplementary Fig. 49b), indicative of efficient charge transfer.

We next examined the role of restricted intramolecular motion in the fluorescence response of our probes using glycerol-MeOH mixtures (0–100%), where glycerol provides a dense, viscous environment. Theoretically, a dense environment enhances emissivity in molecular rotor probes, possibly by lowering nonradiative decay. Representative probe I-43 exhibited weak emission at 645 nm (Fig. 2f) in low glycerol content (≤50%), possibly due to strong intermolecular hydrogen bonding. Beyond 50% glycerol content, I-43 behaved as a molecular rotor probe, showing gradual fluorescence increment with emission maxima at 705 nm, indicating the dominance of viscosity effects. These findings support that the fluorescence response of I-43 upon Aβ binding arises from the restriction of intramolecular motion and microenvironmental polarity.

Subsequently, I-43 also exhibited a marked fluorescence enhancement (****P≤0.0001; Fig. 2a, b) with fibrillar Aβ 1-40 species (another key pathogenic AD biomarker), compared to its monomeric and oligomeric forms, highlighting its affinity for the aggregated species. Following this, to quantify binding strength, saturation binding affinity of the probe 1-32, 1-38, and I-43 in concentration-dependent titration assay against Aβ 1-42 aggregates (5 µM). Among the tested probes, I-43 displayed the lowest dissociation constant (Kd = 104 ± 25 nM) compared to I-38 and I-32 (Kd = 279 ± 79, and 286 ± 93 nM for I-38 and I-32, respectively; Supplementary Fig. 50a). Notably, I-43 also showed higher binding affinity to Aβ 1-40 and Aβ 1-42 fibrils (Kd = 58.19 ± 9.7 nM and 104 ± 25 nM, respectively; Fig. 2g and Supplementary Fig. 50a) compared to their oligomeric forms (Kd = 213 ± 44 nM and 337 ± 112 nM, respectively; Supplementary Fig. 50b). With a low binding constant, I-43 joins the club of Aβ aggregate specific NIR dyes with ≤200 nM affinities, such as DANIR-2c, CRANAD-2, MCAAD-3, DNTPH, and QM-FN-SO3 with Kd = 26.9, 38.69, 106, 143, and 170 nM, respectively29,35,46–48. The fluorescence response of I-43 (5 µM) with Aβ 1-40 fibrils demonstrates a strong quantitative correlation (r² = 0.97), as shown in Fig. 2i, with corresponding fluorescence spectra in Fig. 2h.

ThT-based assays are a widely used method for tracking time-dependent fibril growth and assessing the anti-amyloidogenic potential of various probes49–51. In a subsequent experiment, the ability of I-43 to attenuate the aggregation of monomeric Aβ 1-40 and Aβ 1-42 peptides into aggregated species was evaluated. Interestingly, I-43 displayed minimal inhibitory activity on the conversion of Aβ 1-42 monomers to aggregates, as evident through the ThT fluorescence assay (Supplementary Fig. 51). While TEM images (Fig. 3a) showed that at a twofold concentration of I-43: Aβ 1-40 formed amorphous clusters instead of apparent fibrils. At fourfold the concentration of I-43, mostly short or broken fibrils appeared, suggesting that I-43 can interfere with fibril formation, especially at a 4:1 probe-to-peptide ratio. These results are consistent with a significant reduction in ThT fluorescence intensity (****P<0.0001) compared to untreated samples (Fig. 3b1, b2), with a similar trend observed in the ThT-based aggregation kinetics study conducted over 72 h (Fig. 3b3). While in vitro experiments provide useful insights, further in situ and in-vivo studies are essential to validate these findings in a biological context.

Similar to AD pathogenesis, the accumulation of misfolded proteins into aggregates is indeed a key feature in several neurodegenerative diseases, such as α-synuclein in Parkinson’s disease (PD), and tau oligomerization, or transactive response DNA/RNA-binding protein (TDP-43) in frontotemporal dementia52. Therefore, to assess selectivity, fluorescence spectra of I-43 were recorded with various amyloidogenic proteins. As shown in Fig. 3c, d, I-43 displayed significantly higher fluorescence intensity with Aβ 1-40 fibrils (****P<0.0001) compared to monomeric and fibrillar forms of tau and α-synuclein, confirming its preferential affinity for β-sheet amyloid aggregates. DFT calculations using Gaussian 16 were performed to examine electronic transitions and ICT behavior in I-32, I-38, and I-43. A lower energy gap between frontier orbitals, i.e., highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), is often associated with bathochromic shifts in emission maxima, bringing fluorescence into the NIR region of fluorescence spectroscopy53. Also, the energy gap between these orbitals ascertains the stability and chemical reactivity of the compounds54,55. Therefore, geometry optimization for the representative probes I-32, I-38, and I-43 was performed using B3LYP functional with 6-311G++(d,p) basis sets in the gas phase, followed by their frequency calculation (HOMO and LUMO states; Supplementary Table 6) to validate the ICT phenomenon and global minimum isomer of I-43, depending on the E and Z orientation of the double bonds (Supplementary Fig. 52). The data presented in Fig. 3e and Supplementary Table 6 strongly suggested that in all the probes, the electron distribution is primarily localized on extended polydienes with maximum electron density residing to the dimethylaniline (donor fragment) in HOMO states, which is delocalized towards cyano (acceptor) in LUMO state corresponds to an ICT event within designed D-π-A scaffold. The energy gap between the orbitals increases in the order I-43 < I-38 < I-32, indicating that I-43 has the lowest stability and, thus, the highest reactivity, consistent with the Kd values.

In Aβ aggregates, the fibril core is predominantly composed of hydrophobic residues (Leu17, Phe19, Phe20, Val24, Ala30, Ile31, Ile32, Leu34, Met35, Val36, Val39, Ile41), while hydrophobic patches on the fibril surface are formed by residues Val18, Ala21, Val40, and Ala4256. To investigate the binding mechanism, electrostatic potential mapping of the optimized I-43 conformer was generated using DFT, vividly illustrating its polarization (Fig. 3f). The hydrophobic benzothiazole cyanate ester fragment generates repulsive interactions (red arrowheads), while the hydrophilic dimethylaniline fragment results in attractive interactions (blue arrowheads). Mechanistically, this unique characteristic suggests that the hydrophobic interactions, driven by the benzothiazole cyanate ester fragment, likely facilitate initial contact with the hydrophobic regions of the aggregates. Concurrently, the hydrophilic dimethyl aniline fragment enhances binding stability through polar interactions with the surrounding aqueous environment or other polar residues in the Aβ structure. These dual interactions improve the specificity and detection property of I-43 in targeting and binding to Aβ aggregates, making it a promising candidate for further investigation in amyloid imaging and potential therapeutic applications. Othersalient features, such as ≥80% cell viability measured in PC12 cells up to 10 µM after 24 h of incubation in MTT assay (Fig. 3g) and ≥95% photostability in physiological buffer (pH 7.4) for up to 6 h (Supplementary Fig. 53), corroborate the safety and biocompatibility of I-43 in the living organism.

Methods
Synthesis and characterization of compounds
A detailed description of the synthesis, experimental procedures, characterization data (Supplementary Figs. 1–41 and Supplementary Table 1), and purity analyses (Supplementary Fig. 42 and Supplementary Table 2), is provided in the Supplementary method section of Supplementary Information.

Protein aggregation assays
Preparation of oligomeric and fibrillar Aβ species. Aβ 1-42 fragments (lyophilized powder, purity >95%, CAS Number: DG-105020; DG peptide Co. Pvt. Ltd., China) were treated as described previously, with minor modifications86. Briefly, a solution of Aβ 1-42 peptide 1.0 mg/mL in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; TCI) was aliquoted into ten microcentrifuge tubes and dried under a gentle nitrogen stream to remove HFIP. The resulting peptide films were stored at −78°C until use. Immediately prior to oligomer preparation, the HFIP pretreated and dried peptide film was dissolved in 60 mM NaOH (4°C), and the concentration was determined by absorbance (NanoDrop™ 2000/2000c; Thermo Scientific). The stock was then diluted in 20 mM phosphate-buffered saline (PBS; pH 7.4) to a final concentration of 20 µM and incubated at room temperature for 12 h without agitation to favour oligomer formation. The sample was filtered through a 0.2 µm membrane filter (Millipore), and the filtrate was collected as smaller Aβ aggregates87. For Aβ 1-42 aggregation experiments, the peptide stock solution was diluted to 20 µM in 20 mM PBS (pH 7.4) and incubated at 37°C for 72 h under continuous agitation (300 rpm) in an orbital shaker (Eppendorf ThermoMixer C) to promote fibril formation. Changes in aggregation state were evaluated using the ThT fluorescence assay (λex = 445 nm; emission scan 475–650 nm; BioTek Synergy H1 microplate reader) and TEM.

Preparation of oligomeric and fibrillar Aβ species. The well-optimized protocol from the following reference article was used88. Briefly, 1.0 mg of Aβ 1-40 peptide was dissolved in 1.0 mL HFIP, aliquoted into ten microcentrifuge tubes, and dried under nitrogen, followed by storage at −78°C until further use. For oligomer formation, the peptide solution was diluted with PBS buffer (pH 7.4) to achieve a final concentration of 20 µM and left at room temperature for 12 h without agitation. TEM analysis revealed the conversion of monomeric species to oligomers or small aggregate formation. For fibrillization, dissolve the dried peptide film in a 1% ammonium hydroxide (NH4OH; Cat. No. 221228-25ML, Sigma-Aldrich) solution to a concentration of 1 mM. Vortex briefly to ensure complete dissolution. The peptide solution was diluted to a concentration of 20 µM with 20 mM PBS (pH 7.4) and stirred at 37°C with constant shaking (300 rpm) for 72 h. The formation of aggregates was confirmed using TEM and a ThT fluorescence assay.

Tau aggregation protocol. Following a modified protocol, E. coli-derived human Tau protein (Cat. # SP-495-100, R&D Systems, USA) was used for aggregation89. An assembly buffer consisting of 10 mM HEPES, DL-dithiothreitol (7.7 mg; CAS 3483-12-3; SRL, India), and NaCl (58.44 mg) was prepared. A 50 µM Tau protein stock solution was subsequently diluted in this buffer to obtain a 21.79 µM working solution (60 µL). Aggregation was induced using heparin (108.9 µM, 3 µL; CAS 9041-08-1; Tocris Bioscience, UK), and the resultant mixture contained 19.8 µM Tau and 4.95 µM heparin. The sample was incubated at 37°C for 72 h with continuous agitation (300 rpm) in a thermomixer to promote fibrillization, and aggregation was confirmed using TEM analysis.

α-synuclein aggregation protocol. For α-synuclein aggregates, E. coli-derived human α-synuclein protein (Met1-Ala140, Cat. No. SP-485-500, batch: DRDU0223121, R&D Systems) was used following a reported protocol with minor modifications90. The assembly buffer was composed of 10 mM Tris-Cl (pH 7.4), 1 mM EDTA (Cat. No. E9884; Sigma-Aldrich), and 10 mM NaCl. The protein was diluted to a final concentration of 100 µM in this buffer and incubated at 37°C for 10 days with continuous agitation at 300 rpm in a thermomixer. Aggregation was confirmed using TEM analysis.

TEM study. The TALOS S transmission electron microscope (200 kV; Thermo Fisher Scientific) was used to examine the morphological characteristics of the samples and is housed at the Sophisticated Analytical Instrumentation Facility (SAIF), AIIMS, New Delhi, India. Samples were diluted to 5 µM in their respective assembly buffers. Subsequently, 5 µL of each sample was applied to glow-discharged, carbon-coated copper grids (300 mesh; Cat. No. 01843, Ted Pella) and allowed to adsorb for 30 min at room temperature, after which excess solution was removed by blotting. Finally, the grids were briefly treated with 2% uranyl acetate dihydrate (Cat. No. 81405, SRL Chem) for 10 s to provide negative contrast for TEM imaging. After blotting and air-drying, the grids were imaged using TEM.

In vitro fluorescence response of probes with Aβ species
To the 40 µM solutions of test probes (30 µL) in 1× PBS (pH 7.4) with 10% DMSO as a cosolvent, various forms of Aβ 1-40 or Aβ 1-42 (30 µL, 20 µM) peptides suspension in PBS (pH 7.4), or with a freshly prepared solution of BSA (Cat. No. MB083, HIMEDIA) in PBS (30 µL; 25 µg/mL) was added. The probe and proteins were mixed in a 2:1 molar ratio in the final solution and incubated for 1 h at 37°C with gentle, constant shaking at 300 rpm. Subsequently, the mixture was transferred to a black 384-well plate (Genetix, Biotech Asia Pvt. Ltd) and subjected to fluorescence reading on a multi-mode microplate reader (SpectraMax i3× Platform, Molecular Devices). The tested probes in 1× PBS alone were also measured to record spectra under the same parameters. For fluorescence characterization, the excitation wavelength (λex) was set at 490 nm and spectral graphs were record over a range from 550 to 850 nm.

Comparative fluorescence analysis of I-43 with Aβ fibrils, tau, and α-synuclein. Aβ 1-40 fibrils, tau proteins, and α-synuclein proteins were each diluted to a final concentration of 10 µM in their preparation buffers (pH 7.4). Stock solutions of I-43 were prepared in DMSO (Cat. 34869-100 mL, Sigma-Aldrich) and added to the protein solutions to obtain mixtures containing 5% (v/v) DMSO and a 2:1 molar ratio of probe to protein. The samples were incubated for 1 h at 37°C with orbital shaking at 300 rpm to ensure equilibrium binding. Fluorescence emission spectra were recorded on SpectraMax i3× reader in black 384-well plates. Measurements were performed at an excitation wavelength of 490 nm, and emission spectra were collected from 550 to 850 nm. Spectra of the probe alone in PBS were recorded under identical conditions as controls.

Saturation binding affinity assay
Saturation binding analysis was conducted according to the protocol optimized in our laboratory91. The binding constant (Kd) of lead probes I-32, I-38, and I-43 for Aβs (oligomers and fibrils of Aβ 1-40 or Aβ 1-42) was measured in PBS solutions containing 10% DMSO as a cosolvent. Briefly, various Aβ aggregated species (5 µM) were admixed with varying concentrations of the probes (5–0.001 µM) and incubated at 37°C with constant and gentle shaking on an orbital shaker (300 rpm) for 1 h. Fluorescence emission at 690 nm (λex = 490 nm) was recorded using a SpectraMax i3× plate reader. Binding constants (Kd) were obtained by nonlinear regression (one-site binding model) in GraphPad Prism 8.0, following the equation: y = Bmax × x / (Kd + x) (1), where the probe concentration (nM) is represented by x, the fluorescence signal by y, and the maximal binding by Bmax. Identical conditions were used to measure the affinity of I-43 for Aβ 1-40 fibrils and Aβ 1-40 oligomers.

Brain tissue sections preparation and staining protocol
APP-PS1 and C57BL/6 mice, used for brain tissue slides preparations as well as for the in vivo and ex vivo fluorescence studies, were obtained from the NII, New Delhi. All animal experiments were performed in accordance with institutional ethical guidelines and approved by the Institutional Animal Ethics Committee (IAEC; Reg. No. 38/GO/ReBi/SL/99/CPCSEA; dated 20 March 2017; IAEC approval #512/19). The animals were maintained in the animal housing facility in well-ventilated cages under standard conditions (22 ± 2°C, 50–60% humidity, 12-h dark-light cycle) with ad libitum food and water supply. Sex was not used as a biological variable in any aspect of the study design or analysis. For brain tissue specimens, intact brains were excised from 6- and 13-month-old APP-PS1 mice and 13-month-old C57BL/6 wild-type (WT or B6) mice after transcardial perfusion with ice-cold 1× PBS (pH 7.4), followed by 4% paraformaldehyde (PFA). After ward, the brains were post-fixed in 4% PFA for 72 h at 4°C, then cryoprotected in 15% and subsequently 30% sucrose in PBS at 4°C until tissues sank. Samples were embedded in optimal cutting temperature (OCT) compound, cryosectioned horizontally at a thickness of 12–15 µm (unless specified) using a Leica CM1520 cryostat, and mounted onto glass slides. Finally, slides were air-dried and stored at −20°C until staining.

Histochemical (HC) staining in mouse brain sections. Cryosection tissues were brought to room temperature and rehydrated through graded ethanol (100%, 90%, 75%, 50%, and 30% in deionized water; 5 min each) followed by PBS. Sections were first stained with reference dyes for 30 min and then washed with 50% and 30% ethanol (each for 3 min). Working concentrations were: Thioflavin T (ThT; 0.1% aqueous; Cat. No. T3516-25G, Sigma-Aldrich), Thioflavin S (ThS; 0.1% aqueous; Cat. No. T1892, Sigma-Aldrich), CRANAD-2 (20 µM in water with 10% DMSO as cosolvent; gift from Prof. Ran Chongzhao, Hravard Medical School), Congo Red (0.1 mg/mL in 50% ethanol/water; Cat. No. C6277-25G, Sigma-Aldrich), and DANIR-2c (10 µM in water with 10% DMSO as cosolvent; in-house synthesized). Sections were then counterstained with test probes (25 µM; compounds 1-32, I-38, or I-43 prepared in deionized water containing 5% DMSO and 1.5% Cremophor EL) for 1 h, washed as described above, and mounted in an antifade medium comprising 2.5% (w/v) 1,4-diazabicyclo[2.2.2]octane (DABCO; Cat. D27802-25G, Sigma-Aldrich) in 90% glycerol and 10% PBS (pH 7.4) before glass cover mounting. In select experiments, variable concentrations of I-43 (0.01–10 µM prepared in 10 mM PBS pH 7.4 and 5% DMSO as a cosolvent) were used as specified in the figure legends. Confocal imaging was performed using an LSM780 laser-scanning microscope (Carl Zeiss) equipped with an Imager.Z2 stand and an AxioCam MRc camera, with fluorophore-appropriate excitation and emission settings, unless specified. Congo Red-stained sections were imaged on a Nikon fluorescence microscope (NIS-Elements BR 4.30.00, 64-bit) using differential interference contrast (DIC) and TRITC filter sets. All images were acquired with consistent exposure and detector settings and processed uniformly using Zeiss ZEN (v3.10.103) software.

Methenamine silver staining. Methenamine-silver staining was performed based on the protocol by Haga et al. (1994) with slight modifications92. After oxidation in 0.25% potassium permanganate for 10 min, sections were reduced in 1% sodium metabisulfite and then cleared in 0.5% oxalic acid, followed by rinsing. Sections were incubated with I-43 (10 µM, 1 h) and then rinsed three times with H2O (5 min each). Silver impregnation was performed using a freshly prepared working solution of 3% methenamine (Cat. No. 398160-5G, Sigma-Aldrich) and 5% silver nitrate (AgNO3; Cat. No. 94118, SRL) in buffer (prepared as described in Haga et al.), and developed at ~75°C until reaction was visible. Slides were rinsed, fixed in 1% sodium thiosulfate (1 min), and treated with a solution of 0.3% gold(III) chloride (Cat. No. MB291, HIMEDIA) in 1× PBS to enhance contrast. Thesections were rinsed with distilled water, dehydrated, cleared, and mounted. Visualization was performed using a DIC filter for silver stain and a TRITC filter for I-43 fluorescence (NIS-Elements BR 4.30.00 64-bit).

Immunohistochemical (IHC) staining in mouse brain sections. Cryosections were rehydrated and subjected to antigen retrieval in 70% formic acid for 20 min, followed by quenching in 0.3% hydrogen peroxide (H2O2) for 10 min. Sections were permeabilized with phosphate-buffered saline containing 0.4% Triton X-100 (PBST) for 20 min and blocked in 5% BSA with 0.3% Triton X-100 in 1× PBS (pH 7.4) for 2 h at room temperature. Primary antibodies (sources and dilutions in Supplementary Table 7) were applied overnight at 4°C in blocking buffer for 14–16 h. The following day, sections were washed three times for 10 min each with PBST and then incubated with fluorophore-conjugated secondary antibodies for 2 h at room temperature. After another three PBST washes (each for 5 min), the sections were incubated with I-43 (10 µM, in 5% DMSO and 1.5% Cremophor EL in water) for 1 h, washed, and then mounted using mounting medium. In addition, a 1 µg/mL solution of 4′,6-diamidino-2-phenylindole (DAPI; Cat. D9542-1MG, Sigma-Aldrich) in 1× PBS (pH 7.4) was used for nuclear staining in triple-staining experiments. Sections were incubated with DAPI for 5 min at room temperature, followed by three washes in PBS prior to mounting, where applicable.

HC and IHC staining of human post-mortem brain tissues. Formalin-fixed, unstained post-mortem brain sections from donors diagnosed with Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), and non-demented controls (CT) samples were received from the Human Brain Tissue Repository (Brain Bank), NIMHANS, Bangalore, India, for academic research use. To maintain donor anonymity while distinguishing biological replicates, each specimen was assigned a unique identifier: AD-1 to AD-5 (five independent AD cases), FTD-1 and FTD-2 (two FTD cases), PD-1 and PD-2 (two PD cases), and CT-1 to CT-4 (four non-demented control samples). Each label corresponds to tissue obtained from a different individual donor. The brain bank obtained ethical approval from the Institutional Ethics Committee (IEC No. NIMHANS/IEC(BS&NSDIV.) 2023) and written informed consent for autopsy and research use from the next of kin. The sampled brain regions, donor age (in years), and sex (female, F; male, M) are reported in the relevant figure panels and legends in both the main manuscript and Supplementary Information. The study was sex-unbiased, and additional clinical metadata were not provided by the brain bank. HC and IHC staining were performed following established protocols93.

The typical HC staining of autopsy brain sections involved routine deparaffinization in xylene (three times, 10 min each), rehydration through graded alcohols, and rinsing in PBS. To minimize tissue autofluorescence (e.g., lipofuscin), sections were oxidized with 0.25% potassium permanganate for 10 min, reduced in 1% sodium metabisulfite, and cleared in 0.5% oxalic acid. For comparison, a subset of sections was processed without this oxidation step to assess potential interference and probe affinity toward endogenous auto-fluorescent deposits. The next step involved PBS rinsing, followed by sequential staining with reference dyes (ThT, Congo red, and CRANAD-2) and then test probes, using the same staining conditions described for mouse brain sections. Following the oxidation-reduction-clearing procedure to reduce endogenous autofluorescence, IHC staining on human sections was performed using the same protocol described above for mouse brain IHC, with identical conditions for antigen retrieval, blocking, antibody incubation, and washing. Confocal imaging was performed using an LSM780 laser-scanning microscope (Carl Zeiss) equipped with an Imager.Z2 stand and an AxioCam MRc camera, with fluorophore-appropriate excitation and emission settings, unless specified. Congo Red-stained sections were imaged on a Nikon fluorescence microscope (NIS-Elements BR 4.30.00, 64-bit) using DIC and TRITC filter sets (to locate I-43). All images were acquired with consistent exposure and detector settings and processed uniformly using Zeiss ZEN software.

For α-synuclein IHC staining. Sections were incubated with the primary antibody (p-Ser129-αSyn; Cat. PA5-37740, Thermo Fisher Scientific) for 16 h at 4°C and then washed in Tris-buffered saline (pH 7.4). Briefly, the primary antibody was applied following antigen retrieval and blocking as described above. Antigen-antibody complexes were visualized using the VECTASTAIN® ABC-HRP Kit (Peroxidase, Rabbit IgG; Vector Laboratories, Cat. No. PK-4001), and the signal was developed with 3,3′-diaminobenzidine (DAB; Cat. D12384, Sigma-Aldrich) as the chromogenic substrate. The next step involved staining with I-43 using the above-mentioned protocol.

Fluorescence imaging in Drosophila melanogaster
We procured the Drosophila melanogaster strains UAS-Aβ42/CyO and ey-GAL4/CyO (eye-specific driver line) from the Bloomington Drosophila Stock Center (Indiana, USA). Male ey-GAL4/CyO flies were crossed with virgin UAS-Aβ42/CyO females to generate F1 progeny (ey-GAL4>UAS-Aβ42/CyO) that exhibit the Aβ42-induced eye degeneration phenotype. The ey-GAL4/CyO line was used as the corresponding genetic control. All flies were reared on standard cornmeal-agar medium and maintained at 28 ± 1°C in a BOD incubator under controlled environmental conditions with a 12-h day-night cycle.

For ex vivo imaging, third-instar larvae were dissected in chilled 1× PBS (pH 7.4) to isolate the eye imaginal discs. The dissected tissues were fixed in 4% PFA for 15 min at room temperature, permeabilized with 0.2% PBST for 30 min, and incubated with I-43 (10 µM in 50% ethanol/water) for 1 h in the dark prior to imaging. After 1× PBS and 50% aqueous ethanol wash (3 times each, 5 min), discs were mounted on glass slides in DABCO-based antifade medium and imaged using a Zeiss LSM780 confocal microscope. Orthogonal views were generated using ZEN (v3.10.103).

For in vivo fluorescence imaging, 15-day-old wild-type (Oregon R+) and AD model (ey-GAL4-UAS-Aβ42/CyO) flies (n ≥ 30 per group) were kept fasting for 2 h, followed by transfer to corn-agar plates containing I-43 (50 µM) supplemented with yeast and maintained at 28 ± 1°C. Intact eye fluorescence was imaged after 2 h of feeding using a Nikon Ri-1 color CCD camera equipped with the Unique Extended Depth of Focus module to capture a fully focused composite view of the entire eye. ROI measurements, including fluorescence intensity and area, were quantified using NIS-Elements (BR) software.

Fluorescence imaging and biodistribution analysis in mice model
For in vivo fluorescence imaging, 6- and 13-month-old APP-PS1 mice and 13-month-old B6 mice, serving as controls, were obtained from the NII, New Delhi. Animals (n = 3 per group) had their fur shaved on the scalp to minimize interference and were administered 100 µL of I-43 (0.8 mg/kg prepared in 1× PBS and 7.5% DMSO, 2.5% Cremophor® EL, and 90% PBS) administered intravenously through tail vein injection using a 30G insulin syringe. Imaging was performed on a LAGO X optical imaging system (Spectral Instruments Imaging, USA) equipped with a high-sensitivity, deep-cooled CCD camera, using 500/690 nm excitation/emission filters, and an exposure time of 30 s under identical acquisition parameters for all groups. Animals were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg), which provided a stable surgical plane of anesthesia for approximately 120 min. Throughout the procedure, body temperature was maintained at 37°C using a temperature-controlled heating platform. Repeated anesthetization was applied for sequential imaging when necessary. Images were acquired, and regions of interest (ROIs) were quantified using Aura Analysis Software (version 4.5.0).

For ex vivo fluorescence brain imaging, 13-month-old APP-PS1 mice and age-matched C57BL/6 wild-type (WT) controls received I-43 (0.8 mg/kg, i.v.). Twenty minutes after probe administration, the animals were anesthetized and transcardially perfused first with 50 mL of ice-cold 1× PBS, followed by 50 mL of ice-cold 4% PFA. Brains were removed, rinsed briefly in PBS to remove residual fixative, and then post-fixed for 15 min in 4% PFA at 4°C. They were stored on ice prior to imaging. Whole-brain fluorescence imaging was performed using the LAGO X optical imaging system (Spectral Instruments Imaging) under standardized acquisition conditions (excitation: 500 nm; emission: 690 nm; binning: 8; exposure: 30 ms; f-stop: 2; field-of-view: 10; excitation power: 10%; object height: 1.5 cm). Image acquisition and ROI-based quantification were conducted using Aura Analysis Software (version 4.5.0), with identical settings applied to all samples. Following ex vivo imaging, brains were cryosectioned as described above, counterstained with DAPI, and subjected to confocal microscopy (LSM 780, Carl Zeiss) to evaluate the distribution of amyloid-β plaques and the localization of I-43 in WT vs APP-PS1 brain tissues.

For organ-level biodistribution, 6-month-old APP-PS1 mice (n = 4 independent animals; either sex) received I-43 (0.8 mg kg−1, i.v.). After 20 min, the mice were perfused under anesthesia conditions exactly as above. Major organs (liver, spleen, kidney, brain, lung, and heart) were dissected, rinsed in PBS, post-fixed for 15 min at 4°C in 4% PFA, and kept on ice until imaging. Intact organs were imaged using the same LAGO X setup and acquisition parameters described in the brain-imaging section. ROIs were manually drawn around each organ, and total radiant efficiency values were then extracted using identical quantification settings for all samples.

In vitro plasma and liver microsomal stability of I-43
For plasma stability assays, I-43 (25 µM) was incubated with 200 µL of mouse plasma (pooled Swiss Albino plasma containing heparin as anticoagulant) at 37°C for 10, 60, 120, and 150 min in capped microcentrifuge tubes. At each time point, reactions were quenched with 800 µL of acetonitrile (ACN; ice-cold), vortexed, and centrifuged at 3370×g for 10 min at 4°C (Remi C-24 Plus). The supernatant was passed through a 0.2 µm syringe filter (13 mm; Thermo Fisher Scientific, Code LSPV2013NS) and analyzed immediately by HPLC (Agilent Technologies, USA). Chromatographic separation was performed using a Shodex C18-4e column with a mobile phase of acetonitrile/water (88.5:11.5, v/v) at a flow rate of 0.65 mL min−1, injection volume of 20 µL, and detection at 470 nm (run time 6 min). The flow rate was adjusted to improve the resolution between I-43 and its metabolite, Met-1, resulting in a minor shift in the retention time of I-43 compared to previous runs.

Liver microsomal stability studies were performed by TCG Lifescience (Chembiotek, Kolkata) on a payment basis. I-43 (10 µM) was incubated at 37°C with inactivated MLM and fresh MLM (1 mg/mL, pooled) in the presence and absence of NADPH cofactor (2.4 mM) in PBS (100 mM, pH 7.4) at different time points. To terminate the reaction, three volumes of ice-cold acetonitrile were added, and the mixture was adequately mixed. After centrifugation (4000 r.p.m. at 15°C for 20 min), the supernatant was transferred to a DW96 plate, evaporated under nitrogen, and reconstituted with a mixture of acetonitrile/methanol in a 1/2 ratio (v/v). Matrix blank samples (Matrix Blank) containing all the constituents without the test compound and a 60-min sample without the NADPH cofactor (60 NCF) were also prepared and analyzed by LC/MS and LC/MS/MS. The analysis condition is as follows: Analysis platform: API 6500+QTRAP, Shimadzu LC, PAL autosampler. Source polarity: +ESI & −ESI. LC conditions: Kinetex biphenyl 4.6×50 mm, 2.6 µ and YMC C18 2.0×10 mm, 4 µ. A: H2O with 0.1% formic acid. B: 60/30/10; methanol/acetonitrile/water with 0.1% Formic acid and 1.25 mM Ammonium acetate. Flow rate: 0.900 ml/min; Elution type: Gradient elution; Injection volume: 5–10 µL. Wash Solvent: Weak Wash: 40/60 ratio of methanol/water (v/v) containing 0.1% Formic acid. Strong Wash: 40:40:20 ratio of methanol/acetonitrile/water (v/v/v) with 5 mM ammonium acetate.

Ex vivo fluorescence response of the developed probes against cholinesterase enzymes
PC12 cells were differentiated with nerve growth factor-β (NGF-β, 75 ng mL−1; Sigma, H9666) for 5–6 days. Differentiated cells were gently dissociated and seeded onto Nunc™ 12-mm glass-based dishes (Thermo Fisher Scientific, Cat. No. 150680) and allowed to adhere overnight prior to experiments. Assessment of intracellular acetylcholinesterase (AChE) activity was performed by adapting the apoptosis-induction procedure of Tang et al. (2019) with minor modifications23. Cells were treated with phorbol-12-myristate-13-acetate (PMA, 1 µg mL−1, 60 min), followed by incubation with I-43 or I-42 (10 µM, 60 min). For inhibitor controls, cells were pre-incubated with 10 µM solution of Donepezil hydrochloride (DPZ; Sigma-Aldrich, Cat. No. D6821-10MG) for 60 min prior to probe loading. In parallel, cells treated with I-43 alone (10 µM, 60 min) served as baseline fluorescence controls. Confocal fluorescence and DIC imaging were performed on a ZEISS LSM980 microscope (AxioObserver. Z1/7) using a 40× immersion Plan-Apochromat objective (water or oil, as per sample requirements) together with Nomarski optics for DIC. I-43 or I-42 was excited at 577 nm using a tunable laser line, and fluorescence was collected at 561–695 nm using GaAsP-PMT detectors. Integrated fluorescence density was quantified from confocal images processed in ZEN Blue (Zeiss) with all acquisition parameters (laser power, gain, and pinhole) kept strictly constant across all constructs and experimental replicates. Images were exported as uncompressed TIFF files for downstream analysis. Fluorescence quantification was performed in ImageJ (v1.54g; NIH, USA) using identical analysis parameters for all samples. For each construct, the corresponding red-channel fluorescence images were opened individually in ImageJ. Region of interest (ROIs) were defined using a fixed (50×50) pixel square selection box, which was applied uniformly across all images. Four cells per image were selected based on clear visualization of cell boundaries without saturation artifacts. For each ROI, Integrated Density (sum of pixel intensities), Area, and Mean Gray Value were recorded using the “Measure” function. A total of n=3 independent images per construct were quantified, yielding 12 ROIs per construct (4 cells × 3 images). All quantification steps were performed blind to sample identity. Data were compiled in source data file and analyzed using GraphPad Prism.

Scopolamine-induced cognitive impairment models. Cognitive behavior was assessed using the Y-maze and Morris Water Maze (MWM) paradigms in a mouse model of scopolamine-induced amnesia. Cognitive impairment was produced by intraperitoneal (i.p.) administration of 3 mg/kg scopolamine hydrochloride (SP; Cat. No. S0929-1G; Sigma-Aldrich). DPZ (1 mg/kg, i.p.; Sigma-Aldrich, Cat. No. D6821-10MG) was used as the reference therapeutic compound. Test compounds I-42 and I-43 were formulated in 0.5% Tween-80 in deionized water and the resultant suspension was administered intraperitoneally at 5 or 10 mg/kg. Swiss albino mice (33±3g; n=6 per group, either sex) were randomly allocated into eight experimental groups: untreated control, vehicle, scopolamine alone, donepezil, I-43 (5 or 10 mg/kg), and I-42 (5 or 10 mg/kg). Treatments were given once daily for seven consecutive days. On day 7, animals were administered scopolamine 30 min after the last treatment (excluding the control and vehicle groups). Spatial working memory was evaluated using the Y-maze 15 min later. The maze consisted of three identical arms (A, B, and C) arranged at 120° angles. Each mouse was placed at the center and allowed to explore freely for 5 min, while the sequence of arm entries was recorded. An arm entry was scored when all four paws of the mouse entered the arm. Spontaneous alternation, defined as consecutive entries into three different arms without repetition (e.g., ABC, ACB, BAC, BCA, CAB, CBA), was analyzed from video recordings by an investigator blinded to treatment. Total arm entries were plotted to verify that locomotor activity was unaffected across groups. The spontaneous alternation (%SA) percentage was calculated as: %SA = (Number of alternation / Total arm entries - 2) × 100 (2).

In a separate cohort of B6 mice (n = 6 per group), the same dosing schedule was used for vehicle, DPZ, SP, and I-43 (10 mg/kg). The Y-maze test was conducted on day 4, followed by MWM training on days 5–6 (three trials/day). Escape latency (the time it took to find the submerged platform) was averaged across trials, and a probe trial on day 7 assessed memory retention by measuring the time spent in the target quadrant. Data are presented as mean ± SD and analyzed using one-way ANOVA followed by Tukey’s post hoc test (GraphPad Prism v8.0.1). Escape latency was calculated as the mean latency across trials, where t mean is mean escape latency, ti = time taken by the mouse in trial, and n = total number of trials. t mean = (Σ n i=1 t i) / n (3).

Statistics and reproducibility
Statistical analyses were performed using GraphPad Prism v8.0.1, and plots were generated using OriginPro v9.0. Data are presented as mean ± SD or SEM, with the number of independent experiments (n) indicated in the corresponding figure legends. Unless otherwise specified, each experimental condition was replicated in at least three independent experiments to ensure robustness and reproducibility. In addition, the statistical tests used for each analysis (one-way ANOVA or t-test, as appropriate) are indicated in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Aβ and AChE play important roles in the development of AD, and the theranostic probes could provide early diagnosis and\nmitigate or slow down late-stage severity in AD. In this manuscript, the author developed a D-π-A based NIR-I probe I-43\nthat shows a good affinity towards Aβ and a moderate AChE inhibition (IC50 = 0.38 µM). Also, it displays a desirable NIR\nimaging potency and improved cognitive function, showing its potential in integrating diagnosis and treatment of AD. The\nauthor also conducted the brain permeability, acute oral toxicity study and behavioral study of I-43. Overall, this is a\nsystematic work, and suitable for publication in Nature Communications after addressing the following questions.\n(1) The lead probe I-43 has the strong affinity for Aβ plaques, however, what is it affinity for soluble Aβ species? Whether the\nprobe could attenuate the propagation of soluble Aβ species into Aβ aggregates? And, the binding mechanism of the probe\nI-43 to Aβ plaques needs to be discussed.\n(2) Structurally, the probes developed by the author in the manuscript is very similar to 7 or 3h (Seo et al. 2016), so the\nauthor should also test the effects of these probes against tau. And, more species should be evaluated for the selectivity of\nthe probes, especially those proteins related to neurodegenerative disease.\n(3) The distribution and principal metabolic pathway of the probe I-43 should be investigated. Quantitative uptake value of\nthe probe I-43 in the brain should also be measured.\n(4) Although the probe I-43 has a good photostability, it will ultimately be used for the diagnosis and treatment of AD. So the\nauthor should explore the stability of the lead probe I-43 in vivo.\n(5) Page 2, line 52. The structures of Quinolinium-based carbazole and naphthalene cyanine probes, hybrid probes (PiB-C\nand TBT) should be provided in Figure 1.\n(6) The cis and trans conformations of the structures in Figure 1B and 1C are different. Please correct.\n(7) Page 7, line 190. The higher reactivity and stability seem to be contradictory.\n(8) Compound I-40 contains a CF3 in its structure, and its 13C NMR exhibits splitting. Please supplementary coupling\nconstant.\n(9) Reference 28 is incomplete, and reference 33 does not have an abbreviated journal name.", "reviewer_2": "The author presented a comprehensive manuscript on the development of novel NIRF probe for detecting amyloid-beta and\ncholinesterase in Alzheimer disease models. The author include molecular docking, toxicity, in vitro, in vivo assessment of\nthe new probe. Despite the extensive data and merits of the manuscript, there are a few concerns regarding the probe.\nMain points:\n1. Fig. 2 The fold change is about 50% increase as shown in Page 7 comparing binding to aggregated Amyloid-beta fibril\nwith monomeric Amyloid-beta and BSA. BSA is in high abundance in living mice. This increase is not sufficient in my\nopinion (and comparing to the other amyloid NIRF probes have been used). Can the author please compare the probe with\nprevious published amyloid NIR probes such as AOI-987, HS-169, CRANAD-2, ZW800–1C.\n2. The detailed information regarding the demographics (age, sex) as well as pathological features of AD brains are not\npresented. Please follow the ‘Sex and Gender Equity in Research – SAGER – guidelines’ and to include sex and gender\nconsiderations for studies involving humans, vertebrate animals and cell lines where relevant to the topic of study. The\nauthor says that postmortem AD human brain tissues (92-year-old, male and female, n=5) were used. It is confusing to\nreader if all the cases are 92 year old? In addition, only one staining of amyloid-beta/ThT was presented for the postmortem\nhuman brain without quantification. Please provide a quantification of the amyloid-beta (sensitivity and specificity) in 5 AD\ncases and control cases.\n3. Please provide staining using 6E10 or 4G8 with the probe on the mouse and human brain. ThT and ThS are not specific\nto amyloid-beta. It is known that tau inclusion is also stained positive in ThT and ThS staining. If the author need to prove\nbinding to amyloid fibrillar structures, please also include OC staining. A11 staining that has been used in the manuscript is\nknown to be quite non-specific in the staining. In addition, please include DAPI channel as well in the staining shown.\n4. It is known that AD brain have both amyloid-beta, tau pathologies (sometime also alpha-synuclein pathology). Please\ninclude staining of the probe with AT-8 for phospho-Tau to show that the probe does not detect other fibrils such as tau. To\nour knowledge, the CRANAD-2 like structures detect also alpha-synuclein fibril. Can the author provide further proof of the\nspecificity of the probe in this regards. In addition, staining on the non-demented control case is also critical to assess the\nspecificity of the probe. Please include this information.\n5. It is known that there is age-dependent accumulation of lipofuscin in human brain tissue and (in aged mouse brain).\nPlease include staining showing that the probe does not bind to lipofuscin or has other off-target in the brain tissue.\nAutofluorescence assessment is also needed on these brain tissue slices.\n6. Please include an ex vivo multiphoton images for the mice underwent in vivo imaging with probe.\n7. Does the probe bind to Cerebral amyloid angiopathy or only parenchymal plaque?\n8. The solubility of the probe is going to be complicated as 7.5% DMSO, 2.5% Cremophor EL is used in solvent. Even\nthough the author showed the low toxicity, the longitudinal application might be an issue.\n9. It is important to show that the probe can monitor the increase in amyloid-beta during the disease development. Please\nassess longitudinally the change of amyloid load in the mouse brain to demonstrate the robustness of this approach.\nMinor points:\n1. The age of mice used the in the study is not consistent (sometime 13 month, sometimes 14 month). Please double check\n2. The author wrote that the brain slices of mice shown in Fig 3, SFig 56, SFig 57 are coronal slices. However the slices are\nclearly horizontal. Please correct.\n3. in Fig 3 i. The author wrote frontoparietal cortex of mouse brain. Can the author indicate where it is in the overview image.\n4. It would be best not to use words like “remarkably” in the manuscript. Please check through the manuscript.\n5. Fig. 3 (iii). The ROI for data analysis is missing. Please add on the image for illustration\n6. Page 6. The author mentioned that the probe was injected into Tg-AD mice. Please be specific. i.v. injection?\n7. Please be consistent when describing the mice. Sometime the author use Tg-AD mice, sometimes APP-PS1 mice."}, "author_response": "Thank you for reviewing our manuscript titled \"Discovery of Novel NIRF Theranostic Probes Targeting Amyloid-β Fibrils and Cholinesterases in Alzheimer's Disease Models\" (NCOMMS-23-59606) by Rai, H. et al. We appreciate the opportunity to resubmit the revised manuscript. We thank the reviewers and editorial board for their unanimous endorsements of the significance and novelty of our manuscript. We have carefully studied the reviewers' insightful comments and incorporated them into the revised manuscript. The Reviewers’ comments are in italics. The necessary revisions, based on your suggestions, are highlighted in red in both the main text and supplementary information for easy viewing. Our detailed point-by-point response is listed below for your assessment.\nThank you for considering this resubmission.\nSincerely yours,\nGyan Modi\nThe Reviewers’ comments are in italics.\nReviewer #1:\nAβ and AChE play important roles in the development of AD, and the theranostic probes could\nprovide early diagnosis and mitigate or slow down late-stage severity in AD. In this manuscript,\nthe author developed a D-π-A based NIR-I probe I-43 that shows a good affinity towards Aβ and\na moderate AChE inhibition (IC50 = 0.38 µM). Also, it displays a desirable NIR imaging potency\nand improved cognitive function, showing its potential in integrating diagnosis and treatment of\nAD. The author also conducted the brain permeability, acute oral toxicity study and behavioral\nstudy of I-43. Overall, this is a systematic work, and suitable for publication in Nature\nCommunications after addressing the following questions.\nResponse: We appreciate the reviewer's concise analysis and ardent encouragement of our\nefforts.\n1) The lead probe I-43 has the strong affinity for Aβ plaques, however, what is it affinity for\nsoluble Aβ species? Whether the probe could attenuate the propagation of soluble Aβ species\ninto Aβ aggregates?\nResponse: Thank you for your comment. As suggested, we conducted an in-vitro fluorescence\nassay of I-43 for soluble species (oligomers), now included in Fig. 2a-b of the revised manuscript\n(page # 8). We prepared Aβ oligomers using the described protocol1 and confirmed them\n1–42\nthrough ThT and TEM analysis (Supplementary Fig. 45a-b; Page #64). Given that Aβ is a crucial\n40\n4\nbiomarker in Aβ pathology, we also examined I-43 for Aβ oligomers and fibrils (prepared as\n1-40\nper the reported protocol2 and confirmed via ThT and TEM analysis; Supplementary Fig. 45a-b;\nPage # 64). Our results indicates that I-43 shows a detection signal for oligomeric Aβ and Aβ\n1–42 1–\nbut relatively insignificant (****P) compared to Aβ and Aβ fibrils. Furthermore, binding\n40 1–42 1–40\naffinity (K ) of I-43 was found to be 337 ± 113 and 213 ± 44 nm for oligomeric Aβ and Aβ\nd 1–42 1–40\nspecies, respectively, which is relatively less compared to fibrillar species (K = 58.19 ± 9.7 and\nd\n104 ± 25 nM for Aβ and Aβ fibrils, respectively). On further exploration, I-43 exhibited a\n1–40 1–42\nsignificant increase (approximately 13-fold and 6.2-fold for Aβ and Aβ fibrils, respectively)\n1–40 1–42\nin fluorescence intensity, compared to probe alone. The revised data, including Fig. 2a-c (Page #\n9), and a brief discussion, have been incorporated into the revised manuscript (Page #6). Further,\nthe fluorescence response of I-43 with changes in fibrillar Aβ concentrations demonstrate its\n1–40\nutility for quantitatively estimation of Aβ aggregates load in in vitro studies (Fig. 2d; Page 9).\nFig. 2. (a) Representative spectral data and a statistical bar graph of I-43 (20 µM) upon incubation\nwith various Aβ species (10 µM; monomers, oligomers, and fibrils) and freshly prepared BSA (25\nµg/mL). (b) In the bar graph, the lowercase roman numerals correspond to: (i) I-43 alone in PBS\n(pH 7.4); (ii) Aβ monomer + I-43; (iii) Aβ oligomers + I-43; (iv) Aβ fibrils + I-43; (v) freshly\n1–42 1–42 1–42\nprepared BSA + I-43; (vi) Aβ monomer + I-43; (vii) Aβ oligomers + I-43; and (viii) Aβ fibrils\n1–40 1–40 1–40\n+ I-43. (c) Fluorescence (FL) intensity plot with gradient concentrations ranging from 5 to 0.03 μM\nof I-43 upon incubation with Aβ fibrils (5 µM), with nonlinear curve fitting using one-site\n1-40\nbinding regression (Ex/Em = 490/690 nm). (d) A correlation plot between fluorescence intensity\nof I-43 (10 µM) vs decreasing concentrations of Aβ species, showing a good correlation with a r²\nvalue of 0.97 in the inset.\n) .u .a\n(\ny\n1\n2\n5\n0\n0\n0\n0\n0\n0\n(\ny\n1\n2\n5\n0\n0\n0\n0\n0\n0\nt is 10000 t i s10000\nn n\ne e\nt n i\nL F\n500\n0\nB\nd\nm\n=\nax\n3\n=\n3\n1\n7\n40\n\n4\n1\n5\n12.90 nM t n i\nL F\n500\n0\nB\nd\nm\n=\nax\n2\n=\n1\n1\n3\n56\n\n3\n4\n6\n4 nM\n0 1000 2000 3000 4000 5000 6000 0 1000 2000 3000 4000 5000 6000\nConcentration (nM) Concentration (nM)\nnonlinear curve fitting drawn in GraphPad Prizm using one-site binding regression.\nNext, as suggested by the reviewer, we evaluated I-43's ability to attenuate the\npropagation of soluble Aβ species into Aβ aggregates. Interestingly, I-43 could moderately inhibit\nconversion of Aβ monomers to Aβ aggregates as evident through the ThT fluorescence assay\n1-42 1-42\n(Supplementary Fig. 54 or SI-54; Page 75 of revised SI). Fascinatingly, I-43 can also slow down the\naggregation of Aβ peptides at both 4:1 and 2:1 probe-to-protein ratio, as confirmed by TEM\n1-40\nimaging and ThT fluorescence assays (Fig. 2e; Page 8 of revised MS). Thus, our in-vitro studies\nshow that I-43 has high affinity for aggregated Aβs, can quantitatively estimate Aβ load, and slows\nAβ aggregation at both 4:1 and 2:1 probe-to-protein ratio. Further extensive studies are\n1-40\nneeded to confirm its potential as an Aβ aggregation inhibitor in pre-clinical studies.\nFig. SI-54. Spectral data represents the change in the fluorescence intensity of the ThT upon\nincubation with Aβ aggregates and with the preparation containing a pre-incubated (for 72h)\n1-42\nmixture of I-43 and Aβ aggregates in fluorescence-based ThT assay. The measurements were\n1-42\nperformed with excitation at 445 nm, and the resulting spectra were recorded within the 460-\n600 nm range using a SpectraMax-i3x (Molecular Devices) microplate reader. (n = 3 independent\nexperiments in triplicates. Fig. 2(e) TEM micrographs demonstrating mitigation of Aβ fibril\n1-40\ngrowth (10 μM) upon treatment with 2- and 4-fold concentrations of I-43 μM for 72 hours, and\nThT based fluorescence study (20 μM).\n2) Structurally, the probes developed by the author in the manuscript is very similar to 7 or 3h\n(Seo et al. 2016), so the author should also test the effects of these probes against Tau. And,\nmore species should be evaluated for the selectivity of the probes, especially those proteins\nrelated to neurodegenerative disease.\nResponse: Thank you for this comment. We agree with the reviewer's point and have investigated\nthe affinity of I-43 for pathogenic Tau and α-synuclein-crucial markers related to the accumulation\nof misfolded proteins in neurodegenerative diseases like FTD and PD. We generated aggregated\nforms of full-length recombinant Tau and α-Synuclein (Met1 - Ala140) according to reported\nliteratures3, 4 and confirmed their morphological states using TEM analysis (Supplementary Fig.\n45c; Page # 64). We measured the in-vitro comparative fluorescence (Fl) response of I-43 after\n6\nincubation with monomers and fibrils of Tau and α-synuclein and compared it to Aβ fibrils.\nThe\n1-40\nresults are shown in Fig. 2f, with the signal intensity plot presented in Fig. 2g of the revised\nmanuscript (Page # 9). This study indicates relatively insignificant change in fluorescence intensity\nof I-43 for fibrillar Tau and α-synuclein, compared to Aβ and Aβ fibrils.\n40 42\nFig. 2. (f, g) Present representative spectral data and a statistical bar graph of I-43 (10 µM)\nincubated with recombinant Tau and α-Synuclein proteins (5 µM; monomers and fibrils),\ncompared to a unified concentration of Aβ fibrils (5 µM).\n1-40\nTo further validate the selectivity of I-43 towards Aβ species, immunohistochemistry of\npostmortem brain tissue specimens from AD, PD, and FTD patients was performed. These tissues\nwere first stained with I-43 and then dual-stained with anti-Tau-specific antibodies (AT8 and\nTau46; Fig. 6f on page # 18 of revised MS and Supplementary Fig. 64 on page # 90) and α-\nSynuclein-specific antibodies (Met1 - Ala140 peptide length α-Syn; Fig. 6b on page # 18 of revised\nMS). The results indicate that I-43 exhibited minimal affinity for these species.\ntau tangles in brain specimens from AD and FTD diagnosed patients. The tissues were first stained\nwith I-43 to assess its affinity for tau tangles, followed by staining with tau tangle-specific\nantibodies (Tau46 and AT8) to confirm the presence of neurofibrillary tangles.\nfrontotemporal dementia (FTD) patients were stained with I-43 and counter staining was\nperformed with AT8 anti-Tau antibody to locate tau tangles. Confocal images were capture using\n488/520 nm channel to detect Alexa FluorTM 488 tagged secondary antibody conjugated to AT8\nprimary antibody and NIR filters were use to detect I-43 associated signals using LSM-780 (Carl\nZeiss) confocal microscope.\nshowing α-Synuclein associated deposits and counterstained with I-43 to determine its affinity\nfor α-Synuclein.\n3) The distribution and principal metabolic pathway of the probe I-43 should be investigated.\nQuantitative uptake value of the probe I-43 in the brain should also be measured.\nResponse: Thank you for your insightful suggestion. A biodistribution study of I-43 was\nconducted, and fluorescence imaging of vital organs (Lv = liver, Sp = spleen, Kd = kidney, Br = brain,\nLu = lung, Hr = heart) has been incorporated into the revised manuscript (Fig. 5e-f on page # 16\nof revised MS). Biodistribution analysis revealed that metabolic organs like the kidney and liver\nhad comparable or higher I-43 signals than the brain, while the spleen, lungs, and heart showed\nminimal retention, indicating low non-specific tissue interactions. These findings confirm the\nefficacy of I-43 as an NIR probe for monitoring Aβ accumulation with minimal toxicity due to its\nrapid clearance. This makes I-43 a valuable tool for longitudinal studies in experimental research.\nwas administered intravenously, and major organs (Abbreviations: Lv = liver, Sp = spleen, Kd =\nkidney, Br = brain, Lu = lung, Hr = heart) were resected at 20 minutes, and used for fluorescence\nimaging. (f) The bar graph shows fluorescence signal intensity profiling (photon count) of I-43 (ns,\nnot significant; **P = 0.0025; ****P < 0.0001 by two-way ANOVA with Dunnett’s multiple\ncomparison test).\nWe also investigated metabolic studies of I-43 in mice liver microsomes, and the data\nindicates that I-43 undergoes rapid metabolism to produce an ester-hydrolyzed acid fragment\n(Supplementary Fig. 75a-c on pages # 105-106). This fragment then undergoes secondary\nmetabolism, resulting in an O-demethylated derivative (Supplementary Fig. 75e). This study was\noutsourced from the industry. Details of the LC-MS/MS data, along with the raw data files, will be\navailable for further assessment.\n11\nf\nSupplementary Fig. 75: (a-b) Representative HPLC chromatogram showing I-43 content in the rat\nliver microsome incubated for various time points (0 and 1.5 h). (c) LC/MRM analysis showed\npotential metabolites (product-1 and product-2) during primary transformations from I-43 after\nincubation for 120 minutes, indicating metabolic activity producing the same metabolites\nobserved in the plasma stability study. (d) Proposed metabolic fragmentation of I-43. (e) LC-MS\ntrace of secondary metabolic transformation of I-43. (f) AUC indicating conversion of I-43 to\nproduce product-1 (acid fragment), which subsequently converting in to product-3\n(demethylation of acid fragment) upon incubation with liver microsomes with or without cofactor\non time scale.\nTo measure the quantitative uptake value of the probe I-43 in the brain, quantitative\nestimation of I-43 in the brain was performed using HPLC, and a brief discussion has been\nsupplemented to the revised manuscript on Page # 23 and Supplementary Figs. 72 and 73 and\nSupplementary Table 12 on page # 101-102. Our data suggested that I-43 exhibited substantial\ninitial brain uptake of 0.59% ID/g at 5 minutes post-injection and was cleared from the brain with\na rapid elimination rate of 3.4 (brain ), potentially crucial for NIR imaging with better S/N\n5min/60min\nratio (Supplementary Table 12 and Supplementary Fig. 73, Page # 101).\nplain brain homogenate methanolic extract. (b) HPLC chromatogram showing I-43 content in the\nbrain homogenates at various time points; herein, the purple line peak represents 1 µM of I-43\nadmix with brain homogenates.\nSupplementary Table 12: Quantification of I-43 in brains of ICR mice.\nProbe Time after injection (% ID/g)\nBrain /Brain\n5min 60min\n5 min 10 min 30 min 60 min\nI-43 0.59 ± 0.11 0.37 ± 0.15 0.26 ± 0.05 0.17 ± 0.01 3.4\n13\n)\ng\n/ 0.8\nD\nI\n%\n(\nn\n0.6\no\ni\na\nr t 0.4\nn\ne\nc\nn\no 0.2\nc\nn\ni\na 0.0\nr\nB\n0 15 30 45 60\ntime after i.v. injection (min)\nSupplementary Fig. 73: The % ID/g of I-43 in excised mouse brain homogenates at different time\npoints post i.v. dosing via the tail vein. Data are expressed as mean ± SD (n = 3).\n4) Although the probe I-43 has a good photostability, it will ultimately be used for the diagnosis\nand treatment of AD. So, the author should explore the stability of the lead probe I-43 in vivo.\nResponse: We sincerely appreciate your valuable suggestion. First, we conducted an in vitro\nplasma stability assay of the lead probe I-43 before proceeding with in vivo studies and observed\nthat the compound undergoes hydrolysis of its ester bond, resulting in the formation of an acid\nmetabolite (Met-1; Supplementary Fig. 74a-d on Page # 102), as substantiated by high-resolution\nmass spectrometry (HRMS) data. Furthermore, microsomal stability studies revealed that I-43 is\nrapidly metabolized to produce the acid derivative, which subsequently undergoes secondary\nmetabolism via O-demethylation (as evident in the work done to address a previous comment).\nIn-vitro plasma stability of I-43 using HPLC\n14\ne\nSupplementary Fig. 74: (a-d) Representative HPLC chromatogram showing I-43 content in the rat\nplasma incubated for various time points (10, 60, 120 and 150 min). (e) HRMS analysis to identify\npotential metabolites (Met-1) in the plasma fraction incubated for 150 minutes, indicating\nmetabolic activity.\nNext, as recommended, the in-vivo stability of I-43 was evaluated. Blood samples from\nhealthy male Wistar rats were collected from the retro-orbital plexus at representative time\npoints following intraperitoneal (i.p.) administration of I-43 at a 5 mg/kg dose. The\npharmacokinetic profile of I-43 is summarized in Supplementary Table 13 (Page # 107), with a\nbrief discussion on Page # 24 of revised MS. The resultant data shows that I-43 is rapidly absorbed,\nhas moderate bioavailability, and optimal half-life suggesting potential for therapeutic effects.\nHowever, the moderate C may limit efficacy depending on the required therapeutic threshold,\nmax\nand successive dosing may be necessary to achieve clinically significant effects.\nthe compound I-43.\nSupplementary Table 13: Important pharmacokinetic parameters of I-43 (at 5mg/kg\nadministered via i.p. route)\nParameter Unit Values obtaineda\nt min 171.4 ± 61\n1/2\nT min 10\nmax\nC μg/ml 0.41 ± 0.08\nmax\nAUC μg/ml*min 13.59 ± 1.4\n0−t\nAUC μg/ml*min 26.60 ± 0.08\n0-inf\nMRT min 228.17 ± 76\n0-inf\naData are expressed as mean ± SEM (n = 3 independent rat). Plasma concentrations of I-43 were\ndetermined by HPLC. PK parameters were derived from AUC values using non-compartmental\nanalysis with the linear trapezoidal method in PK Solver 2.0 software.\nTo determine whether the behavioral recovery in a scopolamine-induced model was due\nto I-43 or its biotransformation-generated acid fragment, Met-1 was synthesized. We confirmed\nits structure using 1H NMR (Supplementary Fig. 79 on Page # 111 of revised SI), and assessed for\nAChE inhibition (IC ) using Ullman calorimetry. Notably, Met-1 demonstrated 47.39 ± 0.094%\n50\nbenzothiazole fragment of I-43 showed 22.187 ± 0.81% AChE inhibitory activity at the same\nconcentration. In contrast, I-43 exhibited 61.32 ± 0.64% AChE inhibitory activity at 10 μM, with\nan IC value of 0.38 ± 0.01 μM—approximately 26-fold more potent than Met-1, emphasizing the\n50\nimportance of our design for cholinesterase modulation. Moreover, according to spectral analysis\n(as shown in Supplementary Fig. 80 on page 112 of the revised SI), the absorbance peak maxima\nin PBS (pH 7.4) and DMSO were 400 nm and 430 nm, respectively, whereas the emission maxima\nwere 725 nm and 650 nm.\n7.4) at representative concentrations.\nOur developed probes efficiently achieve simultaneous in vitro and in vivo detection of Aβ\nand ChEs, a capability not offered by existing probes best to our knowledge. However, the lead\ncompound I-43, despite its excellent NIR imaging properties, requires further structural\nmodifications to improve ChE inhibition efficacy, metabolic stability, and physicochemical\nproperties for potential therapeutic application in AD. Future research will focus on enhancing\nthese aspects through targeted SAR studies.\n5) Page 2, line 52. The structures of Quinolinium-based carbazole and naphthalene cyanine\nprobes, hybrid probes (PiB-C and TBT) should be provided in Figure 1.\nResponse: Thank you for the valuable suggestion. As requested, the structures of the\nQuinolinium-based carbazole and naphthalene cyanine probes, as well as the hybrid probes (PiB-\nC and TBT), have been incorporated to Figure 1 on page # 4 of the revised MS.\n17\n6) The cis and trans conformations of the structures in Figure 1B and 1C are different. Please\ncorrect.\nResponse: We thank the reviewer for pointing out the discrepancy. As suggested, we have\ncorrected the conformations of the structures in Fig. 1C, which is now Fig. 1d in the revised\nmanuscript (Page # 4).\n7) Page 7, line 190. The higher reactivity and stability seem to be contradictory.\nResponse: Thank you for pointing out our mistake. We fully agree with the reviewer comment\nthat higher reactivity and stability are contradictory. The correct statement should be “the higher\nreactivity and lower stability of I-43” rather than “the higher reactivity and stability of I-43.” This\nhas been corrected in the revised manuscript to: “The energy gap between the orbitals increases\nin the order I-43 < I-38 < I-32, indicating that I-43 has the lowest stability and thus the highest\nreactivity.” on Page # 8 of the revised MS.\n8) Compound I-40 contains a CF3 in its structure, and its 13C NMR exhibits splitting—please\nsupplementary coupling constant.\nResponse: We appreciate your valuable suggestion. In response, we have conducted 13C NMR\nanalysis of compound I-40 at 600 MHz, and the corresponding data has been incorporated into\nthe revised Supplementary Information (SI). Specifically, we observed splitting at the\ncharacteristic peak of 127.02 (q, J = 4.1 Hz) as shown in revised Supplementary Fig. 33 (Page 44\ncf3\nof the revised SI, with the coupling constant values now provided on Page 10 of the revised SI.\nHowever, the other two peaks in the 122.9–129.1 ppm region were not clearly resolved due to\ninterference from other carbon peaks. Additionally, we have done 19F NMR data for further\nstructural confirmation of the CF group [19F NMR (471 MHz, DMSO) δ -60.64, Supplementary\nwelcome any further suggestions you might have on this matter.\n9) Reference 28 is incomplete, and reference 33 does not have an abbreviated journal name.\nResponse: We feel sorry for our mistake and thank you for the suggestions. As suggested, the\nreferences 28 and 33 have been revised as suggested, which is now references 29 and 32 in the\nrevised manuscript on Page # 33. We have also critically reviewed the other references to avoid\nsimilar mistakes.\nThe author presented a comprehensive manuscript on the development of novel NIRF probe for\ndetecting Aβ and cholinesterase in Alzheimer disease models. The author includes molecular\ndocking, toxicity, in vitro, in vivo assessment of the new probe. Despite the extensive data and\nmerits of the manuscript, there are a few concerns regarding the probe.\nResponse: We are grateful to the reviewer for appreciating our work and for the detailed review,\nwhich has significantly enhanced the quality of our manuscript.\nMain points:\n1) Fig. 2 The fold change is about 50% increase as shown in Page 7 comparing binding to\naggregated Aβ fibril with monomeric Aβ and BSA. BSA is in high abundance in living mice. This\nincrease is not sufficient in my opinion (and comparing to the other amyloid NIRF probes have\nbeen used). Can the author please compare the probe with previous published amyloid NIR\nprobes such as AOI-987, HS-169, CRANAD-2, ZW800–1C.\nResponse: Thank you for your insightful comment. We acknowledge that the 50% increase in fold\nchange observed in Fig. 2, comparing binding to aggregated Aβ fibrils with monomeric Aβ and\nBSA, is modest. BSA's high abundance in living mice could influence this comparison. Despite this,\nour data indicates a notable difference in fluorescence signal intensity between Aβ aggregated\nspecies vs BSA. The development of an effective theragnostic agent for AD depends on achieving\nbalanced NIRF Aβ probes, such as CRANAD-2, ZW800-1C, AOI-987, and HS-169 (Supplementary\nTable 14; Pages 112–113), which show promise for in vitro and in vivo imaging of amyloid-beta\n(Aβ) plaques, meeting several critical requirements. However, NIR dyes are not yet clinical tools,\nemphasizing the need for the development of theranostic probes with high selectivity and\nenhanced therapeutic efficacy. Our probes, especially I-43, effectively detect amyloid-beta\nplaques in histological brain tissue samples (mice, and human), as confirmed by dual-\nimmunostaining assays. Additionally, they show moderate therapeutic efficacy in modulating\nAChE activity, supporting their potential as valuable diagnostic and therapeutic tools in AD. We\nare hopeful that further systematic SAR exploration of our designed scaffold will yield promising\nleads as potential pre-clinical theranostic tools for screening and assessment in AD in near future.\nAs suggested by the reviewer, following an extensive literature review related to previous\npublished amyloid NIR probes such as AOI-987, HS-169, CRANAD-2, ZW800–1C, we found that\nonly CRANAD-2 has been reported for fluorescence-based studies in the presence of BSA as well.5\nResearch work on CRANAD-2 demonstrating it well-suited for in-vitro and in-vivo imaging of\namyloid-beta deposits in AD mouse models.5, 6 CRANAD-2 shows a 70-fold fluorescence\nenhancement upon binding to Aβ fibrils and has a binding affinity of 38.69 μM, making it\n1-42\nsuitable for in-vitro and in-vivo imaging of amyloid-beta deposits in AD mouse models.7 Ni et al.\n19\n(2021) reported that CRANAD-2 exhibits significantly higher fluorescence with Aβ fibrils\n1−42\ncompared to α-synuclein, insulin, lysosome fibrils, and Aβ monomers, but also generates 30%\n1−42\nof the fluorescence signal with BSA.5 Similarly, ZW800–1C, a deep-NIR dye, exhibits a 3.9-fold\nfluorescence enhancement when bound to Aβ aggregates and a 1.8-fold enhancement with\n42 40 42\nwith binding constants (K ) of 43.7 ± 5.8 nM for Aβ and 78.9 ± 12.3 nM for Aβ .8 Conversely,\nd 40 42\nAOI-987 demonstrates a binding affinity of 0.22 ± 0.13 μM for Aβ aggregates.9 Our data indicate\n40\nthat I-43 exhibits comparable binding affinities, with K values of 58.19 ± 9.7 nM for Aβ fibrils\nd 1–40\nand 104 ± 25 nM for Aβ fibrils, respectively, and shows a fluorescence intensity fold change of\n1–42\napproximately 13-fold for Aβ fibrils and 6.2-fold for Aβ fibrils. The revised data, including\n1-40 1-42\nFig. 2a-c (Page # 9 of the revised MS), and an updated discussion on Page # 6 of the revised MS,\nhave been incorporated into the manuscript to provide a more comprehensive analysis. As\nsuggested, we have added a comparative table in the Supplementary Information\n(Supplementary Table 13; Page 112-113 of revised SI) to compare the photophysical and biological\nproperties of the suggested probes (AOI-987, HS-169, CRANAD-2, and ZW800–1C) with lead\nprobe I-43. Also, a brief discussion based on experimental evidence is incorporated in the revised\nMS (Page 29). In addition, histological dual staining with CRANAD-2 using I-43 as a counterstain\nin brain tissue from APP-PS1 mice (Fig. 3b; Page # 12 of revised MS) and postmortem brain\nspecimens from clinically diagnosed AD patients (Fig. 6a; Page # 18 of revised MS) provides an\nevidence of high S/N ratio fluorescence intensity of I-43, comparable to CRANAD-2, in in-vitro\nstaining. We hope we have addressed the reviewer’s concerns and welcome additional\nsuggestions to further elevate the quality of our research.\nFig. 2. (a) Representative spectral data and a statistical bar graph of I-43 (20 µM) upon incubation\nwith various Aβ species (10 µM; monomers, oligomers, and fibrils) and freshly prepared BSA (25\nµg/mL). (b) In the bar graph, the lowercase roman numerals correspond to: (i) I-43 alone in PBS\n(pH 7.4); (ii) Aβ monomer + I-43; (iii) Aβ oligomers + I-43; (iv) Aβ fibrils + I-43; (v) freshly\n1–42 1–42 1–42\nprepared BSA + I-43; (vi) Aβ monomer + I-43; (vii) Aβ oligomers + I-43; and (viii) Aβ fibrils\n1–40 1–40 1–40\n+ I-43. (c) Fluorescence (FL) intensity plot with gradient concentrations ranging from 5 to 0.03 μM\n20\nof I-43 upon incubation with Aβ fibrils (5 µM), with nonlinear curve fitting using one-site\n1-40\nbinding regression (Ex/Em = 490/690 nm).\nFig. 3. (b) Brain sections of 13-month-old APP-PS1 mice stained with ThS (FITC filter) and CRANAD-\n2 (CD-2; TRITC filter) (white arrowheads) were counterstained with I-43 to reaffirm I-43's affinity\nfor neuritic plaques. Scale bar = 20 μm. The signal intensity plot (Fig. b3) shows I-43 has low\nbackground noise and lower signal intensity than CD-2, with perfectly superposed signals.\ntissue specimens of AD patients using CD-2 reference dyes. The image in the middle panel\nrepresents counter staining with I-43 exhibited colocalization with the standard dyes revalidates\nthe data of histological staining of mice brain sample experiments.\n2) The detailed information regarding the demographics (age, sex) as well as pathological\nfeatures of AD brains are not presented. Please follow the ‘Sex and Gender Equity in Research\n– SAGER – guidelines’ and to include sex and gender considerations for studies involving\nhumans, vertebrate animals and cell lines where relevant to the topic of study. The author\nsays that postmortem AD human brain tissues (92-year-old, male and female, n=5) were used.\nIt is confusing to reader if all the cases are 92-year-old? In addition, only one staining of\nAβ/ThT was presented for the postmortem human brain without quantification. Please provide\na quantification of the Aβ (sensitivity and specificity) in 5 AD cases and control cases.\nResponse: Thank you for your valuable feedback. We acknowledge the importance of adhering\nto the SAGER guidelines and the necessity of providing detailed demographic and pathological\ninformation in our study (Supplementary Table 9 on page # 83 of the revised SI).\nWe apologize for any confusion regarding the age of the cases. The representative\nfluorescence image is from a 92-year-old female patient. We have added the patient details (age\nand sex) to their corresponding fluorescence micrographs throughout the manuscript and\nsupplementary information to avoid any ambiguity. The postmortem AD human brain tissues\nused in our study included both male and female subjects with an age range of 82-92 years in the\ncase of AD, 65 and 76 in the case of FTD, 74 and 86 in the case of PD, and 65-92 in the case of\nnon-demented patients (Supplementary table 9 on page # 83 of revised SI).\nWe recognize the importance of quantification in providing a comprehensive analysis.\nFigures 3c and 3e (Page 12) and Supplementary Figures 56-57 (Pages 81-82) present\nrepresentative fluorescence micrographs of APP-PS1 brain tissues dual-stained with test\ncompound I-43. Counterstaining was performed using conventional Aβ-specific reagents (Congo\nRed or CR, silver staining) and anti-Aβ antibodies (D54D2 and OC). Additionally, Fig. 6a and 6c on\npage # 18 of the revised MS and Supplementary Figures 58, 60-63 on pages # 84, 86-88 of the\nrevised SI include representative dual staining confocal and fluorescence imaging micrographs of\nI-43 using conventional congophilic Aβ-specific dyes (CR) and anti-Aβ antibodies as\ncounterstaining agents (D54D2, 6E10, OC and A11). This revalidates the affinity and selectivity of\nthe developed compound I-43.\nWe measured Aβ lesions associated fluorescence integrated density (quantification) of I-\n43 in NIR filter (633/ 700 nm) vs 6E10 anti-Aβ antibody in FITC filter (488/520 nm) from ≥ 8\n22\nsamples from 5 AD patients using ImageJ tool. This demonstrates the potency of I-43 in\ndetermining the extent of Aβ pathology corresponding to the 6E10 antibody stain (Fig. 6d; Page\n# 18 of revised MS). Furthermore, dual staining IHC of brain specimens from AD cases and control\ncases by I-43 following OC anti-Aβ antibody were also quantified for FL integrated density. this\ndemonstrated the robustness of the I-43 response to Aβ quantification in AD vs non-demented\npatient (NDP; control) brain specimens (Fig. 6e; Page # 18 of revised MS). We presented the\nresults using appropriate statistical analysis to support our findings.\nFig 6. (d,e) Semiquantitative analysis via fluorescence (FL) integrated density plot measured using\nImageJ tool. Statistical analysis for image d was conducted using a two-tailed unpaired t-test (n =\n13; nsP = 0.466), while image was analyzed using a one-way ANOVA with Sidak's multiple\ncomparisons test (n = 9; *P = 0.0442 and ****P < 0.0001).\n3) Please provide staining using 6E10 or 4G8 with the probe on the mouse and human brain. ThT\nand ThS are not specific to Aβ. It is known that tau inclusion is also stained positive in ThT and\nThS staining. If the author needs to prove binding to amyloid fibrillar structures, please also\ninclude OC staining. A11 staining that has been used in the manuscript is known to be quite\nnon-specific in the staining. In addition, please include DAPI channel as well in the staining\nshown.\nResponse: Thank you for your insightful comments. We agree with your suggestion\nregarding the non-specific binding associated with ThT, ThS, or the A11 antibody, apart from Aβ\ndeposits. We appreciate your recommendations and have incorporated additional staining\nexperiments to enhance the rigor and specificity of the developed probe. Fig. 6c (Page # 18 of\nrevised MS) and Supplementary Fig. 62-63 on pages 88-89 of revised SI show representative\nconfocal images of autopsy brain tissue specimens from AD patients, dual-stained with I-43 and\nanti-Aβ antibodies (6E10 and OC) using immunohistochemistry.\namyloid-beta (Aβ) aggregates, it was strategically employed in dual immunohistochemical (IHC)\nstaining of brain tissue specimens from APP-PS1 transgenic mice. This approach was used to\nsubstantiate the findings of our study. Supplementary Figure 57a (Page 82 of the revised SI)\nillustrates our results. The dual immunohistochemical staining with I-43 alongside 6E10 and OC\nantibodies corroborates the probe's specificity and reproducibility in targeting amyloid-beta\nplaques within human and mice brain tissues. These findings highlight the diagnostic potential of\nI-43 as a reliable molecular imaging agent for delineating amyloid pathology in Alzheimer's\ndisease.\nFig 6. (c) Dual stained fluorescence images of brain tissue specimens from AD patients by I-43\n(NIR channel) and anti-Aβ antibodies (6E10, and OC; green channel).\nstaining spots for OC (green channel; Ex/Em = 488/520 nm) and I-43 (NIR channel; Ex/Em =\n633/700 nm) in three distinct regions marked by a square box with the dotted line and annotated\nas a1, a2, and a3. The scale bar represents the 10 µm length in the subregions (generated using\nthe freeware ImageJ tool).\nWe conducted additional dual staining immunohistochemistry using the D54D2\nantibodies alongside our probe on both APP-PS1 mice and human brain tissues. The D54D2\nantibody recognizes endogenous Aβ , Aβ , Aβ , Aβ , and Aβ , making it a versatile tool for\n42 40 39 38 37\nstaining amyloid deposits in both human and mouse tissues. This addition enhances our ability to\ndetect a broad spectrum of Aβ forms and improves the robustness of our findings. We also\nincluded DAPI staining in the fluorescence images for both mouse and human brain tissue staining\nexperiments to provide a nuclear counterstain and a better context for the Aβ deposits\nlocalization. The updated figures are included in the revised manuscript as Figures 3e (Page # 11\nof revised MS), 6c (Page # 18 of revised MS), and Supplementary Fig. 61 (Page # 87 of revised SI).\nThe overlap of staining signals observed in the green (anti-Aβ antibodies) and NIR channels\nindicates the high affinity and selectivity of I-43 for Aβ deposits.\nby counterstaining with I-43. The enlarged views e1 and e2 are from merged sections of Fig. 3e\n(white square box, dotted line)—scale bar: 50 μm in e panel images and 20 μm in e1 and e2\nimages.\n(NIR channel) and anti-Aβ antibodies (D54D2; green channel).\n4) It is known that AD brain has both Aβ, tau pathologies (sometime also alpha-synuclein\npathology). Please include staining of the probe with AT-8 for phospho-Tau to show that the\nprobe does not detect other fibrils such as tau. To our knowledge, the CRANAD-2 like structures\ndetect also alpha-synuclein fibril. Can the author provide further proof of the specificity of the\nprobe in this regard. In addition, staining on the non-demented control case is also critical to\nassess the specificity of the probe. Please include this information.\nResponse: Thank you for your valuable feedback. We understand the importance of\ndemonstrating the specificity of our probe in distinguishing between Aβ and other pathologies,\nsuch as tau and alpha-synuclein. We have incorporated AT-8 staining, which detects\nphosphorylated tau, alongside our I-43 probe. This dual staining demonstrates that the I-43 probe\nproduces inconspicuous signals for tau tangles, as evidenced by the lack of overlap between the\nI-43 and AT-8 signals (lower panel of Fig. 6f on Page # 18 of the revised MS and Supplementary\nFig. 64 on Page # 90 of the revised SI).\nFig. 6(f): Brain specimens from FTD-diagnosed patients were stained with the NFTs-specific\nantibody AT8 (green channel) and then dual-stained with I-43 to assess its affinity for tau tangles.\nbetween I-43 and AT8-stained tangles.\nSupplementary Fig. 64: Deparaffinized brain slices from frontal cortex (Ft. Cr.) regions of\nfrontotemporal dementia (FTD) patients were stained with I-43 and counter staining was\nperformed with AT8 anti-Tau antibody to locate tau tangles. Confocal images were captured using\na 488/520 nm channel to detect Alexa FluorTM 488 tagged secondary antibody conjugated to AT8\nprimary antibody, and NIR filters were used to detect I-43 associated signals using LSM-780 (Carl\nZeiss) confocal microscope.\nTo address concerns regarding potential cross-reactivity with alpha-synuclein, we have\nincluded staining with an alpha-synuclein antibody (P-Ser129α-Synuclein) in conjunction with the\nI-43 probe. Our results show that the I-43 probe exhibits signals for alpha-synuclein plaques that\nare below the detection limit, indicating its minimal cross-reactivity with alpha-synuclein\npathology (Fig. 6b on Page # 18 of the revised MS).\nshowing α-Synuclein associated deposits and counterstained with I-43 to determine its affinity\nfor α-Synuclein.\nWe have also included staining of non-demented control brain tissues with the I-43 probe\nto further validate its off-target (Supplementary Fig. 58-59; Page # 84-85 of revised SI) staining\naffinity. The tiny stains observed in the 488/520 nm (FITC filter) are not localized with the I-43\nmarked patterns in the NIR filter (633/700 nm), except for a few instances. We suspect this might\nbe autofluorescence from lipofuscin deposits.\nSupplementary Fig. 58: Representative dual stained deparaffinized brain slices (Hip or\nhippocampal region) from control patients (92-year-old female) acquired using the confocal\n29\nmicroscope. ThT as amyloid beta-specific reference dye (0.1% concentration; green channel; FITC\nor 488/520 nm) following lead probes staining I-38 (upper panel) and I-43 (lower panel) and\nconfocal images were acquired using a NIR filter (25 μM; 633/700 nm)—scale bar: 20 μm length\nsize.\nand frontal cortex (Ft. Cr.) regions of non-demented patients (NDP; n = 4 independent samples)\nstained with I-43 alone, and confocal images were taken in green (488/520 nm) and NIR filters\n(633/700 nm)—scale bar: 20 μm length size.\n5) It is known that there is age-dependent accumulation of lipofuscin in human brain tissue and\n(in aged mouse brain). Please include staining showing that the probe does not bind to\nlipofuscin or has other off-target in the brain tissue. Autofluorescence assessment is also\nneeded on these brain tissue slices.\nResponse: To address the concern of off-target binding, particularly to lipofuscin, we have\nconducted a comprehensive fluorescence-based study in both aged mouse models and human\nbrain specimens. Specifically, we examined brain tissue from 13-month-old wild-type (WT) and\nAPP/PS1 transgenic mice (Fig. 3d, Page # 11 in the revised MS), as well as human brain specimens\n(Fig. 6g on Page # 18 in the revised MS and Supplementary Figs. 59, 63, and 65 on Pages # 86, 89\nand 91 in the revised SI.\nFor these studies, we incubated tissue sections with our probe, I-43 alone, and acquired\nfluorescence data using both FITC (488/520 nm) and NIR (633/700 nm) filters. Our results\nrevealed numerous tiny autofluorescent spots observed under the FITC filter, indicating\nlipofuscin and other endogenous fluorophores. However, these autofluorescent signals did not fully\noverlap with those detected in the NIR filter. Notably, cerebral amyloid angiopathy (CAA) is stained with\nI-43 in Fig. 6g, as shown by the red arrows, in the NIR filter. This selective binding confirms that I-\n43 effectively targets Aβ deposits while avoiding non-specific binding to autofluorescent materials\nlike lipofuscin. Supplementary Figs. 59, 63, and 65 also demonstrate that I-43 signals have minimal\nor no overlap with autofluorescent materials. Thus, our data demonstrate that probe I-43 does\nnot exhibit significant off-target binding to lipofuscin or other autofluorescent components in the\nbrain tissue, ensuring the specificity and reliability of our staining method for detecting Aβ\ndeposits.\nusing confocal microscopy. The green channel (488/520 nm) highlighted auto-fluorescent\nlipofuscin debris (indicated by yellow arrowheads), while the NIR filter was used to detect I-43-\nstained spots.\nFig. 6g: Representative confocal images of brain tissue from AD postmortem specimens stained\nwith I-43 provide a detailed view of the probe's specificity. In the green channel confocal images\n(488/520 nm), autofluorescent lipofuscin debris is indicated by white arrowheads. Additionally,\n32\nother endogenous autofluorescent deposits, marked by yellow arrows, are visible in the FITC filter\nbut are not detected in the NIR filter, demonstrating no affinity for I-43.\nCr.) regions of non-demented patients (n = 4 independent samples) were stained with I-43 alone,\nand confocal images were taken in green (488/520 nm) and NIR filters (633/700 nm).\nSupplementary Fig. 65: Deparaffinized brain slices from hippocampal (Hip) regions of AD patients\nstained with I-43 alone, and confocal images were taken in green (488/520 nm) and NIR filters\n(633/700 nm). Scale bar: 50 μm length size\n34\n6) Please include an ex vivo multiphoton images for the mice underwent in vivo imaging with\nprobe.\nResponse: Thank you for your suggestion. Currently, we do not have access to multiphoton\nimaging facilities. Despite our efforts to perform this analysis at other institutions, we\nencountered significant constraints, including the need for ethical approval and access to\ntransgenic mice facilities. Although some colleagues agreed to assist with the experiment, they\nalso faced similar limitations related to time (breeding and aging of APP-PS1 mice for appropriate\nage) and ethical concerns.\nAs an alternative, we have conducted ex vivo fluorescence confocal imaging of 40-\nmicrometer tissue sections from 13-month-old APP-PS1 mice to assess the visualization\ncapabilities of I-43 in labeling amyloid deposits (Fig. 4f-g; Page # 14 of revised MS). Confocal z-\nstack imaging revealed pronounced I-43 emissions (λex/em = 633/700 nm) in plaques (Fig. 4f)\nand in dual-staining with the OC antibody (Fig. 4g), with color-coded projections (Figs. 4f1 and\n4g1) showing signals detectable even beyond 45 μm. A brief discussion has been added to Page\n13 of the revised manuscript. We hope this serves as an appropriate replacement for the\nsuggested study. Further suggestions are welcome.\nFig. 4: (f) Schematic illustration to show fluorescence signal intensity of I-43 associated with Aβ\nplaques in z-stack projections up to a depth of 60 μm from APP-PS1 mice (13 months old) stained\nwith I-43 alone and dual-stained sections with I-43 + OC. (g) The f1 and g1 images are color-coded\nprojections of f and g, respectively, measuring the fluorescence signal strength of I-43 associated\nwith amyloid plaques from the upper layers to deeper tissue layers using λex/em = 633/700 nm.\nThese projection images are derived using Zeiss Zen 3.10 software, a freeware tool to analyze\nconfocal fluorescence imaging data.\n7) Does the probe bind to Cerebral amyloid angiopathy or only parenchymal plaque?\nResponse: Thank you for your query. We investigated whether our probe binds specifically to\ncerebral amyloid angiopathy (CAA) or only to parenchymal plaques. To address this question, we\nconducted a dual staining experiment using Congo Red, a dye known to detect CAA, alongside\nlead probe I-43 efficiently. The results, shown in Figure 6a (Page # 18 of revised MS) and\nlocalizes with the Congo Red stain, indicating that our probe binds to both parenchymal plaques\nand cerebral amyloid angiopathy.\nFig. 6a: Congophilic angiopathy or CAA was confirmed using CR dyes in the AD brain, which also\noverlap upon staining with the I-43. Congo red dye staining was recorded in the DIC filter and I-\n43 in the TRITC filter using the NIS-Elements BR 4.30.00 64-bit fluorescence microscope.\nSupplementary Fig. 60: Deparaffinized brain slices from frontal cortex (Ft. Cr.) regions of AD\npatients were stained with I-43, and counterstaining was performed with Congo Red (CR) to locate\ncongophilic amyloid deposits or cerebral amyloid angiopathy (CAA; indicated by yellow\n36\narrowheads). In the lower panel, the white arrowhead indicates Aβ plaque in both DIC and TRITC\nfilters. Fluorescence images were taken using the NIS-Elements BR 4.30.00 64-bit fluorescence\nmicroscope—scale bar: 20 μm.\nAdditionally, a panel from Fig. 6g (Page # 18 of revised MS) and Supplementary Fig. 61 (Page # 87\nof revised SI) from IHC staining with OC and D54D2 anti-Aβ antibodies following I-43 staining\nshowed similar results, revalidating our earlier findings. These consistent results confirm that the\nI-43 probe effectively detects both parenchymal Aβ plaques and CAA, highlighting its reliability\nand specificity for Aβ detection in AD brain tissue.\nFig. 6g: Congophilic angiopathy or CAA was confirmed using fibril-specific OC Anti-Aβ antibody\n(FITC filter) in AD brain also overlapped upon staining with the I-43 (NIF filter).\nSupplementary Fig. 61: Deparaffinized brain slices from the hippocampal (Hip) region and frontal\ncortex (Ft. Cr.) of AD patients were stained with I-43, and counterstaining was performed with\nD54D2 anti-Aβ antibody to locate amyloid deposits. Confocal images were captured using a\n488/520 nm channel to detect Alexa FluorTM 488 tagged secondary antibody conjugated to D54D2\nprimary antibody, and an NIR filter was used to detect I-43 associated signals using an LSM-780\n(Carl Zeiss) confocal microscope. Autofluorescence associated with Aβ deposits (blue) is noticed\nin the DAPI filter, an inherent property of Aβ, as mentioned in the previously reported article.10\n37\n8) The solubility of the probe is going to be complicated as 7.5% DMSO, 2.5% Cremophor EL is\nused in solvent. Even though the author showed the low toxicity, the longitudinal application\nmight be an issue.\nResponse: We appreciate your concerns regarding the solubility and formulation of our probe,\nnoting that the current vehicle (7.5% DMSO and 2.5% Cremophor EL) is suboptimal for clinical\nuse. We emphasize that this study involves the early-stage exploration of newly designed scaffolds\nfor the detection of Aβ and simultaneous ChEs detection for its application in the investigational\npathogenesis of AD. Investigational studies revealed that I-42 and I-38, structural analogs of lead\ncompound I-43, offer NIR detection for Aβ and ChEs with moderate AChE inhibition. This suggests\nthat targeted structural modifications could substantially enhance therapeutic efficacy with\ndesirable metabolic stability and aqueous solubility while maintaining selectivity for amyloid\nspecies. Our research group is actively working in this direction, using the I-43 structural template\nto explore these avenues and develop more effective refined probes in our future research. We\nhope the reviewer and potential readers will understand that our primary focus in these studies\nis to develop a novel scaffold using a rational design approach with dual affinity for Aβ and ChEs.\nOur future goal is to expand this scaffold through SAR exploration, aiming to create theragnostic\nprobes with high affinity and desirable physicochemical and pharmacokinetic properties.\n9) It is important to show that the probe can monitor the increase in Aβ during the disease\ndevelopment. Please assess longitudinally the change of amyloid load in the mouse brain to\ndemonstrate the robustness of this approach.\nResponse: Thank you for your valuable suggestion. We agree that demonstrating the ability of\nour probe to monitor the increase in Aβ during disease development is crucial. To address this,\nwe have conducted both in-vitro and in-vivo studies to evaluate the robustness of the I-43 probe\nin tracking Aβ load over time.\nWe performed histological staining of brain sections from APP-PS1 mice at 6 months and\n13 months of age and 13-month WT mice as control. These studies demonstrated that I-43\neffectively tracks Aβ burden, with an observable increase in Aβ deposits as the disease progresses\n(Fig. 4a-e; Pages # 13-14 of revised MS). Quantitative measurements were included in our\nrevision, showing that changes in Aβ count and density can be effectively determined using I-43.\nThese results are presented in Figures 4d-e of the revised manuscript.\nacross different ages. (a) WT mice (13 months old) had no plaque deposits (a1; magnified view),\nconfirmed via both OC and I-43 staining. Brains of APP-PS1 mice at different time points (6- and\n13-months old) revealed denser plaques compared to the younger littermates (b1 and c1;\nmagnified view), confirmed via both OC and I-43, which perfectly colocalized. Scale bar: 500 μm\nfor tile scan in (a), (b), and (c), and 100 μm for corresponding zoom in views (a1), (b1), and (c1),\nrespectively. (d) I-43 and OC-stained brain sections were quantified for labeled plaques using\nImageJ software. Statistical data in the bar graph are mean ± SD, n =10 independent biological\nsamples using ordinary one-way ANOVA; *P = 0.0437, and ****P < 0.0001. (e) Integrated FL\nintensity of I-43 in both 6- and 13-month age groups of APP-PS1 mice and statistical difference of\ndata set are mean ± SD determined using two-tailed unpaired t-test.\nWe also conducted in-vivo live imaging to measure the difference in radiant efficiency of\nthe I-43 probe between 6-month-old and 13-month-old APP-PS1 mice (Fig. 5a-c; Page # 15 of\nrevised MS and Supplementary Fig. 71 on Pages # 98-99 of revised SI). This imaging study provided\nquantitative data on the probe’s performance and ability to detect changes in Aβ load\nlongitudinally. We are assuming that these studies give an early overview that I-43 can be an\neffective NIR dye in tracking the progression of Aβ pathology in pre-clinical studies as a reliable\ntool for monitoring disease development.\nInitially, the whole brain area was considered for radiant efficiency counts on time scale.\nHowever, based on expert advice, we revised the ROI, now highlighted in a black square in all\ngroups, to unify quantitative signal estimation. We also incorporated data from two additional\n39\nanimals in each group and revised the supplementary data accordingly. We believe this study is\ncrucial and welcome any valuable suggestions based on the updated data.\nand 6-month-old APP-PS1 and 13-month-old WT mice after 0.8 mg/kg intravenous administration\nup to 150 minutes. Scalp imaging was performed at representative time points (n = 3 biologically\nindependent mice; Ex/Em 490/690 nm; Lago-X from Spectral Instruments Imaging, equipped with\ndesirable filter sets). (b) The radiance efficiency vs. time plot showed higher signal intensity in\nAPP-PS1 mice brains compared to WT mice throughout the study. (c) The bar graph showed\nsignificant differences in fluorescence intensity (**P =0.028) between APP-PS1 mice (13-month-\nold; n = 3) and WT mice at 60 minutes post i.v. administration of I-43, while significant difference\nin comparison to Bkg (*P = 0.048, **P = 0.002, and ***P = 0.0001, determined using one-way\nvivo fluorescence imaging was conducted on (d) 13-month-old APP-PS1 (n = 3), (e) 6-month-old\nAPP-PS1 (n = 3), and (f) 13-month-old WT (n = 3) mice. Scalp regions were shaved to capture\nradiance fluorescence signals at 10, 30-, 60-, 120-, and 150 minutes post-tail vein injection of I-43\n(0.8 mg/kg). Fluorescence images were acquired using the (Lago X - Spectral Instruments Imaging)\nsystem, with excitation at 470 nm and emission detection at 690 nm. Regions of interest (ROIs)\nwere marked as indicated in each image (Aura software; Version 4.5.0) for data processing, with\nresults presented as mean ± SD. (Note: The animal data of two other animals from different\ngroups were incorporated in Supplementary Fig. 71a-c on Page # 99 of the revised SI).\nMinor points:\n1) The age of mice used the in the study is not consistent (sometime 13 months, sometimes 14\nmonth). Please double check.\nResponse: We apologize for the oversight regarding the age of the mice used in our study. We\nconfirm that we are using 13-month-old mice from both the APP-PS1 and WT groups during the\nscreening process. We appreciate your attention to this detail, and thank you for pointing out the\ninconsistency. We have corrected this information in the Supplementary Information section,\nspecifically in Fig. S55-S57, which is now updated as Supplementary Fig. 55- 56 on Pages # 80-81\nin the revised SI.\n2) The author wrote that the brain slices of mice shown in Fig 3, SFig 56, SFig 57 are coronal\nslices. However, the slices are clearly horizontal. Please correct.\nResponse: We apologize for the mistake in describing the brain slices shown in Fig. 3a and\nSupplementary Fig. 56-57, which are 55-56 on Pages # 80-81 in the revised SI. As suggested, the\ncorrect orientation of these slices is horizontal, not coronal. We appreciate your attention to this\ndetail and have corrected the description in the revised manuscript and Supplementary\nInformation. Thank you for bringing this to our attention.\n3) in Fig 3 i. The author wrote the frontoparietal cortex of mouse brain. Can the author indicate\nwhere it is in the overview image.\nResponse: Thank you for your suggestion. We have highlighted the frontoparietal cortex region\nin Fig. 3i by marking it with a white dotted line square in the tissue section. This update, now\nreflected in Fig. 3a of the revised manuscript, should make the specified region more apparent to\nthe reader in the overview image.\n4) It would be best not to use words like “remarkably” in the manuscript. Please check through\nthe manuscript.\nWe have revised \"Remarkably, improved cognitive function in a scopolamine-induced amnesia\nmodel may be attributed to the potent anti-AChE activity\" statement from the abstract to\n\"Additionally, I-43 improved cognitive function in a scopolamine-induced amnesia model due\nto its potent anti-AChE activity.\" We have also reviewed the entire manuscript to ensure that\nsimilar subjective terms have been appropriately revised. Thank you for your valuable input.\n5) Fig. 3 (iii). The ROI for data analysis is missing. Please add on the image for illustration.\nResponse: Thank you for pointing out the missing ROI for data analysis in Fig. 3 (iii). We have now\nhighlighted the ROI area with a black square box in Fig. 3(iii), which is now Fig. 5a on Page # 15 of\nthe revised manuscript and rest two animal data represents in Supplementary Fig. 71a-c on Pages\n# 98-99 of the revised SI. This addition will help illustrate the area used for data analysis. We\nappreciate your attention to this detail.\n6) Page 6. The author mentioned that the probe was injected into Tg-AD mice. Please be specific.\ni.v. injection?\nResponse: Thank you for highlighting the need for specificity regarding the injection method. We\nhave revised the statement \"injected into Tg-AD mice\" on Page 6 to \"was administered\nintravenously to 6- and 13-month-old APP-PS1 mice,\" which is now reflected on Page 14 in the\nrevised manuscript. We have cross-verified the entire manuscript draft to ensure that details\nregarding the administration route are clearly provided, aiming to avoid confusion. We appreciate\nyour feedback.\n7) Please be consistent when describing the mice. Sometime the author uses Tg-AD mice,\nsometimes APP-PS1 mice.\nResponse: Thank you for pointing out the inconsistency in describing the mice. To ensure\nconsistency, we have replaced \"Tg-AD mice\" with \"APP-PS1 mice\" throughout the manuscript. We\nappreciate your attention to this detail.\nReferences:\n43\n1. Lim, C. Z. J. et al. Subtyping of circulating exosome-bound amyloid β reflects brain plaque\ndeposition. Nat. Commun. 10, 1144 (2019).\n2. Yang, J. et al. Bioluminescence Imaging with Functional Amyloid Reservoirs in Alzheimer’s Disease\nModels. Anal. Chem. 95, 14261-14270 (2023).\n3. Maeda, S. et al. Granular tau oligomers as intermediates of tau filaments. Biochem. 46, 3856-3861\n(2007).\n4. de Oliveira, G. A. P., Silva, J. L. Alpha-synuclein stepwise aggregation reveals features of an early\nonset mutation in Parkinson’s disease. Commun. Biol. 2, 374 (2019).\n5. Ni, R. et al. In-vitro and in-vivo characterization of CRANAD-2 for multi-spectral optoacoustic\ntomography and fluorescence imaging of amyloid-beta deposits in Alzheimer mice. Photoacoustics\n23, 100285-100285 (2021).\n6. Zhang, X. et al. Near-infrared fluorescence molecular imaging of amyloid beta species and\nmonitoring therapy in animal models of Alzheimer’s disease. Proc. Natl. Acad. Sci. USA 112, 9734-\n9739 (2015).\n7. Ran, C. et al. Design, Synthesis, and Testing of Difluoroboron-Derivatized Curcumins as Near-\nInfrared Probes for in Vivo Detection of Amyloid-β Deposits. J. Am. Chem. Soc. 131, 15257-15261\n(2009).\n8. Hou, S. S. et al. Near-infrared fluorescence lifetime imaging of amyloid-β aggregates and tau fibrils\nthrough the intact skull of mice. Nat. Biomed. Eng. 7, 270-280 (2023).\n9. Hintersteiner, M. et al. In vivo detection of amyloid-β deposits by near-infrared imaging using an\noxazine-derivative probe. Nat. Biotechnol. 23, 577-583 (2005).\n10. Gao, Y., Liu, Q., Xu, L., Zheng, N., He, X., Xu, F. Imaging and Spectral Characteristics of Amyloid\nPlaque Autofluorescence in Brain Slices from the APP/PS1 Mouse Model of Alzheimer's Disease.\nNeurosci. Bull. 35, 1126-1137 (2019).\n44"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised manuscript, the authors have gone a long way to meeting the critiques of the reviewers, and have addressed\nmost of reviewer's concerns. Especially, they conducted an in-depth study on the metabolic properties of the probe.Although\nthe hydrolysis of probe I-43 exists, this does not affect the fact that compound I-43 is a probe with good optical activity and\nbiological activity. This is a systematic work, and suitable for publication in Nature Communications.There's just one\nquestion that I want to go back to. In the response letter (Q4), they author claim \"The resultant data shows that I-43 is rapidly\nabsorbed, has moderate bioavailability...\", I think it's not inaccurate. The bioavailability (F%) should be calculated based on\nintravenous and intraperitoneal blood concentrations. Bioavailability cannot be quantified by intraperitoneal blood\nconcentrations alone.But that doesn't matter, because this statement is not included in the manuscript.Overall, I look forward to\nseeing this work appearing in Nature Communications.", "reviewer_2": "The authors have greatly improved the manuscript and addressed all the questions. I recommend the acceptance of the\nmanuscript.", "reviewer_3": "Overall, the manuscript represents a substantial body of work. However, there are major issues listed below that need to be\naddressed:\n1. Working premise: Authors highlight the significance in the dual nature of theranostic on targeting both amyloid beta and\nacetylcholinesterase. While it is agreeable that targeting acetylcholinesterase (AChE) and amyloid beta aggregation are two\nmajor strategies in treating Alzheimer's disease (AD), the introduction is confusing for the role of acetylcholinesterase in AD.\nFurthermore, it is unclear whether the same compound can be used to differentially detect the alteration in AChE and\namyloid beta aggregates in the same sample. If so, it should be highlighted.\n2. Presentation style: This manuscript seems to be put together from multiple reports. Frequently, the results are presented in\nthe manner that does not provide the meaningful conclusion and in some cases, such as the docking and MD simulations,\nnot enough detail. Furthermore, much of the data presented in this paper is accompanied by only technical description with\nno biological interpretation. This would make readers to wonder why the data was presented and why the readers should\ncare. Consequently, this makes the manuscript to be read as a technical report with a collection of data, instead of a\ncoherent investigation for the broad audiences.\n3. The construction of the manuscript: There are many seemingly minor issues but they amount to the perception that the\nmanuscript is poorly constructed. The following are a few examples:\nA. Some symbols in figure 1 are missing. Furthermore, some reference is not cited (e.g., Yang et al 2016 in references).\nB. Missing figure citation in main text, e.g., Figure 2C and supplemental figures 49-53.\nC. Missing description in Figure legend, e.g., Figure 6 i-j, Figure 79.\nD. Figures are of low resolution.\nE. Supplemental figure 54 is uninterpretable due to insufficient labeling.\n4. The interpretation of results and necessary controls: There are quite a bit of data interpretation issues in part due to the\npresentation and hyperbolic statement. Below are a few examples:\nA. page 6 line 2-6, 12-15: The Abeta40 and Abeta42 solutions were prepared following different methods. How do the\nauthors confirm that NH4OH (or other solvent differences) in the Abeta40 solution does not enhance compound\ninteraction/fluorescence? Subsequent data indicate that the fluorescence properties of I-43 can be altered by changes in\nsolvent composition.\nB. page 6 about that “I-43 can also slow down the aggregation of Abeta1-40 peptides”: This statement can be interpreted as\ninferred change in the rate of Abeta aggregation. However, the endpoint TEM and ThT assays do not measure the rate of\naggregation. Aggregation rates need to be measured directly in a kinetic ThT assay. The example of such rate analysis can\nbe found in Shoffner, S. K., and Schnell, S. (2016) Estimation of the lag time in a subsequent monomer addition model for\nfibril elongation. Phys Chem Chem Phys 18, 21259-21268\nC. page 11, second paragraph: Statements regarding specificity and overlap are overstated. “overlap exactly” “perfect\ncolocalization”, these statements are not true and should be toned down. There appear to be substantial regions stained by\nD54D2 and OC that are not stained by I-43, likewise, there are substantial regions stained by I-43 that are not stained by\nD54D2 and/or OC (Fig. 3e1/2)\nD. page 15 third paragraph on benefit of the rapid clearance: What is the support for this statement? The authors do not\npresent any data relating to CNS-associated toxicity. How does I-43 “improve temporal resolution” and “enhance\nspecificity”? The authors need to provide a comparison or reference point if they wish to make these statements.\nE. page 21 the end of second paragraph on imaging AChE activity (“We posit that this observation indicates the efficiency of\nthe probes in effectively imaging the ChE activity within a biological system”: Nothing the authors have presented\ndemonstrate any indication that the fluorescence of I-43 correlates with ChE activity. All of their ChE fluorescence data is\nmissing a vital control to demonstrate the fluorescence of I-43 in the presence of ChE without substrate. Without that control,\nit appears that I-43 fluorescence corresponds to the presence of the ChE protein, not it’s enzymatic activity. In fact, this\nappears to be supported by panels 7d,e which show increasing fluorescence of I-43 as the amount of AChE increases.\nF. The results in figures 4 and 5 are presented without context. Some statement or control experiment is needed to establish\nhow the I-43 results compare to currently available tools used for similar purposes.\nG. Panels A, B, and C in figure 6 all display fluorescent clusters stained with I-43 that are not stained by the control dye (with\nthe exception of CRANAD-2, which stains much more than I-43). The authors do not address this apparent non-specific\nstaining, and it appears to contradict their earlier claims of enhanced specificity of I-43. This trend continues in\nsupplementary figures 61,62, additionally, the authors state: Autofluorescence associated with amyloid-beta deposits (blue) is\nobserved in the DAPI filter, an inherent feature of Aβ plaques, as discussed in an earlier report.10 In supplementary figure\n61, yet these autofluorescent amyloid plaques are not stained by I-43 or D54D2. If the indicated structures are truly amyloid\nplaques, then neither staining method appears to be very good, and the author’s statement that I-43 can be applied as a\nsensitive marker for detecting β-459 amyloid deposits in AD pathology is dubious, at best. In the discussion, the authors\nmention that I-43 suffers from poor aqueous solubility, are these fluorescent cluster merely I-43 aggregates?\n5. Docking and MD simulation: There are a number of issues in the approach and execution in elucidating the molecular\nbasis for the interaction of I-43 with AChE or Abeta fibrils.\nA. The major conclusion for the interaction between I-43 and AChE is derived from docking and then short MD simulation\n(100 ns). It is curious that authors observed >5 angstrom RMSD for ligand yet only ~2 angstrom RMSD for protein (assuming\nmainchain) (Figure 9e). Giving such large RMSD, it is difficult to relay on the original docking. However, it is unclear whether\nauthors use the model original docking or the final MD simulation for their interpretation.\nB. From the checklist for MD simulation, authors marked NO on both 1c and 1d, meaning that the MD simulation was not\ndone as rigor as it should.\nC. 3c in the checklist for MD simulation should be NO, instead of yes."}, "author_response": "Response Letter\nThank you for reviewing and sharing the comments of the reviewers on our manuscript titled\n\"Discovery of Novel NIRF Theranostic Probes Targeting Amyloid-β Fibrils and Cholinesterases in\nAlzheimer's Disease Models\" (NCOMMS-23-59606). We appreciate the opportunity to resubmit\nthe revised manuscript. We also thank the reviewers and editorial board for their unanimous\nendorsements of the significance and novelty of the work carried out in our manuscript. We have\ncarefully gone through the reviewers' insightful comments and tried our level best to address the\ncomments in the revised manuscript. The Reviewers' comments are in italics. The necessary\nrevisions, based on the suggestions, are highlighted in red or blue in both the main text and\nsupplementary information for easy viewing. Our detailed point-by-point response is listed below\nfor your assessment.\nWe sincerely believe the above responses and further clarifications will adequately address all the\nissues raised by the reviewers. We sincerely hope you will allow us to discuss any other issues\nregarding this manuscript.\nRespectfully awaiting your decision.\nSincerely,\nGyan Prakash Modi\nThe Reviewers' comments are in italics.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this revised manuscript, the authors have gone a long way to meeting the critiques of the\nreviewers, and have addressed most of reviewer's concerns. Especially, they conducted an in-\ndepth study on the metabolic properties of the probe. Although the hydrolysis of probe I-43 exists,\nthis does not affect the fact that compound I-43 is a probe with good optical activity and biological\nactivity. This is a systematic work, and suitable for publication in Nature Communications. There's\n1\njust one question that I want to go back to. In the response letter (Q4), they author claim \"The\nresultant data shows that I-43 is rapidly absorbed, has moderate bioavailability...\", I think it's not\ninaccurate. The bioavailability (F%) should be calculated based on intravenous and\nintraperitoneal blood concentrations. Bioavailability cannot be quantified by intraperitoneal\nblood concentrations alone. But that doesn't matter, because this statement is not included in the\nmanuscript. Overall, I look forward to seeing this work appearing in Nature Communications.\nResponse: We sincerely appreciate the reviewer's recognition of our efforts to address the\nsuggested comments and positive assessment of our revised manuscript. We appreciate your\nattention to detail regarding the statement in our response letter (Q4) claiming that \"I-43 is\nrapidly absorbed, has moderate bioavailability.\" We agree with your point that bioavailability\n(F%) should be calculated using both intravenous and intraperitoneal blood concentrations. We\nfeel sorry for any ambiguity in our response letter. As you noted, this statement is not included\nin the manuscript itself, and we have ensured that all claims in the manuscript are supported by\nappropriate data and analyses. We are grateful for your recommendation for acceptance of our\nmanuscript in Nature Communications and look forward to seeing our work published.\nReviewer #2 (Remarks to the Author):\nThe authors have greatly improved the manuscript and addressed all the questions. I recommend\nthe acceptance of the manuscript.\nResponse: We sincerely thank the reviewer for their positive evaluation and recommendation\nfor acceptance of our manuscript. We appreciate the valuable feedback that has helped to\nimprove our manuscript and are pleased that our revisions have satisfactorily addressed all\nconcerns.\nReviewer #3 (Remarks to the Author):\nOverall, the manuscript represents a substantial body of work. However, there are major issues\nlisted below that need to be addressed:\n1. Working premise: Authors highlight the significance in the dual nature of theranostic on\ntargeting both amyloid beta and acetylcholinesterase. While it is agreeable that targeting\nacetylcholinesterase (AChE) and amyloid beta aggregation are two major strategies in\ntreating Alzheimer's disease (AD), the introduction is confusing for the role of\nacetylcholinesterase in AD. Furthermore, it is unclear whether the same compound can be\nused to differentially detect the alteration in AChE and amyloid beta aggregates in the same\nsample. If so, it should be highlighted.\nWhile AChE is primarily known for its cholinergic function, emerging evidence suggests its\ninvolvement in amyloid plaque formation and disease progression. Accordingly, we have\nrevised the introduction (pages # 2–3 of the revised manuscript) to provide a more focused\nand comprehensive discussion of the pathological role of cholinesterases (ChEs) in AD.\nWe are also thankful to the reviewer for insightful comments regarding the potential utility\nof our compound for differentially detecting alterations in AChE and amyloid-β aggregates\nwithin the same sample. In this study, we screened newly designed scaffolds to detect\namyloid-β aggregates and modulate AChE activity under separate experimental conditions.\nAChE activity was evaluated through enzymatic assays and cell-based models, while amyloid-\nβ binding was assessed via fluorescence imaging and staining. In the previous version of the\nmanuscript, the combined detection of both biomarkers in the same sample was not\nincluded. As suggested, this has now been clearly acknowledged in the revised Discussion\n(page 34 of revised MS). In response to your suggestion, we carried out a few initial\nexperiments to detect both AChE and amyloid-β simultaneously in the same sample. While\nthese early attempts didn’t produce clear results, they gave us useful insights that are shaping\nour future projects. We recognize the strong potential of dual-target detection and are now\nactively exploring this direction as part of our ongoing research.\n2. Presentation style: This manuscript seems to be put together from multiple reports.\nFrequently, the results are presented in the manner that does not provide the meaningful\nconclusion and in some cases, such as the docking and MD simulations,\nnot enough detail. Furthermore, much of the data presented in this paper is accompanied by only technical\ndescription with no biological interpretation. This would make readers to wonder why the\ndata was presented and why the readers should care. Consequently, this makes the\nmanuscript to be read as a technical report with a collection of data, instead of a\ncoherent investigation for the broad audiences.\nResponse: We appreciate your thorough review, which has undoubtedly enhanced the\nquality of our manuscript. We feel sorry that the previous version of the manuscript came\nacross as fragmented and overly technical, with insufficient biological context. After carefully\nre-reading the manuscript, we've made a concerted effort to revise and streamline the\npresentation to ensure the results are better connected and the biological relevance of our\nfindings is clearly communicated. Some of them are as follows:\n1) We have completely redesigned Figure 1 (page # 3 of the revised manuscript) to make it\nmore visually engaging and easier to follow. We've streamlined the content to reduce\nclutter, carefully checked all details to ensure accuracy, and organized the elements to\nbetter reflect the logic behind our probe design. The updated figure now provides a\nclearer connection to the hypothesis we’re proposing.\n3\n2) To improve clarity, we have divided the original Figure 2 into two separate figures—now\nFigures 2 and 3 (pages 8 and 11 in the revised manuscript)—due to the high number of\ngraphs. Additionally, we reorganized the spectroscopic data presentation to better reflect\nthe updated discussion, enhancing the connection between the results and their\nbiological significance.\n3) We have revised the remaining figures in the main manuscript (Figures 4–10) and the\nSupplementary Information in accordance with the journal’s formatting guidelines.\n4) We have also corrected and rephrased several statements to improve tone, eliminate\noverstatements, and remove unnecessary wording to ensure conciseness and compliance\nwith the journal’s word limit. These edits do not alter the original meaning or scientific\ncontent and therefore have not been highlighted. Only major revisions, including newly\nadded experimental data, are marked in red or blue in the revised manuscript.\nNext, in response to the valuable feedback on lacking in meaningful conclusion especially as\nthe docking and MD simulations, we have thoroughly revised the docking and molecular\ndynamics (MD) simulation sections to enhance clarity, depth, and interpretability.\nSpecifically:\n1) The original Fig. 9e has been replaced with an updated and more informative version\n(now Fig. 10g in the revised manuscript on page #32), clearly separating the RMSD profiles\nof the hAChE protein and the ligand I-43. This revision improves the visual presentation\nand better reflects the structural stability of both the protein and ligand, thereby\nsupporting the observed binding behavior.\n2) We have also added new complementary simulation data (Figs. 10h–i, page 32) including\nRMFS analyses and interaction persistence maps, which together provide mechanistic\ninsight into the stability and specificity of I-43 binding within the AChE active site. The\nassociated Results and Discussion sections have been expanded to contextualize these\nfindings (page # 31-33 of revised MS, in blue) and convey a more meaningful and\nevidence-based conclusion. As per the journal’s word limit guidelines, we have presented\nthis data in a concise yet meaningful manner, aiming to maximize scientific depth while\nmaintaining brevity.\nWe hope these revisions satisfactorily address your concerns and contribute to a clearer and\nmore cohesive manuscript. We sincerely appreciate your insights, which have helped us\nimprove the quality and presentation of our work for the benefit of the readers.\n3. The construction of the manuscript: There are many seemingly minor issues but they amount\nto the perception that the manuscript is poorly constructed. The following are a few examples:\n4\na. Some symbols in figure 1 are missing. Furthermore, some reference is not cited (e.g.,\nYang et al 2016 in references).\nResponse: We thank the reviewer for identifying these issues, and we feel sorry for these\nmistakes. As suggested by the reviewer, we have made the necessary corrections in the\nrevised manuscript.\n1) We have completely redesigned Figure 1 (page # 3 of the revised manuscript) to make it\nmore visually engaging and easier to follow. We've streamlined the content to reduce clutter,\ncarefully checked all details to ensure accuracy, and organized the elements to better reflect\nthe logic behind our probe design. The updated figure now provides a clearer connection to\nthe hypothesis we’re proposing.\n2) We have reviewed the reference list and added the missing citation for Yang et al., 2016\n(reference 38 in the revised MS) in the appropriate section of the manuscript.\nb. Missing figure citation in main text, e.g., Figure 2C and supplemental figures 49-53.\nResponse: Thank you for pointing out the missing figure citations. We have now addressed\nthis issue in the revised manuscript. Specifically, Fig. 2c (now revised as Fig. 2g) has been cited\nappropriately in the main text on page 8. In addition, the following Supplemental Figures have\nbeen correctly cited in the respective sections of the text:\n Supplemental Figs. 49–50 (now revised as 48–49) on page 7\n Supplemental Fig. 51 (now revised as 52) on page 9\n Supplemental Fig. 52 (now revised as 75a) on page 29 and highlighted in blue\n Supplemental Fig. 53 on page 10\nWe appreciate your attention to these details. The updated manuscript with all corrected\nfigure citations has been attached for your kind review.\nc. Missing description in Figure legend, e.g., Figure 6 i-j, Figure 79.\nResponse: Thank you for pointing out the missing details in the figure legend. As suggested,\nthe legend for Figure 6i–j (now revised as Figure 7j–k in the updated manuscript on page 23)\nhas been thoroughly revised to include the previously omitted descriptions. The updated\nlegend now clearly explains the imaging setup, fly genotypes, treatment conditions, ROI\nselection, and fluorescence quantification parameters. These corrections have been\nincorporated in the revised manuscript and are highlighted in blue on page 24 for your\nconvenience.\nAdditionally, we have revised the figure description for Supplementary Fig. 79 on page # 112-\n13 of revised SI. We have now revised the figure legends accordingly to ensure clarity and\ncompleteness. We appreciate your careful review and suggestions.\nd. Figures are of low resolution.\nResponse: Thank you for your feedback. We acknowledge the issue with the figure resolution.\nWe have now replaced the low-resolution images with high-resolution versions to ensure\n5\nclarity and readability. The revised figures have been updated in the manuscript and\nsupplementary materials for better visualization. We appreciate your attention to this detail.\ne. Supplemental figure 54 is uninterpretable due to insufficient labeling.\nResponse: Thank you for pointing this out. We agree that the original Supplementary Figure\n54 lacked clear labeling, which made it difficult to interpret. We've now updated the figure\nwith proper axis labels, legends, and annotations to make it much clearer. The revised version\nappears as Supplementary Fig. 51 on page 72 of the updated supplementary file. Also, to\nclarify—the change in figure numbering in both the main manuscript and supplementary\ninformation is due to minor revisions we made to the discussion section, to better align the\ntext with the experimental results. We really appreciate your helpful feedback-it allowed us\nto improve the clarity of the data presentation.\nSupplementary Fig. 51: ThT fluorescence spectra for aliquots (i)–(iv): (i) ThT alone (20 μM),\n(ii) ThT + Aβ (2:1, 72 h), (iii) ThT + I-43 + Aβ (2:4:1, 72 h), and (iv) ThT + I-43 + Aβ (2:2:1,\n1-42 1-42 1-42\n72 h). Measurements were performed with excitation at 445 nm, and emission spectra were\nrecorded from 460–650 nm using a SpectraMax-i3x microplate reader (Molecular Devices). (n = 3\nindependent experiments in triplicate)\n4. The interpretation of results and necessary controls: There are quite a bit of data\ninterpretation issues in part due to the presentation and hyperbolic statement. Below are a\nfew examples:\na. page 6 line 2-6, 12-15: The Abeta40 and Abeta42 solutions were prepared following\ndifferent methods. How do the authors confirm that NH4OH (or other solvent differences)\nin the Abeta40 solution does not enhance compound interaction/fluorescence?\nchanges in solvent composition.\nResponse: Thank you for your insightful comment. To address the concern regarding the\npotential influence of NH₄OH or other solvent conditions on the fluorescence properties\nof I-43, we performed an additional Aβ₁₄₀ aggregation experiment under two different\n-\nconditions:\n 20 mM phosphate buffer (pH 7.4; molecular biology grade)\n 1% NH₄OH (w/v) in 1× PBS, with pH adjusted to 7.5–8.0\nIn both cases, Aβ₁₄₀ monomer solutions were incubated at 37 °C with constant shaking\n-\nfor 72 hours to induce fibril formation. Following incubation, fibril formation was\nconfirmed (ThT based assay), and fluorescence measurements with I-43 were carried out\nto assess its response under both the conditions. These results allowed us to evaluate\nwhether NH₄OH influences the fluorescence behavior of I-43 in the context of Aβ fibril\nbinding. As shown in Fig. 1, results revealed a slight decrease in fluorescence intensity for\nAβ aggregates formed in PBS alone compared to those formed using the NH₄OH\n1–40\nprotocol. This difference may be attributed to variations in the aggregate morphology or\nstructure under different incubation conditions. Nevertheless, in both cases, I-43\nexhibited a significant fluorescence response (****P < 0.0001), confirming its robust\nability to detect Aβ aggregates regardless of the solvent environment.\n1–40\nFig. RL-1: Fluorescence (Fl) spectra of I-43 (20 μM) were recorded for the following\naliquots: (i) I-43 + Aβ monomers (10 μM) in 1× PBS (pH 7.4); (ii) I-43 + Aβ fibrils\n1–40 1–40\n(10 μM) formed after 72 h incubation in 1× PBS (pH 7.4); and (iii) I-43 + Aβ fibrils\n1–40\n(10 μM) formed after 72 h incubation using the NH₄OH protocol. Fluorescence\nmeasurements were carried out using a SpectraMax i3x microplate reader (Molecular\nDevices) with excitation at 490 nm and emission recorded from 525 to 850 nm. Data are\n7\npresented as mean ± SD (n = 3 independent experiments, each performed in triplicate).\nStatistical analysis was performed using one-way ANOVA; ****P < 0.0001.\nb. page 6 about that \"I-43 can also slow down the aggregation of Abeta1-40 peptides\": This\nstatement can be interpreted as inferred change in the rate of Abeta aggregation.\nHowever, the endpoint TEM and ThT assays do not measure the rate of aggregation.\nAggregation rates need to be measured directly in a kinetic ThT assay. The example of such\nrate analysis can be found in Shoffner, S. K., and Schnell, S. (2016) Estimation of the\nlag time in a subsequent monomer addition model for fibril elongation.\nResponse: Thank you for your insightful comment. As suggested, ThT based time\ndependent aggregation kinetic was performed. Briefly, the monomeric Aβ peptide was\n1-40\ndiluted in PBS (20 mM phosphate, pH 7.4) to a final concentration of 10 μM in a total\nvolume of 1.0 mL for each of three experimental conditions: Aβ alone, Aβ with I-43\n1-40 1-40\nat 1:2 molar ratio (20 μM), and Aβ with I-43 at 1:4 molar ratio (40 μM), each prepared\n1-40\nin triplicate. The final mixtures contained 10% DMSO (v/v), accounting for I-43 stock\nsolutions (5 mM in DMSO) and adjusted accordingly to maintain consistent solvent\nconditions. All solutions were vortexed and incubated at 37 °C with constant shaking at\n300 rpm to initiate aggregation. At selected time points (0, 2, 4, 8, 12, 24, 36, 48, 60, and\n72 h), 90 μL aliquots were collected from each replicate and immediately mixed with\nThioflavin-T (ThT) to achieve a final ThT concentration of 10 μM. The resulting samples\nwere transferred to a black 384-well plate (30 μL per well, three technical replicates per\nsample), and fluorescence intensities were recorded using a multimode microplate\nreader. ThT fluorescence was measured at 445 nm excitation and 485 nm emission, while\nI-43 fluorescence was monitored at 490 nm excitation and 690 nm emission to\nsimultaneously track aggregation kinetics and probe interaction.\nA brief discussion with slight modification is added “TEM images from Fig. 3a\nshowed that at 2-fold concentration of I-43, Aβ formed amorphous clusters instead of\n1-40\nwell-defined fibrils, and at 4-fold the concentration, mostly short or fragmented fibrils\nwere observed. This suggests that I-43 may interfere with fibril formation, particularly at\na 4:1 probe-to-peptide ratio. These observations are consistent with ThT fluorescence and\ntime-dependent aggregation kinetics over 72 hours (Fig. 3b3). While in vitro experiments\noffer valuable insights, further in situ studies are needed to validate these findings in a\nbiological context.”\nThe kinetic ThT assay results in the provided figure substantiate the claim that I-\n43 slows down Aβ aggregation, by demonstrating a significant reduction in ThT\n1-40\nfluorescence over time in the presence of I-43. Specifically, condition (ii) Aβ monomer\n1-40\nalone showed a rapid increase in ThT fluorescence, plateauing around 24 h, indicative of\ntypical sigmoidal fibril formation kinetics involving nucleation, elongation, and saturation\nphases. In contrast, samples containing I-43 at 4:1 (iii) and 2:1 (iv) molar ratio exhibit\n8\nmarkedly suppressed ThT fluorescence across all time points, with minimal increase even\nafter 72 h, indicating inhibition of β-sheet-rich fibrillar aggregation. Importantly, this data\ndirectly addresses the reviewer’s concern that endpoint assays (e.g., 72 h TEM or final-\npoint ThT) do not measure aggregation rates. Here, the full kinetic profile was recorded\nat defined time intervals (0–72 h), permitting time-dependent observation of aggregation\nprogression. The absence of a typical exponential rise or sigmoidal curve in I-43-treated\ngroups supports a kinetic delay or suppression of fibril formation, thus reflecting a slowed\naggregation rate.\nWe would like to acknowledge that the primary objective of the current study was\nto develop theranostic probes targeting amyloid-β and cholinesterases. Our probe design\nstrategy was based on creating a novel scaffold capable of detecting amyloid-β and\ntargeting the cholinergic system for theranostic applications. In response to the earlier\nreviewer’s suggestion to investigate Aβ aggregation modulatory activity, we conducted\nadditional experiments. The results demonstrated that I-43 can selectively modulate Aβ\n1-\naggregation in vitro, although this effect was observed only at a relatively higher\n40\nconcentration (four times the concentration of the peptide). While these findings are\npreliminary and do not fully replicate the complexity of the biological environment,\nfurther in-depth investigations are necessary to establish a correlation between the\ntherapeutic applicability of I-43 as a Aβ modulator. We are grateful for this valuable\nsuggestion and intend to explore it as part of our future work.\n9\ny tis\nn\n1.2 ThT + A42(Mono)\ne tn0.9 ThT + A42(Mono) & I-43\ni lF in 1:4\nT h0.6\nT\nd\ne zila 0.3\nm\nro0.0\nN\n0 20 40 60 80\nTime (h)\nFig. 3. Panel b3 shows ThT and I-43 Fl intensity measurement to record aggregation\nkinetics, depicting the time-dependent conversion of monomeric Aβ peptides into\n1-40\naggregates in the presence or absence of I-43 over 72 h. In the graph, roman values\nrepresent for the following conditions: (ii) ThT + Aβ (2:1, 72 h); (iii) ThT + Aβ + I-43\n1-40 1-40\n(2:1:4, 72 h); and (iv) ThT + Aβ + I-43 (2:1:2, 72 h).\n1-40\nc. page 11, second paragraph: Statements regarding specificity and overlap are overstated.\n\"overlap exactly\" \"perfect colocalization\", these statements are not true and should be\ntoned down. There appear to be substantial regions stained by D54D2 and OC that are not\nstained by I-43, likewise, there are substantial regions stained by I-43 that are not stained\nby D54D2 and/or OC (Fig. 3e1/2).\nResponse: We thank the reviewer for this valuable observation. We accept the opinion\nthat the interpretation regarding the exact co-localization between the reference dyes\nand I-43 may have been overstated, and we regret any confusion this may have caused.\nIn response, we have revised the text to reflect that I-43 exhibits partial colocalization\nwith D54D2- and OC-positive amyloid deposits, as shown in Figs. 4g–h and insets g1 and\nh1. We have also acknowledged the presence of non-overlapping regions, indicating that\nI-43 and these antibodies may target different conformational states or regions within\nthe Aβ aggregates. This distinction is further supported by additional observations in\nSupplementary Fig. 56a3 (In SI on page #82), where certain areas stained by OC were not\nlabeled by I-43 and vice versa. The revised text now accurately represents these findings\n(page # 15-16 of revised MS) and avoids overstatements regarding colocalization.\nAfter reviewing Figure 3e1/2 again, which is now Fig. 4g–h in the revised manuscript (page\n# 14 of revised MS), we have updated the figure to include merged images alongside\nindividual fluorescence channels for D54D2 and OC to facilitate easier interpretation of\ncolocalization.\n(white square box, dotted line)—scale bar: 50 μm in e panel images and 20 μm in g1 and\nh1 image.\nThese variations likely reflect the distinct binding profiles of the antibodies and the\nmolecular probe. D54D2, which targets the N-terminal region of Aβ, binds a broad\nspectrum of Aβ species, including monomers, oligomers, and fibrils. In contrast, OC is a\nconformation-specific rabbit polyclonal antiserum that selectively binds to fibrillar Aβ and\nsimilar aggregates formed by other amyloidogenic peptides, but not to monomers or\nprefibrillar oligomers (https://doi.org/10.1074/jbc.M114.594846). Nonetheless, we\ncannot exclude the possibility that these differences stem from epitope masking or\nartifacts introduced during tissue processing.\nTo reflect this more accurately in the manuscript, we have revised the text on page 11 of\nour earlier draft from: “Fig. 3e and their magnified insets (Figs. 3e1, and 3e2) show that\nD54D2 and OC-positive amyloidogenic deposits overlap exactly with I-43 labeled stains,\nconfirming I-43’s affinity for Aβ deposits in APP-PS1 mice. Regions a1, a2, and a3 from\nSupplementary Fig. 56a (page #82) of a 13-month-old APP-PS1 mouse brain showed\nperfect colocalization of I-43 and OC anti-Aβ antibody, indicating equipotency of I-43\ncorresponding to OC with high specificity.” to the following: “I-43 exhibits substantial\n11\nspatial overlap with D54D2- and OC-positive amyloid plaques in APP-PS1 brain sections,\nhighlighting its potential to target Aβ pathology. However, magnified views (Figs. 4g–h,\ninsets g1 and h1) reveal partial colocalization, suggesting differences in binding specificity\nor epitope accessibility. Furthermore, analysis of regions a1, a2, and a3 in Supplementary\nFig. 56a shows that dense-core plaques were co-labeled by both OC and I-43. However,\ncertain plaque regions stained by OC lacked corresponding I-43 signal, and vice versa\n(Supplementary Fig. 56a3), further supporting the notion that I-43 may bind to specific\nAβ conformations distinct from those recognized by OC.”\nThis revision is now clearly reflected on page 15 of the updated manuscript. We believe\nthese adjustments align with the data and enhance the manuscript's accuracy. We\nwelcome the reviewer's suggestions for additional measures to further validate these\nobservations.\nd. page 15 third paragraph on benefit of the rapid clearance: What is the support for this\nstatement? The authors do not present any data relating to CNS-associated toxicity. How\ndoes I-43 \"improve temporal resolution\" and \"enhance specificity\"? The authors need to\nprovide a comparison or reference point if they wish to make these statements.\nResponse: We thank the reviewer for this thoughtful comment. We agree that\nstatements regarding rapid clearance and its benefits should be supported by more direct\nevidence, such as brain pharmacokinetic data, rather than relying solely on in vivo\nfluorescence imaging. In the previous version of manuscript (page 15), the statement “Its\nrapid clearance reduces background signal, lowers CNS-associated toxicity, improves\ntemporal resolution, and enhances specificity…” was intended as a summary\ninterpretation of our in vivo imaging and ex vivo biodistribution results. Specifically, we\nobserved that I-43 exhibited strong brain fluorescence within 30 minutes post-injection,\nfollowed by a steep decline, approaching baseline by ~150 minutes. This rapid decline\nsuggests fast washout of the probe from the brain. We recognize that rapid clearance\ninferred from imaging kinetics may not directly equate to reduced toxicity without formal\ntoxicological assessments. However, from an imaging standpoint, such washout behavior\nenhances temporal resolution, enabling tracking of dynamic signal changes within a\nnarrow imaging window. It may also contribute to enhanced specificity by limiting the\nduration for non-specific accumulation. As suggested, we have revised the discussion on\npage 18 of the updated manuscript to reflect interpretations based on observed imaging\ndynamics and biodistribution profiles. Additionally, we have removed conclusive\nlanguage regarding toxicity to avoid overinterpretation to the following section.\nFortunately, we conducted experiments to quantitatively assess the rapid elimination and\npotential CNS-related toxicity of I-43, as described in other sections of the manuscript.\nIn particular, the section titled “Brain permeability, acute oral toxicity study,\npharmacokinetic profiling, and behavioral study in AD mice model” (page 27 of the\n12\nrevised manuscript) presents HPLC-based brain pharmacokinetic data, showing that I-43\nexhibited substantial initial brain uptake (0.59% ID/g at 5 minutes post-injection),\nfollowed by a rapid elimination rate, as indicated by a brain ratio of 3.4. This\n5min/60min\nquantitative profile reinforces the inference drawn from in vivo imaging that I-43 exhibits\nrapid brain washout, supporting its potential for high temporal resolution imaging.\nWhile we did not perform dedicated CNS toxicity assays such as behavioral or\nhistopathological analysis, we do have supporting evidence suggesting I-43 is not overtly\nneurotoxic under the tested conditions. Specifically, in-vitro based MTT assays in cultured\nneuronal cells (PC12 cells; see Fig. 3g on page 11 of revise MS) showed over 80% viability\neven at 10 μM after 24 hours of incubation. Additionally, during acute toxicity evaluation\nin mice (dose: 550 mg/kg; OECD 425; see Fig. 9b discussion on page 29 of revised MS), we\ndid not observe any abnormal behaviors, including, changes in water or food intake,\naberrant behavior such as seizure-like symptoms. We hope the following revision\naddresses the concern raised, and we thank you again for helping us improve the clarity\nof this section.\npage 21 the end of second paragraph on imaging AChE activity (\"We posit that this\nobservation indicates the efficiency of the probes in effectively imaging the ChE activity\nwithin a biological system\": Nothing the authors have presented demonstrate any\nindication that the fluorescence of I-43 correlates with ChE activity. All of their ChE\nfluorescence data is missing a vital control to demonstrate the fluorescence of I-43 in the\npresence of ChE without substrate. Without that control, it appears that I-43 fluorescence\ncorresponds to the presence of the ChE protein, not it's enzymatic activity. In fact, this\nappears to be supported by panels 7d,e which show increasing fluorescence of I-43 as the\namount of AChE increases.\nResponse: We sincerely thank the reviewer for this thoughtful and constructive\ncomment. In response to the raised concern regarding whether the fluorescence of I-43\nreflects cholinesterase (ChE) activity or merely the presence of ChE proteins, we carried\nout additional control experiments. As suggested, we evaluated the fluorescence\nresponse of I-43 in the presence of AChE and BChE alone, i.e., in the absence of their\nrespective substrates (ATCI for AChE and BTCI for BChE). These new data are now included\nin Fig. 8a-d of revised MS on page # 25 (the original Fig. 7a-c on page 23 of the previously\nsubmitted version). Form the study, we found that the presence of ChE proteins\n(highlighted in red in both the spectra a-c and bar graphs b-d) alone upon incubation with\nI-43 does not lead to a significant fluorescence enhancement. In contrast, substantial\nfluorescence increases are observed only in the presence of both the enzyme and its\ncorresponding substrate. These findings confirm that the fluorescence turn-on effect of I-\n43 is dependent on enzymatic activity, rather than simple protein binding. We have\nrevised the relevant section of the manuscript accordingly (page 24 of revise MS) to clarify\n13\nthis interpretation and better reflect the activity-dependent nature of I-43 fluorescence.\nAdditionally, we note a slight red shift (~15 nm) in the emission maxima observed in these\nexperiments compared to earlier results. This is likely due to the use of a different plate\nreader—SPARK Microplate Reader (TECAN, Austria)—in this set of experiments, as our\npreviously used SpectraMax i3x instrument is temporarily out of service due to hardware\nissues. We sincerely believe this instrument-related shift does not affect the overall\ninterpretation or conclusions of the experiment.\nWe appreciate the reviewer’s suggestion, which helped us strengthen the mechanistic\ninsight into the probe’s response and validate its specificity for ChE enzymatic activity.\nFig. 8. In-vitro and cell-based fluorescence assay to monitor detection affinity of I-43 for\ncholinesterases (ChEs). (a-b) Fluorescence (Fl) spectra and emission intensity bar graphs\nof I-43 alone, and upon co-incubation with: (i) AChE (2.0 U/mL) alone, (ii) ATCI (1.5 mM)\nalone, and a mixture of (i) + (ii). (c-d) Fluorescence (Fl) spectra and emission intensity bar\ngraphs of I-43 alone, and upon co-incubation with: (iii) BChE (10 U/mL) alone, (iv) BTCI\n(1.5 mM) alone, and a mixture of (iii) + (iv). Data were acquired at 490 nm excitation using\na SPARK Microplate Reader (TECAN, Austria). Values are mean ± SD from three\nindependent experiments in triplicates; unpaired two-tailed t-test, ****P < 0.0001, ***P\n= 0.0003.\n14\ne. The results in figures 4 and 5 are presented without context. Some statement or control\nexperiment is needed to establish how the I-43 results compare to currently available tools\nused for similar purposes.\nResponse: Thank you for your thoughtful comment. We agree that adding context is\nimportant for interpreting the performance of I-43.\nAs suggested, we have revised the sections related to Fig. 4 (updated as Fig. 5) on page\n16-17 of the revised MS. Specifically, we now include a comparative analysis highlighting\nthe advantages of I-43 over conventional Aβ dyes such as ThT, ThS, and Congo Red, which\nare limited by poor selectivity and shallow tissue penetration due to short emission\nwavelengths (500–550 nm). In contrast, I-43 demonstrates reliable longitudinal tracking\nof age-dependent Aβ deposition, as evidenced by semi-quantitative analysis of plaque\nburden in APP-PS1 mice at different ages. Furthermore, its deep-tissue labeling capacity\nis validated through Z-stack confocal imaging and co-localization with the fibrillar Aβ-\nspecific OC antibody. Together, these findings support I-43 as a technically superior\nalternative for Aβ imaging in preclinical and histopathological applications. As such, Fig. 5\nfocuses specifically on I-43’s ability to monitor age-related Aβ burden and tissue\npenetration, and does not duplicate the control comparisons already detailed in other\nparts of the manuscript.\nAs noted, Figure 5 (now revised as Fig. 6) specifically evaluates the in vivo imaging\nperformance of I-43 for semi-quantitative estimation of Aβ burden in live APP-PS1 mice,\nalong with its biodistribution profile. We fully agree that including a direct comparative\nreference with a known Aβ-targeting dye would further strengthen the study. However,\nconducting such in vivo comparative experiments posed significant logistical and ethical\nconstraints. Specifically, acquiring animal ethics approval and aging mice to later stages\nof disease progression required for longitudinal studies exceeded our current project\ntimelines and regulatory allowances. Despite our best efforts, we were unable to secure\nthe necessary permissions within the revision window.\nTo address this limitation, we are now considering alternative strategies that do not\nrequire additional animal experimentation. Moreover, we have incorporated a\nretrospective comparison using literature data on well-established Aβ-targeting NIR\nprobes (CRANAD-2, ZW800–1C, AOI-987, and HS-169), provided in Supplementary Table\n14 (pp. 113–114) and highlighted in blue on pages 34 of the revised manuscript. This\ncomparison qualitatively and quantitatively contextualizes I-43’s performance in terms of\nimaging depth, signal specificity, and sensitivity to reported versatile NIR dyes. We would\ngreatly value the reviewer’s feedback on whether this approach would be a suitable\nalternative, or if other ex vivo or computational comparisons might be appropriate.\nDiscussion section and to clearly articulate the rationale for our current design and the\nconstraints faced.\nf. Panels A, B, and C in figure 6 all display fluorescent clusters stained with I-43 that are not\nstained by the control dye (with the exception of CRANAD-2, which stains much more than\nI-43). The authors do not address this apparent non-specific staining, and it appears to\ncontradict their earlier claims of enhanced specificity of I-43. This trend continues in\nsupplementary figures 61,62, additionally, the authors state: Autofluorescence associated\nwith amyloid-beta deposits (blue) is observed in the DAPI filter, an inherent feature of Aβ\nplaques, as discussed in an earlier report.10 In supplementary figure 61, yet these\nautofluorescent amyloid plaques are not stained by I-43 or D54D2. If the indicated\nstructures are truly amyloid plaques, then neither staining method appears to be very\ngood, and the author's statement that I-43 can be applied as a sensitive marker for\ndetecting β-459 amyloid deposits in AD pathology is dubious, at best. In the discussion,\nthe authors mention that I-43 suffers from poor aqueous solubility, are these fluorescent\ncluster merely I-43 aggregates?\nResponse: We acknowledge and appreciate the reviewer’s constructive critique, which\nallowed us to address key points and enhance the scientific quality of our work. First, we\nwould like to note that, in response to comment (#2) regarding presentation style, we\nhave revised Figure 6 and its associated data to provide a more systematic correlation.\nThis is now presented as Figure 7 on page 22 of the updated manuscript.\nTo ensure clarity and avoid any ambiguity, we have outlined our point-by-point revisions\nbelow.\n“Panels A, B, and C in figure 6 all display fluorescent clusters stained with I-43 that are not\nstained by the control dye (with the exception of CRANAD-2, which stains much more than\nI-43). The authors do not address this apparent non-specific staining, and it appears to\ncontradict their earlier claims of enhanced specificity of I-43. This trend continues in\nsupplementary figures 61,62”\nIn Panel A, Thioflavin T (ThT) was used as a counterstain alongside I-43. While ThT is\neffective for detecting β-sheet-rich amyloid plaques, its broader reactivity with non-\namyloid structures—such as DNA base pairs, hyaline cartilage, human serum albumin,\nand α-helices in acetylcholinesterase—can result in staining beyond amyloid aggregates\n(DOI: 10.1038/s41598-024-74359-8). Therefore, we assume that the lack of overlap in a\nfew instances reflects either non-amyloid regions stained by ThT or differences in binding\naffinity to amyloid aggregates between the two dyes. This clarification has now been\nclearly mentioned in the revised manuscript on page 20.\nPanel B of Figure 6 from our earlier submission is now presented as Figure 7f in the revised\nmanuscript. This panel demonstrates I-43’s affinity assessment for α-synuclein aggregates\n16\nin Parkinson’s disease brain sections, using the P-ser129 α-synuclein antibody. The lack of\ncolocalization between I-43 and the antibody indicates that I-43 does not bind to α-\nsynuclein deposits. This finding further strengthens the utility of I-43 to detect Aβ\naggregates with specificity.\nSimilarly, panel C of Figure 6 from our earlier submission is now presented as Figures 7b–\ne in the revised manuscript, showing dual immunohistochemistry staining with various\namyloid-β-specific antibodies (D54D2, 6E10, OC, and A11) alongside I-43. The confocal\nimages reveal strong colocalization of I-43 with cored and diffuse amyloid plaques, except\nwith A11, an oligomer-specific antibody. The minimal staining of oligomers by I-43,\nconsistent with our in vitro data, indicates its preference for fibrillar amyloid structures\nover oligomeric forms. Notably, discrete signals from I-43 or antibody staining, observed\nin a few instances without overlap, suggest differential epitope recognition,\nheterogeneity in plaque composition, or differences in probe binding specificity. This\nrevision is now been clearly reflected in the revised manuscript on page 21.\n“Additionally, the authors state: Autofluorescence associated with amyloid-beta deposits\n(blue) is observed in the DAPI filter, an inherent feature of Aβ plaques, as discussed in an\nearlier report.10 In supplementary figure 61, yet these autofluorescent amyloid plaques\nare not stained by I-43 or D54D2. If the indicated structures are truly amyloid plaques,\nthen neither staining method appears to be very good, and the author's statement that I-\n43 can be applied as a sensitive marker for detecting β-459 amyloid deposits in AD\npathology is dubious, at best.’’\nWe thank the reviewer for their insightful comment regarding the staining of amyloid-\nbeta (Aβ) plaques and the observed autofluorescence in the DAPI filter. We acknowledge\nthe concern about the sensitivity of I-43 as a marker for detecting Aβ deposits in AD\npathology and have conducted additional experiments to clarify this issue. To address\npotential interference of I-43 across different filter sets, we first performed\nhistochemistry (HC) with I-43 alone in the cortex of APP-PS1 mice (Figure RL-2a, shown\nbelow), which indicate fluorescence exclusively in the NIR (λ = 633/700 nm) channel,\nex/em\nwith no detectable signal in either the FITC (λ = 488/520 nm) or DAPI (λ = 405/490\nex/em ex/em\nnm) channels, confirming spectral specificity. Subsequently, we conducted triple staining\nwith DAPI, D54D2, and I-43 in the same model (Figure RL-2b). Our results demonstrate\nthat plaques stained with D54D2 and I-43 are colocalized, with no detectable signal in the\nDAPI channel, suggesting that I-43 staining does not contribute to the observed\nautofluorescence in this model.\nIn human AD brain specimens, the underlying pathology is more complex, often\naccompanied by autofluorescent debris such as lipofuscin, tissue degradation by-\n17\nproducts (altered protein confirmations etc.), and other co-existing neurodegenerative\nchanges that can interfere with fluorescence-based detection. To validate our previous\nfindings and ensure protocol reproducibility, we repeated triple IHC staining (DAPI,\nD54D2, and I-43) on human autopsy samples (Figure RL2c–d). As shown in Figure RL-2c,\ncompact plaques display fluorescence in the DAPI channel, which partially overlaps with\nstaining from D54D2 and I-43. Interestingly, in Figure RL-2d, some diffuse plaques\n(highlighted with yellow circles) stained by D54D2 and I-43 don’t show any DAPI\nfluorescence. This suggests that the autofluorescence is specific to certain plaque\nsubtypes, rather than being a universal characteristic of all Aβ plaques. From the following\ndata, we are confident that I-43 has potential to detect amyloid beta plaques in HC and\nused protocol has reproducible outcomes.\nAfter an extensive review of the literature, we found relevant studies reporting\nthat autofluorescence in compact Aβ plaques within the blue spectral range (detected via\nthe DAPI filter) is an intrinsic feature of specific plaque subtypes rather than an artifact\n(e.g., https://doi.org/10.1007/s12264-019-00393-6 ; Fig. 5 of\nhttps://doi.org/10.3390/ijms252212211 ). For instance, Xie et al. (2014, Neurobiology of\nDisease, https://doi.org/10.1016/j.nbd.2014.07.011 ) showed diffuse DAPI-positive DNA\nstaining that colocalizes with M78 immunoreactivity at the center of neuritic plaques,\nindicating the presence of nucleic acid components within the plaque core. Similarly, Fu\net al. (2022, https://doi.org/10.1002/1873-3468.14549 ) demonstrated that these plaque\ncores are enriched in blood-derived metabolites, encased by vascular and glial proteins,\nand exhibit Hoechst-independent blue autofluorescence as a characteristic feature.\nSupporting this, Zhang et al. (2022, Theranostics, https://doi.org/10.7150/thno.68743 )\nobserved blue autofluorescence signals within Aβ plaques when imaged using the DAPI\nfilter (Fig. 3a2–c2), reinforcing the notion that such signals are not staining artifacts but\nrather an inherent property of certain Aβ deposits.\nWe believe these results and additional experiments strengthen the case for I-43 as a\nsensitive marker for fibrillar Aβ deposits. That said, we value the reviewer's expertise and\nwould welcome any methodological suggestions such as alternative counterstains or\nimaging conditions to further refine our approach and ensure robustness. We are happy\nto incorporate such recommendations and clarify these points in the revised manuscript.\nWe also welcome the opportunity to clarify the solubility of I-43 in the context of our\nexperiments.\nmouse shows fluorescence exclusively in the NIR channel (633/700 nm), with no\n19\ndetectable signal in the DAPI (405/490 nm) or FITC (488/520 nm) channels. (b) Triple IHC\nstaining in the cortex of a 13-month-old APP-PS1 mouse brain with DAPI (blue), D54D2\n(green), and I-43 (magenta) shows co-localization of I-43 and D54D2 with amyloid\nplaques. (c) Triple IHC staining in the hippocampus of an AD-1 (92F) human brain\nspecimen shows co-localization of I-43 and D54D2 in compact plaques. (d) Triple IHC\nstaining in the frontal cortex of an AD-3 (92F) human brain specimen indicates co-\nlocalized I-43 and D54D2 signals in compact plaques; DAPI autofluorescence is also\nobserved in some plaques (yellow circle), suggesting intrinsic blue-channel emission\nunrelated to I-43.\nTo address the raise concern “In the discussion, the authors mention that I-43\nsuffers from poor aqueous solubility, are these fluorescent cluster merely I-43\naggregates?”, whether the observed NIR signals arise from I-43 aggregation or specific\nAβ binding, we first determined the critical micelle concentration (CMC) of I-43 in PBS\n(10 mM, pH 7.4, 1% DMSO as cosolvent), which was ~24 µM (Supplementary Fig. 57a on\npage 83 of Revised SI). Importantly, for histological staining, we used a formulation\ncontaining 5% DMSO and 1.5% CremophorEL, which enhances aqueous solubility and\nmaintains the probe in solution for extended periods. Next, histochemical staining with\n10 µM I-43 on APP-PS1 brain sections showed fluorescence exclusively in the NIR (λ\nex/em\n= 633/700 nm) channel, with no detectable signal in either the FITC (λ = 488/520 nm)\nex/em\nor DAPI (λ = 405/490 nm) channels, confirming spectral specificity (Supplementary\nex/em\nFig. 57b). Following this, concentration-dependent staining using I-43 from 10 µM down\nto 0.01 µM (Fig. 5c), along with DAPI counterstaining to confirm that nuclear signals in the\nDAPI channel are distinct from I-43 labeling. Notably, I-43 produced clear, plaque-specific\nfluorescence in the NIR channel even at 0.1 µM (well below its critical micelle\nconcentration), which is distinct from DAPI staining (indicated by dotted circles), strongly\nindicating that the observed signals arise from specific binding to Aβ plaques rather than\nfrom nonspecific probe aggregation (see Supplementary Fig. 57c). Even at a concentration\nof 0.01 µM, I-43 produced signals clearly distinguishable from background in amyloid-\nbeta plaques. Some DAPI signals were observed to coexist (highlighted by yellow circles)\nwith a distinct profile from the NIR channel, indicating that the signals in the DAPI channel\ndo not originate from I-43. Based on the following data, we strongly believe that the\nobserved fluorescence reflects specific biological binding, not probe clustering.\nWe also acknowledge the reviewer’s concern regarding our note on I-43's poor aqueous\nsolubility on page 34 of revised MS. As described in the discussion, this limitation was\nmentioned specifically in the context of administering higher doses (5 mg/kg and\n10 mg/kg) for preliminary therapeutic evaluation. In those cases, suitable formulation\nstrategies were used to ensure solubilization and systemic bioavailability. Importantly, in\nall imaging and histological experiments, I-43 was used at concentrations ranging from\n20\n10-25 µM with nearly 5% DMSO as cosolvent, where it remains monomeric in solution.\nFortunately, in the course of addressing the reviewer’s suggestion, we found that I-43 can\neffectively label amyloid plaques in histochemical staining at 1.0 µM, yielding results\ncomparable to those observed at 10 µM. To prevent any confusion between solubility-\nrelated issues at therapeutic versus imaging doses, we have clarified this distinction in the\nrevised manuscript. Earlier statement “However, we acknowledge that despite\nsuccessfully detecting targeted AD biomarkers with high specificity and selectivity in both\ninvitro and in vivo studies, our designed probes exhibit limitations, including poor\naqueous solubility….’’ is replaced with ‘’However, we acknowledge that while our probes\ndemonstrated efficient detection of targeted AD biomarkers in both in vitro and in vivo\nstudies, their application in therapeutic evaluations revealed certain limitations:\nspecifically, poor aqueous solubility at therapeutic dose.’’ highlighted on page 34 of revise\nMS.\nSupplementary Fig. 57: (a) Critical micelle concentration (CMC) of I-43 determined in PBS\n(10 mM, pH 7.4, 1% DMSO), showing a CMC of ~23.94 µM. (b) Confocal images of APP-PS1\ncortex section stained with 10 µM I-43, showing exclusive fluorescence in the NIR channel (λ\nex/em\n21\n= 633/700 nm) and no signal in FITC or DAPI channels. (c) Concentration-dependent staining\n(10–0.01 µM) of APP-PS1 hippocampal sections with I-43 and DAPI counterstain, demonstrating\nAβ plaque labeling in the NIR channel down to 0.1 µM—well below the CMC—supporting\nspecific, aggregation-independent binding.\n5. Docking and MD simulation: There are a number of issues in the approach and execution in\nelucidating the molecular basis for the interaction of I-43 with AChE or Abeta fibrils.\na. The major conclusion for the interaction between I-43 and AChE is derived from docking\nand then short MD simulation (100 ns). It is curious that authors observed >5 angstrom\nRMSD for ligand yet only ~2 angstrom RMSD for protein (assuming mainchain) (Figure 9e).\nGiving such large RMSD, it is difficult to relay on the original docking. However, it is unclear\nwhether authors use the model original docking or the final MD simulation for their\ninterpretation.\nb. From the checklist for MD simulation, authors marked NO on both 1c and 1d, meaning\nthat the MD simulation was not done as rigor as it should.\nc. 3c in the checklist for MD simulation should be NO, instead of yes.\nPoint to point response:\na. The major conclusion for the interaction between I-43 and AChE is derived from docking\nand then short MD simulation (100 ns). It is curious that authors observed >5 angstrom\nRMSD for ligand yet only ~2 angstrom RMSD for protein (assuming mainchain) (Figure 9e).\nGiving such large RMSD, it is difficult to relay on the original docking. However, it is unclear\nwhether authors use the model original docking or the final MD simulation for their\ninterpretation.\nResponse: We sincerely appreciate the reviewer’s observation regarding the apparent\ndiscrepancy in RMSD values between the ligand and protein in Figure 9e.\nThe original Figure 9e, which is Fig. 10g in revised MS (Page # 32) presented ligand RMSD\nvalues which were calculated using the Lig fit Prot metric in Desmond, where the ligand\nRMSD was determined after aligning the trajectory to the protein’s backbone. This\napproach often results in higher-than-expected RMSD values due to internal ligand\nfluctuations and local rearrangements within the binding pocket, even when the ligand\nremains stably bound. The primary goal of including this plot was to illustrate that ligand\nI-43 did not diffuse out of the binding pocket during the simulation. However, we\nunderstand that this presentation may have been misleading in terms of assessing the\nstability of the ligand binding mode.\nwhich calculates ligand heavy-atom RMSD after aligning the ligand to its own initial\nconformation. This metric provides a more accurate depiction of the ligand’s\nconformational stability throughout the simulation. The revised MS now includes the\nfollowing revision Lig fit Lig RMSD plot, which shows that, “following the initial\nequilibration phase, the RMSD of I-43 stabilizes below 1.7 Å from 40 ns onward. This very\nlow RMSD fluctuation confirmed a stable binding pose. Meanwhile, the protein Cα RMSD\nremains around ~2 Å, verifying the receptor’s structural integrity” reflected on page # 31\nof the revised MS.\nFurthermore, our conclusions regarding the interaction of I-43 with AChE are based on\nthe MD-refined binding conformation obtained post-MD rather than relying solely on the\ninitial docking pose. This final conformation preserves key interactions observed in the\ndocking stage while allowing for relaxation and improved binding complementarity. To\nensure clarity, we have replaced the original RMSD plot in the main figure with the\ncorrected Lig fit Lig and Cα RMSD plots, while moving the Lig fit Prot version to the\nsupporting information as Supplementary Fig. 82d (on page #120 of revised SI) for\nreference.\nFig. 9e in the left panel is RMSD plot from original MS, showing protein backbone Cα\natoms (blue, left Y-axis) and ligand heavy atoms (magenta, right Y-axis) over a 100 ns\nsimulation, aligned to the protein. RMSD values were computed using the Desmond\nsimulation package. While Fig. 10g in the right panel is updated RMSD plot from revised\nMS, displaying separate trajectories of hAChE protein (black) and ligand I-43 (red) over\n100 ns, with the ligand aligned to itself and the protein aligned on Cα atoms. The revised\nplot demonstrates improved trajectory stability and clearer separation of ligand and\nreceptor mobility.\nthat the earlier 100 ns simulation was performed using an older version of Desmond,\nwhich has unfortunately become corrupted for reasons beyond our control. We've also\nencountered technical difficulties in setting up the newer version on our institutional\nsupercomputing server. Since then, our computational workflow has transitioned to\nGROMACS, which is now fully supported. If the reviewer recommends, we’re more than\nwilling to perform extended MD simulations (300–500 ns) using GROMACS to further\nsupport our findings.\nd. From the checklist for MD simulation, authors marked NO on both 1c and 1d, meaning\nthat the MD simulation was not done as rigor as it should.\nResponse: We thank the reviewer for pointing this out. While our simulation results show\nstability and consistency within the 100 ns timeframe, we acknowledge that the\nindependence from initial configuration cannot be conclusively determined without\nadditional simulations starting from different seed velocity.\nAs, the molecular dynamics (MD) simulations were indeed conducted with rigorous standard\nprotocols, following best practices for protein-ligand systems. Specifically, the simulations\nwere performed using the Desmond module in the Schrödinger software package (Maestro\nv2020.1). Prior to the simulation run, the protein-ligand complexes were prepared using\nProtein Preparation Wizard, where missing atoms and loops were modeled using Prime\nrefinement, appropriate bond orders were assigned, and protonation states were optimized\nusing Epik and PROPKA at pH 7.0 ± 2.0. The complexes were solvated in explicit TIP3P water,\nneutralized with Na⁺/Cl⁻ ions, and the system was energy minimized (2000 iterations,\nconvergence <1 kcal/mol/Å). Periodic boundary conditions were applied and the simulation\nwas run for 100 ns in the NPT ensemble using OPLS3e force field. The Martyna–Tobias–Klein\nbarostat and Nosé–Hoover Chain thermostat maintained constant pressure (1 atm) and\ntemperature (310 K), respectively. These steps ensure that the simulation was conducted\nunder thermodynamically appropriate and biologically relevant conditions. However, we\nrecognize that further studies would be required to fully address this aspect, therefore the\nresponse in the checklist was marked as “No” on both 1c and 1d.\ne. 3c in the checklist for MD simulation should be NO, instead of yes.\nResponse: We are thankful to the reviewer. We apologize for the mistake. We agree that\nthe correct response for item 3c should be 'NO.' The checklist has now been updated\naccordingly.\n24"}, {"round": 3, "reviewer_comments": {"reviewer_3": "Overall, this manuscript presents a substantial body of work, and the compound I-43 represents a promising lead as a theranostic probe for investigating both amyloid fibrils and cholinesterase activity. The authors have addressed most of the previous concerns, and the revised manuscript shows significant improvements. However, several new issues require careful attention:\n1. Specificity and Utility of I-43: Concerns remain regarding the specificity of I-43. The relevance of the CMC calculation using bulk absorbance is unclear and does not adequately address questions of aggregation or poor solubility. Furthermore, the buffer conditions significantly influence I-43 fluorescence, and it is not evident how much this will impact practical applications. The co-localization of I-43 with amyloid plaques, when compared to established dyes, appears questionable. While the authors state that I-43’s fluorescence with cholinesterases is activity-dependent, they have not examined how enzymatic products affect I-43 fluorescence. Additionally, the interpretation of the molecular dynamics (MD) results remains ambiguous given these concerns. It would also be informative to determine whether I-43 alters cholinesterase enzyme kinetics. Nonetheless, given that I-43 is an initial lead compound requiring substantial further modification and optimization, these limitations may be less relevant in improved derivatives. Still, the authors should clearly summarize I-43's current limitations in both the abstract and discussion, and explicitly discuss strategies for improvement, rather than promoting I-43 for immediate further validation in in vitro and in vivo AD models. At this stage, such promotion seems premature.\n2. Manuscript Structure: It is unclear why the manuscript contains both a \"Results and Discussion\" section and a separate \"Discussion\" section. The authors should consider either keeping these strictly distinct or combining all relevant content into a single 'Results and Discussion' section for clarity and cohesion.\n3. Writing and Consistency: The revised text appears to incorporate input from multiple authors, resulting in inconsistencies in style and voice. The authors should carefully review the entire manuscript to address such issues. Some specific examples include:\na. The abbreviation \"AD\" is used before line 56, but Alzheimer’s disease is redefined as AD in line 56. This redundancy should be removed.\nb. The sentence on lines 60–62 lacks a main verb (“However, extensive ongoing research for clinical translation, underscoring the need for innovative designs to advance theranostics for effective AD diagnosis and therapy, simultaneously.”). Please revise for clarity and correct grammar.\nc. The sentence in lines 62–65 seems out of place and does not substantially contribute to the surrounding context. Consider deleting or relocating it.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response Letter\nWe thank the reviewers for their unanimous endorsements of the significance and novelty of the\nwork carried out in our manuscript. We have carefully reviewed the reviewers' insightful,\nconstructive comments, which have been fully incorporated into the revised manuscript and have\nstrengthened the rigor and presentation of our study. The Reviewers' comments are in italics. The\nnecessary revisions, based on the suggestions, are highlighted in red or blue in both the main text\nand supplementary information for easy viewing. Our detailed point-by-point response is listed\nbelow for your assessment.\nIn addition, to address the Reviewer's concern regarding the specificity of I-43 for amyloid-\nβ plaques, we performed dual histological staining of brain sections from >17-month-old APP-PS1\nmice using DANIR-2c, a widely recognized NIR probe for Aβ aggregates. Given time constraints in\ncommercial procurement and the straightforward synthetic route, we prepared DANIR-2c in our\nlaboratory following the reported protocol (doi: 10.1021/ja4052922) and verified its structure by\n1H and 13C NMR spectroscopy (spectra provided at the end of the response letter).\nWe sincerely believe the above responses and further clarifications will adequately address all the\nissues raised by the reviewers.\nSincerely,\nGyan Prakash Modi\nThe Reviewer's comments are in italics.\nREVIEWER COMMENTS:\nReviewer #3 (Remarks to the Author):\nOverall, this manuscript presents a substantial body of work, and the compound I-43 represents\na promising lead as a theranostic probe for investigating both amyloid fibrils and cholinesterase\nactivity. The authors have addressed most of the previous concerns, and the revised manuscript\nshows significant improvements.\nResponse: We sincerely thank the Reviewer for the positive evaluation of our revised manuscript\nand for recognizing the significance of our work. We greatly appreciate your encouraging\ncomments regarding the potential of compound I-43 as a promising theranostic probe for both\namyloid fibrils and cholinesterase activity. We are pleased that the revisions have addressed your\nearlier concerns and that the improvements to the manuscript were well received. Your feedback\nhas been instrumental in strengthening the quality and clarity of our work. Thank you once again\nfor your thoughtful review and constructive input.\nHowever, several new issues require careful attention:\n1. Specificity and Utility of I-43: Concerns remain regarding the specificity of I-43. The relevance\nof the CMC calculation using bulk absorbance is unclear and does not adequately address\nquestions of aggregation or poor solubility. Furthermore, the buffer conditions significantly\ninfluence I-43 fluorescence, and it is not evident how much this will impact practical applications.\nThe colocalization of I-43 with amyloid plaques, when compared to established dyes, appears\nquestionable. While the authors state that I-43's fluorescence with cholinesterases is activity-\ndependent, they have not examined how enzymatic products affect I-43 fluorescence. \nAdditionally, the interpretation of the molecular dynamics (MD) results remains ambiguous given\nthese concerns. It would also be informative to determine whether I-43 alters cholinesterase\nenzyme kinetics. Nonetheless, given that I-43 is an initial lead compound requiring substantial\nfurther modification and optimization, these limitations may be less relevant in improved\nderivatives. Still, the authors should clearly summarize I-43's current limitations in both the\nabstract and discussion, and explicitly discuss strategies for improvement, rather than promoting\nI-43 for immediate further validation in in vitro and in vivo AD models. At this stage, such\npromotion seems premature.\nPoint-to-point response: In response to the following comments, we provided further\nclarifications and revisions one by one.\nConcerns remain regarding the specificity of I-43. The relevance of the CMC calculation using bulk\nabsorbance is unclear and does not adequately address questions of aggregation or poor\nsolubility. The colocalization of I-43 with amyloid plaques, when compared to established dyes,\nappears questionable.\nResponse: We thank the Reviewer for this valuable insight and agree that the original manuscript\ncontained limited discussion regarding the specificity of I-43 for amyloid-β. To strengthen the\nanalysis, we have revised the manuscript and incorporated additional data and clarifications, as\noutlined below.\nIn our study, we employed histological staining experiments with widely accepted fibrillar Aβ\nreference dyes (ThT, ThS, Congo Red, and CRANAD-2) to evaluate the selectivity of I-43 for Aβ\nfibrils. While Figure 4 in the original submission highlighted colocalization patterns, the revised\nmanuscript now includes semi-quantitative analyses (scatter colocalization plot; Supplementary\nFig. 55c-d), directly comparing I-43 colocalization with ThT and ThS in brain specimens from ≥17-\nmonth-old APP/PS1 mice, prepared under identical conditions and imaged using the same\nconfocal platform.\nIn the earlier reported data (Figure 4b-d; page # 14 of the revised MS), confocal analyses\nshowed partial colocalization of I-43 with ThT and ThS, and strong colocalization with CRANAD-2\n(r = 0.95), as demonstrated by scatter plots. In the revised manuscript, we have now included\nadditional semiquantitative analyses (scatter plots; Supplementary Fig. 55c-d), directly\ncomparing ThT and ThS colocalization with I-43 in brain specimens from ≥17-month-old APP-PS1\nmice. Furthermore, we have incorporated data with an additional Aβ fibril-specific dye, DANIR-\n2c, which provides further validation of the specificity of I-43.\nConfocal imaging and scatter plot analyses demonstrated that I-43 colocalizes with\nestablished amyloid reference dyes. With ThT, the correlation at the section level was moderate\n(r = 0.72; SI Fig. 55c on pages # 82-83 of revised SI), whereas zooming in on individual plagues\nrevealed a stronger Pearson coefficient of r = 0.87 (SI Fig. 55c1 on page # 82-83 of revised SI). In\nthese regions, colocalization was evident along the plaque core (outlined by the white dotted\nline), with only minor peripheral discrepancies highlighted by yellow arrows, likely reflecting\nlocalized differences in dye affinity. By contrast, I-43 exhibited markedly stronger colocalization\nwith ThS, yielding correlation coefficients of r = 0.89 at the section level (SI Fig. 55d on page # 82-\n83 of revised SI) and r = 0.95 within individual plaques (SI Fig. 55d1 on page # 82-83 of revised\nSI). The high degree of signal overlap with ThS, a well-established marker of fibrillar amyloid,\nsupports the conclusion that I-43 binds to the core of mature Aβ fibrillar plaques with optimal\nspecificity. From these data, it may be inferred that the differential colocalization at certain\nplaces of I-43 with ThT and ThS in mature Aβ fibrils is most likely attributable to specific\nbiochemical affinities arising from distinct Aβ species and their conformational states, rather than\nto variations in experimental conditions or imaging methodologies.\nSupplementary Fig. 55: Colocalization of amyloid-binding dyes with I-43 positive aggregates in\nthe cortex of aged APP/PS1 mice. (c) Representative confocal images of brain cortex sections\n(>17 months old APP/PS1 mice) stained with Thioflavin T (ThT, green) and I-43 (magenta). \nMerged images show colocalization (white). Enlarged region (c1) highlights overlapping ThT and\nI-43 signals (enclosed in white dotted line) and partial on no colocalization of signals, with scatter\nplots confirming a strong correlation (r = 0.72; r = 0.87 for enlarged region). (d) Representative\nimages of cortical sections stained with Thioflavin S (ThS, green) and I-43 (magenta). \nMerged images demonstrate colocalization (white). Enlarged region (d1) reveals substantial overlap\nbetween ThS and I-43 signals, confirmed by scatter plots (r = 0.89; r = 0.95 for enlarged region).\nScale bars: 50 μm (upper panels), 20 μm (lower panels).\nThe subsequent study involved dual histochemical staining using DANIR-2c as a reference\ndye to substantiate the specificity of I-43 to amyloid beta fibrils. In panel (e) of SI Fig. 55 on pages\n# 82-83 of the revised SI, the TRITC channel displays distinct red fluorescent puncta\ncorresponding to amyloid deposits, while the NIR channel shows minimal background, confirming\nnegligible fluorescence interference from DANIR-2c under the selected filter sets. Colocalization\nanalysis in panel (f) of SI Fig. 55 on pages # 82-83 of the revised SI demonstrates substantial spatial\noverlap between DANIR-2c and I-43, supported by high Pearson correlation coefficients (r = 0.93).\nThe zoom-in view (f1) of individual plaque further confirms that I-43 penetrates densely packed\namyloid plaques core and produces fluorescence patterns closely matching those of the fibril-\nspecific dye DANIR-2c. We believe these additions address the Reviewer's concern and provide\ngreater confidence in labeling I-43 for amyloid beta plaques.\nSupplementary Fig. 55. Colocalization of DANIR-2c with amyloid plaques in APP-PS1 mouse\ncortex. (e) Representative images of DANIR-2c staining (TRITC channel) in the cortex region of a\n17-month-old APP-PS1 mouse brain. DANIR-2c-positive aggregates are observed (red), with no\nsignificant signal detected in the near-infrared (NIR) channel, confirming fluorophore specificity.\nScale bar = 50 μm. (f) Dual immunofluorescence staining of DANIR-2c (red) and amyloid-binding\ndye I-43 (magenta) in the cortex of APP-PS1 mice (>17 months). Merged images demonstrate\nstrong colocalization of DANIR-2c and I-43-labeled amyloid plaques. Higher magnification of\nregion f1 highlights dense amyloid deposits with overlapping signals from DANIR-2c and I-43.\nScatter plots show strong positive correlations between DANIR-2c and I-43 intensities (r = 0.93 at\nlow magnification; r = 0.91 at magnified view), confirming colocalization. Scale bars = 50 μm (f),\n10 μm (f1). Magnified images of plaques and colocalization correlation scatter plots were\ngenerated using ImageJ software.\nWe thank the reviewer for giving us the opportunity to clarify this important point. We\nagree that bulk absorbance-based CMC estimation has inherent limitations and cannot fully\nresolve questions of aggregation or solubility. In the manuscript, our reference to poor aqueous\nsolubility pertains specifically to in vivo administration, where intravenous delivery requires\nformulation to enhance solubility. For these experiments, I-43 was injected intravenously at a\ndose of 0.8 mg/kg using a vehicle composed of 7.5% DMSO, 2.5% Cremophor® EL, and 90% 1×\nPBS. Contrary to this, for histological staining, we used I-43 at a lower concentration of 10-25 µM\nin a vehicle containing 5% DMSO and 1.5% Cremophor® EL, which ensures stabilization of the\nprobe in non-aggregated form for extended periods, thereby minimizing the likelihood of\naggregation under the staining conditions. Moreover, our intention in including the CMC analysis\nwas not to suggest that it alone provides a comprehensive solution, but rather to support our\ninterpretation with an established approach recently applied to NIR probes (DOI:\n10.1038/s41467-024-44818-x). The revised result section on page # 16 of the revised MS has\nclearly mentioned this as, “In a subsequent experiment (Supplementary Fig. 57a), the critical\nmicelle concentration (CMC) of I-43 was determined to be 23.94 μM in phosphate buffer (10 mM,\n1% DMSO) as a complementary indicator of the aggregation threshold.” Apart from this, several\nadditional factors strengthen our conclusion: (i) all working concentrations were well below the\nmeasured CMC, (ii) I-43 signals consistently showed colocalization with multiple fibril-specific\ndyes (ThT, ThS, DANIR-2c, CRANAD-2, and Congo Red), (iii) can effectively label amyloid plaques\nin histochemical staining at 1.0 µM, yielding results comparable to those observed at 10 µM,\n(iv) amyloid-negative control specimens displayed no fluorescent clustering, and (v) the use of\nsequential 30% and 50% ethanol washes prior to mounting further minimized any contribution\nfrom unbound dye. Together, these points provide supporting evidence that the observed\nstaining arises from specific amyloid-associated binding rather than probe aggregation or\nnonspecific interactions.\nFurthermore, the buffer conditions significantly influence I-43 fluorescence, and it is not evident\nhow much this will impact practical applications.\nResponse: We are happy to answer the query raised. In the original manuscript, our in-vitro data\nshowed that I-43 alone exhibits minimal fluorescence changes across different buffers, as well as\nwith various tau species and α-synucleins, compared to the pronounced emissivity observed\nupon binding to fibrillar Aβ and Aβ (Fig. 2a,b and Fig. 3c,d). This selectivity is further\n1–40 1–42\nsupported by histochemical staining, where I-43 labeled amyloid-β plaques with a high signal-to-\nnoise ratio and minimal off-target signals (Figs. 4, 5, and 7), as well as by in-vivo imaging,\nwhich showed stronger and more sustained fluorescence in APP-PS1 mice compared to WT controls\n(Fig. 6a,b). Together, these results indicate that although fluorophores can be sensitive to\nmicroenvironmental factors such as ionic strength or polarity (Fig. 2e), I-43 performs robustly\nacross in-vitro and in-vivo experiments, reliably detecting insoluble Aβ species. Finally, the PMA-\ninduced stress model (Fig. 8g–i) further confirmed this behavior, with significant emission\nchanges linked to AChE only in PMA-treated cells, and not in controls.\nFig. 2. Photophysical properties and in vitro fluorescence assays of lead compound I-43. (a,b)\nRepresentative fluorescence spectra and corresponding bar graph of I-43 (20 μM) after\nincubation with various Aβ species (10 μM; monomers, oligomers, and fibrils) and freshly\nprepared BSA (25 μg/mL). Mono, Oligo, and Fibril refer to Aβ monomers, oligomers, and fibrils,\nrespectively. Lowercase Roman numerals correspond to: (i) I-43 alone in PBS (pH 7.4); (ii) I-43 +\nAβ (Mono); (iii) I-43 + Aβ (Oligo); (iv) I-43 + Aβ fibrils; (v) I-43 + freshly prepared BSA;\n1–42 1–42 1–42\n(vi) I-43 + Aβ (Mono); (vii) I-43 + Aβ (Oligo); and (viii) I-43 + Aβ fibrils.\n1–40 1–40 1–40\nFigure 3. (c,d)\nComparative analysis of fluorescence intensity changes (Fl spectral and bar graph) of I-43 (10 μM)\nfollowing incubation with monomeric and fibrillar forms of recombinant tau and α-synuclein\n(5 μM), relative to Aβ and Aβ fibrils (5 μM). Data represented as mean ± SD (n = 3\n1-42 1-40\nindependent experiments in triplicate), with error bars from one-way ANOVA and significance\nlevels: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\nHowever, to ease viewing the possible interference from different buffers vs the I-43\nresponse to targeted key biomarker (Aβfibrils), we conducted another fluorescence-based study\nof I-43 in various buffers used as controls and compared it with Aβ fibrils (10 μM). Our data\n1-40\nsuggested that [Response letter (RL) Figure 1, shown below], although minor variations were\nobserved across buffers, the fluorescence enhancement upon binding to Aβ fibrils was\n1-40\nsignificantly higher (****P < 0.0001) than any buffer-related effects. These findings suggest that\nbuffer interference is minimal, and therefore, I-43 provides a reliable readout for quantitative\nanalysis of the targeted biomarker.\nRL Figure 1. Comparative analysis of fluorescence intensity changes (Fl spectral and bar graph) of\nI-43 (10 μM) following incubation with various physiological buffers (PBS, HEPES, and Tris buffer)\nand with Aβ fibrils (10 μM). Data represented as mean ± SD (n = 3 independent experiments\n1-40\nin triplicate), with error bars from one-way ANOVA and significance levels: ****P < 0.0001.\nAdditionally, the interpretation of the molecular dynamics (MD) results remains ambiguous given\nthese concerns.\nResponse: We thank the reviewer for highlighting the need for greater clarity in the\ninterpretation of our molecular dynamics (MD) analyses. In the revised manuscript (pages # 32–\n34 of revised MS), we have incorporated results from an extended 200 ns MD simulation to\nensure adequate sampling of the conformational landscape. Ensemble-averaged RMSD, RMSF,\nand interaction-fraction profiles have been added to the revised version, accordingly, providing\na clearer view of persistent protein–ligand stability trends. The updated figure (revised Fig. 10g-\nk and Supplementary Fig. 82) now illustrates residue-level flexibility, ligand atom-wise\nfluctuations, and recurrent hydrophobic, hydrogen-bonding, and water-mediated interactions\nstabilizing I-43 within the active-site gorge.\nThe revision is as follows:\nTo evaluate the thermodynamic stability and interaction profile of the docked hAChE-I-43\ncomplex, two independent 200 ns molecular dynamics simulations were carried out using\ndifferent initial velocity seeds. Trajectories were aligned on protein frames on Cα atoms for\nprotein root mean square deviation (RMSD) and on ligand heavy atoms for ligand RMSD. The\nensemble-averaged protein Cα RMSD remained stable around ~2.0 Å (Fig. 10g), indicating\nminimal global conformational changes during the simulation. In contrast, the ensemble-\naveraged RMSD of I-43 displayed higher fluctuations during the initial equilibration phase (0-100\nns). During this period, the 2-methoxy aniline moiety of I-43 repositioned within the PAS pockets\n(1-5 Å range), particularly shifting towards residue Trp286 to establish strong hydrophobic\ninteractions, while the benzo[d]thiazole-2-carbonitrile moiety showed slight adjustments within\nthe CAS pocket. After ~100 ns, the ligand RMSD stabilizes and remains within the acceptable (2.7-\n4.0 Å) range for the remainder of the simulation (Supplementary Fig. 82). Considering the\ninherent flexibility of I-43, such a relatively low RMSD fluctuation suggests a stable binding pose.\nConsistently, the protein fit ligand root mean square fluctuation (RMSF; Fig. 10h) also showed\nlocalized fluctuations in the atoms of the 2-methoxy aniline moiety (atoms 29-33; 2.7-3.5 Å),\nwhereas the rest of the molecules remained largely stable throughout the 200 ns simulation.\nAdditionally, the ensemble-averaged per-residue RMSF profile (Fig. 10i) highlights residue-level\nfluctuations across hAChE over the 200 ns simulation. Key active-site residues (Ser203, Trp286,\nPhe295, and Tyr341) showed limited fluctuations (< 2.0 Å), suggesting local rigidity in the binding\nregion and stable engagement with I-43. These observations confirm the stability of the hAChE-I-\n43 complex over the 200 ns simulations. Interaction analysis of the trajectories (Fig. 10i) revealed\nthat the nitrile moiety of I-43 maintained both direct and water-mediated hydrogen-bonding\ninteractions with Phe295 in the acyl pocket for ~78% of the simulation, suggesting a key role in\nligand stabilization. Additionally, Phe338 and Tyr341 residues located within the catalytic anionic\nsite and mid-gorge regions exhibited persistent π-interactions and hydrophobic contacts with I-\n43 for ~50% and 78% of the trajectory, respectively. Notably, Trp86 and Ser203 from the CAS,\nTrp286 from the PAS, and Phe338 and Tyr341 from the acyl pocket exhibited recurrent\nhydrophobic as well as water-mediated interactions with I-43, while Phe295 and Arg296 provided\ncomplementary hydrogen-bonding and hydrophobic contacts, together reinforcing a\nmultiregional binding profile across the active-site gorge of hAChE (Fig. 10k; Supplementary Fig.\n83c). Collectively, these MD-derived stability metrics, limited local RMSF in key residues, and\npersistent interactions pattern support a stable and specific binding mode for I-43 in hAChE,\nconsistent with the proposed design rationale.\nFig. 10. (g) Ensemble-averaged RMSD plot showing the stability of the I-43-hAChE complex over\n200 ns MD simulations, comparing the Cα RMSD of hAChE backbone (black) and the ligand I-43\n(red). (h) Atom-wise RMSF analysis of I-43 indicates higher mobility at terminal substituents,\nwhereas the central aromatic scaffold remained rigid due to persistent π–π stacking. (i) Per-\nresidue RMSF plot of hAChE highlighting flexible regions during the 200 ns simulation. (j)\nEnsemble-averaged protein-ligand contact histogram showing the frequency and types of\ninteractions (H-bonds, hydrophobic, water bridges) between I-43 and hAChE residues over the\nsimulation trajectory.\nWe hope that our revised MD data effectively answers the concern raised.\nWhile the authors state that I-43's fluorescence with cholinesterases is activity-dependent,\nthey have not examined how enzymatic products affect I-43 fluorescence. It would also be\ninformative to determine whether I-43 alters cholinesterase enzyme kinetics.\nResponse: We sincerely thank the reviewer for this constructive suggestion. The data presented\nin the Figures 8e,f (page 25 of the revised MS) shown that I-43 fluorescence increases\nprogressively with AChE activity, while negligible fluorescence is observed in the absence of\n1-40\nenzyme, indicating that the signal arises from enzymatic turnover rather than buffer, protein, or\nsubstrate alone; and (ii) Figure 8k presents kinetic analysis, where Lineweaver–Burk plots\ndemonstrate that I-43 increases Km in a concentration-dependent manner without significantly\naltering Vmax, consistent with competitive inhibition. Together, these results indicate that I-43\nreports on enzyme activity and interacts with the catalytic site without affecting maximal\ncatalytic turnover. Although the following data partially address the reviewer's query, an\nextensive exploration to fully delineate the mechanistic basis of probe activity will be part of\nfuture studies with optimized leads, as noted on page 36 of the revised manuscript in red.\nFig. 8. (e-f) Spectral data and corresponding emission intensity bar graph showing changes in\nfluorescence intensity of I-43 upon co-incubation with varying concentrations of AChE: (ii) ATCI,\n(v) 2.0, (vii) 0.5, (viii) 0.25, (ix) 0.1, and (x) 0.0 U/mL, in the presence of ATCI (1.5 mM). Values are\nmean ± SD, n = 3, Dunnett t-test using ANOVA, ****P ˂ 0.0001, ***P ˂ 0.0003, *P = 0.0222 and\nns.\nNonetheless, given that I-43 is an initial lead compound requiring substantial further modification\nand optimization, these limitations may be less relevant in improved derivatives. Still, the authors\nshould clearly summarize I-43's current limitations in both the abstract and discussion, and\nexplicitly discuss strategies for improvement, rather than promoting I-43 for immediate further\nvalidation in in vitro and in vivo AD models. At this stage, such promotion seems premature.\nResponse: We thank the Reviewer for this important suggestion. In the revised manuscript,\nwe have now explicitly summarized the current limitations of I-43 in both the Abstract (on page # 1\nof the revised MS) and the Discussion (on page # 34 of the revised MS). In addition, on page 34,\nwe have included a new paragraph outlining literature-supported strategies for structural\noptimization. Specifically, we discuss how modifications such as replacement of the N, N-\ndimethylamine donor with more polar residues, incorporation of heteroaryl thiophene fragments\nto reduce aggregation-prone planarity, and the use of cyano-aryl motifs with polar substituents\nto enhance cholinesterase activity may collectively help improve solubility, selectivity, and\ntherapeutic potential. We believe this addition complies with the reviewer’s suggestion by\nproviding a balanced perspective on I-43, acknowledging its current limitations while also\noutlining potential avenues for future improvement, rather than prematurely positioning it for\nimmediate preclinical validation.\n2. Manuscript Structure: It is unclear why the manuscript contains both a \"Results and Discussion\"\nsection and a separate \"Discussion\" section. The authors should consider either keeping these\nstrictly distinct or combining all relevant content into a single 'Results and Discussion' section for\nclarity and cohesion.\nResponse: We are sorry for the oversight in the original submission and sincerely thank the\nReviewer for pointing it out. As suggested, we have revised the manuscript to present the Results\nand Discussion as two separate sections. We believe this improves the clarity and overall\norganization of the paper.\n3. Writing and Consistency: The revised text appears to incorporate input from multiple authors,\nresulting in inconsistencies in style and voice. The authors should carefully review the entire\nmanuscript to address such issues. Some specific examples include:\na. The abbreviation \"AD\" is used before line 56, but Alzheimer's disease is redefined as AD in line\n56. This redundancy should be removed.\nResponse: We felt sorry for this oversight and sincerely thank the Reviewer for bringing it to our\nattention. As suggested, we have corrected the abbreviation issue and carried out a careful,\nmanuscript-wide review to remove other inconsistencies. As the changes are minor, these were\nnot highlighted in the revised manuscript.\nb. The sentence on lines 60–62 lacks a main verb (\"However, extensive ongoing research for\nclinical translation, underscoring the need for innovative designs to advance theranostics for\neffective AD diagnosis and therapy, simultaneously.\"). Please revise for clarity and correct\ngrammar.\nResponse: We thank the Reviewer for pointing out the grammatical issue in the sentence on lines\n60–62. We agree that the original sentence lacked a main verb and have revised it for clarity and\ngrammatical correctness. The revised sentence is \"However, extensive ongoing research aimed\nat clinical translation underscores the need for innovative designs to advance theranostics for\neffective AD diagnosis and therapy.\", as reflected on page # 2 of the revised MS in blue.\nc. The sentence in lines 62–65 seems out of place and does not substantially contribute to the\nsurrounding context. Consider deleting or relocating it.\nResponse: We thank the Reviewer for this comment and agree that the sentence on lines 62–65\ndoes not substantially contribute to the findings. As suggested, the sentence has been removed\nin the revised manuscript.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely thank Reviewer #4 for the time and effort dedicated to reviewing our\nwork. We greatly appreciate the endorsement of our study along with the constructive and\ninsightful comments, which have significantly improved the presentation and quality of the\nrevised manuscript."}]

Decision letter