bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effects of PDGF-AB/BB against cellular senescence in human intervertebral disc
Authors: Zhang Changli , Diaz-Hernandez Martha Elena , Fukunaga Takanori , Sreekala Shenoy , Yoon Sangwook Tim , Haglund Lisbet , Drissi Hicham
Published: October 2024

Cellular senescence, characterized by a permanent state of cell cycle arrest and a secretory phenotype contributing to inflammation and tissue deterioration, has emerged as a target for age-related interventions. Accumulation of senescent cells is closely linked with intervertebral disc (IVD) degeneration, a prevalent age-dependent chronic disorder causing low back pain. Previous studies have highlighted that platelet-derived growth factor (PDGF) mitigated IVD degeneration through anti-apoptosis, anti-inflammation, and pro-anabolism. However, its impact on IVD cell senescence remains elusive. In this study, human NP and AF cells derived from aged, degenerated IVDs were treated with recombinant human (rh) PDGF-AB/BB for 5 days and changes of transcriptome profiling were examined through mRNA sequencing. NP and AF cells demonstrated similar but distinct responses to the treatment. However, the effects of PDGF-AB and BB on human IVD cells were comparable. Specifically, PDGF-AB/BB treatment resulted in downregulation of gene clusters related to neurogenesis and response to mechanical stimulus in AF cells while the downregulated genes in NP cells were mainly associated with metabolic pathways. In both NP and AF cells, PDGF-AB and BB treatment upregulated the expression of genes involved in cell cycle regulation, mesenchymal cell differentiation, and response to reduced oxygen levels, while downregulating the expression of genes related to senescence associated phenotype, including oxidative stress, reactive oxygen species (ROS), and mitochondria dysfunction. Network analysis revealed that PDGFRA and IL6 were the top hub genes in treated NP cells. Furthermore, in irradiation-induced senescent NP cells, PDGFRA gene expression was significantly reduced compared to non-irradiated cells. However, rhPDGF-AB/BB treatment increased PDGFRA expression and mitigated the senescence progression through increased cell population in the S phase, reduced SA-β-Gal activity, and decreased expression of senescence related regulators including P21, P16, IL6, and NF-κB. Our findings reveal a novel anti-senescence role of PDGF in the IVD, demonstrating its ability to alleviate the senescent phenotype and protect against the progression of senescence. This makes it a promising candidate for preventing or treating IVD degeneration by targeting cellular senescence.

Abstract Figure
Mitochondrial Dysfunction in Alzheimer’s Disease Is Driven By Excess ER-Calcium Release in Patient-Derived Neurons
Authors: Mustaly-Kalimi Sarah , Gallegos Wacey , Marr Robert A. , Peterson Daniel A. , Sekler Israel , Stutzmann Grace E.
Published: October 2024

Tight regulation of mitochondrial Ca2+is essential for neuronal bioenergetics and cellular metabolism. Ca2+transfer from ER-localized ryanodine receptors (RyR) and inositol triphosphate receptors (IP3R) to the mitochondria maintains a steady Ca2+source that fuels oxidative phosphorylation and ATP production. In Alzheimer’s disease (AD), RyR-evoked Ca2+release is markedly increased, contributing to synaptic deficits, protein mishandling, and memory impairment. Here, we demonstrate that dysregulated RyR-Ca2+release directly compromises mitochondrial activity and is an early contributor to AD cellular pathology. We measured an array of mitochondrial functions using fluorescent biosensors and optical imaging in RyR2-expressing HEK cells and iPSC-derived neurons from familial AD and nonAD patients. In neurons from AD patients, resting mitochondrial Ca2+levels were elevated alongside increased free radical production and higher caspase-3 activity relative to nonAD neurons. RyR-evoked Ca2+release further potentiated pathogenic mitochondrial responses in AD neurons, with increased Ca2+uptake and exaggerated membrane depolarization. Additionally, clearance of damaged mitochondria was impaired in AD neurons, demonstrating consequences from dysfunctional lysosomes. Notably, impairments to mitochondria in AD neurons were largely prevented with the RyR negative allosteric modulator, Ryanodex. These findings highlight how excess RyR-Ca2+release broadly contributes to early cellular pathology in AD which includes a cascade of ER, lysosomal, and mitochondrial deficits culminating in neuronal decline and degeneration. Additionally, pharmacological suppression of RyR-Ca2+release preserves mitochondrial, ER and lysosomal function, thus providing a novel and effective therapeutic.

Significance Statement

Mitochondrial dysfunction plays a central role in the cellular pathogenesis of Alzheimer’s disease, yet the upstream mechanisms driving these deficits remain largely unknown. Here, in human neurons derived from Alzheimer’s patients, we reveal how early Ca2+mishandling through the ER-localized ryanodine receptor (RyR) is sensed by the mitochondria and triggers a pathogenic metabolic cascade. Pharmacologically restoring Ca2+homeostasis reversed these deficits, including the increased free radicals and defective mitophagy, and restored the nonAD phenotype. The findings reveal the early neuronal signaling mechanisms that affect ER Ca2+handling, proteolysis, and mitochondrial activity, highlighting the potential role of the dysregulated RyR as a therapeutic target for AD using clinically relevant model systems.
Allele-specific silencing of a dominantSETXmutation in familial amyotrophic lateral sclerosis type 4
Authors: Winkelsas Audrey , Apfel Athena , Johnson Brian , Harmison George , Li Dongjun , Cheung Vivian , Grunseich Christopher
Published: October 2024

Amyotrophic lateral sclerosis 4 (ALS4) is an autosomal dominant motor neuron disease that is molecularly characterized by reduced R-loop levels and caused by pathogenic variants insenataxin(SETX).SETXencodes an RNA/DNA helicase that resolves three-stranded nucleic acid structures called R-loops. Currently, there are no disease-modifying therapies available for ALS4. Given thatSETXis haplosufficient, removing the product of the mutated allele presents a potential therapeutic strategy. We designed a series of siRNAs to selectively target the RNA transcript from the ALS4 allele containing the c.1166T>C mutation (p.Leu389Ser). Transfection of HEK293 cells with siRNA and plasmids encoding either wild-type or mutant (Leu389Ser) epitope tagged SETX revealed that three siRNAs specifically reduced mutant SETX protein levels without affecting the wild-type SETX protein. In ALS4 primary fibroblasts, siRNA treatment silenced the endogenous mutantSETXallele, while sparing the wild-type allele, and restored R-loop levels in patient cells. Our findings demonstrate that mutantSETX, differing from wild-type by a single nucleotide, can be effectively and specifically silenced by RNA interference, highlighting the potential of allele-specific siRNA as a therapeutic approach for ALS4.
Bond-centric modular design of protein assemblies
Authors: Wang Shunzhi , Favor Andrew , Kibler Ryan Daniel , Lubner Joshua Morris , Borst Andrew J , Coudray Nicolas , Redler Rachel , Chiang Huat Thart , Sheffler William , Hsia Yang , Li Zhe , Ekiert Damian Charles , Bhabha Gira , Pozzo Lilo D , Baker David
Published: October 2024

We describe a modular bond-centric approach to protein nanomaterial design inspired by the rich diversity of chemical structures that can be generated from the small number of atomic valencies and bonding interactions. We design protein building blocks with regular coordination geometries and bonding interactions that enable the assembly of a wide variety of closed and opened nanomaterials using simple geometrical principles. Experimental characterization confirms successful formation of more than twenty multi-component polyhedral protein cages, 2D arrays, and 3D protein lattices, with a high (10-50 %) success rate and electron microscopy data closely matching the corresponding design models. Because of the modularity, individual building blocks can assemble with different partners to generate distinct regular assemblies, resulting in an economy of parts and enabling the construction of reconfigurable systems.
Characterization of neural infection by Oropouche orthobunyavirus
Authors: Connors Kaleigh A. , Pedlow Maris R. , Frey Zachary D. , McGaughey Jackson J. , Amarasinghe Gaya K. , Duprex W. Paul , D’Aiuto Leonardo , Wills Zachary P. , Hartman Amy L.
Published: October 2024

Oropouche fever is a re-emerging global viral threat caused by infection with Oropouche orthobunyavirus (OROV). While disease is generally self-limiting, historical and recent reports of neurologic involvement highlight the importance of understanding the neuropathogenesis of OROV. In this study, we characterize viral replication kinetics in neurons and microglia derived from immortalized, primary, and induced pluripotent stem cell-derived cells, which are all permissive to infection. We demonstrate that ex vivo rat brain slice cultures can be infected by OROV and produce antiviral cytokines and chemokines, including IL-6, TNF-α and IFN-β, which introduces an additional model to study viral kinetics in the central nervous system. These findings provide additional insight into OROV neuropathogenesis and in vitro modeling strategies for a newly re-emerging arbovirus.
Reshaping the tumor microenvironment by degrading glycoimmune checkpoints Siglec-7 and -9
Authors: Wang Chao , Hou Yingqin , Zak Jaroslav , Zheng Qinheng , McCord Kelli A. , Wu Mengyao , Zhang Ding , Chung Shereen , Shi Yujie , Ye Jinfeng , Zhao Yunlong , Hajjar Stephanie , Wilson Ian A. , Paulson James C. , Teijaro John R. , Zhou Xu , Barry Sharpless K. , Macauley Matthew S. , Wu Peng
Published: October 2024

Cancer treatment has been rapidly transformed by the development of immune checkpoint inhibitors targeting CTLA-4 and PD-1/PD-L1. However, many patients fail to respond, especially those with an immunosuppressive tumor microenvironment (TME), suggesting the existence of additional immune checkpoints that act through orthogonal mechanisms. Sialic acid-binding immunoglobulin-like lectin (Siglec)-7 and -9 are newly designated glycoimmune checkpoints that are abundantly expressed by tumor-infiltrating myeloid cells. We discovered that T cells express only basal levels of Siglec transcripts; instead, they acquire Siglec-7 and -9 from interacting myeloid cells in the TME via trogocytosis, which impairs their activation and effector function. Mechanistically, Siglec-7 and -9 suppress T cell activity by dephosphorylating T cell receptor (TCR)-related signaling cascades. Using sulfur fluoride exchange (SuFEx) click chemistry, we developed a ligand that binds to Siglec-7 and -9 with high-affinity and exclusive specificity. Using this ligand, we constructed a Siglec-7/9 degrader that targets membrane Siglec-7 and -9 to the lysosome for degradation. Administration of this degrader induced efficient Siglec degradation in both T cells and myeloid cells in the TME. We found that Siglec-7/9 degradation has a negligible effect on macrophage phagocytosis, but significantly enhances T cell anti-tumor immunity. The degrader, particularly when combined with anti-CTLA-4, enhanced macrophage antigen presentation, reshaped the TME, and resulted in long-lasting T cell memory and excellent tumor control in multiple murine tumor models. These findings underscore the need to consider exogenous checkpoints acquired by T cells in the TME when selecting specific checkpoint blockade therapy to enhance T cell immunity.
Identification of functional rare coding variants in IGF-1 gene in humans with exceptional longevity
Authors: Ali Amanat , Zhang Zhengdong , Gao Tina , Aleksic Sandra , Gavathiotis Evripidis , Barzilai Nir , Milman Sofiya
Published: October 2024

Diminished signaling via insulin/insulin-like growth factor-1 (IGF-1) axis is associated with longevity in different model organisms. IGF-1 gene is highly conserved across species, with only few evolutionary changes identified in it. Despite its potential role in regulating life span, no coding variants in IGF-1 have been reported in human longevity cohorts to date. This study investigated the whole exome sequencing data from 2,487 individuals in a cohort of Ashkenazi Jewish centenarians, their offspring, and controls without familial longevity to identify functional IGF-1 coding variants. We identified two likely functional coding variantsIGF-1:p.Ile91Leu andIGF-1:p.Ala118Thr in our longevity cohort. Notably, a centenarian specific novel variantIGF-1:p.Ile91Leu was located at the binding interface of IGF-1 – IGF-1R, whereasIGF-1:p.Ala118Thr was significantly associated with lower circulating levels of IGF-1. We performed extended all-atom molecular dynamics simulations to evaluate the impact of Ile91Leu on stability, binding dynamics and energetics of IGF-1 bound to IGF-1R. TheIGF-1:p.Ile91Leu formed less stable interactions with IGF-1R’s critical binding pocket residues and demonstrated lower binding affinity at the extracellular binding site compared to wild-type IGF-1. Our findings suggest thatIGF-1:p.Ile91Leu andIGF-1:p.Ala118Thr variants attenuate IGF-1R activity by impairing IGF-1 binding and diminishing the circulatory levels of IGF-1, respectively. Consequently, diminished IGF-1 signaling resulting from these variants may contribute to exceptional longevity in humans.
Pre-fusion AAA+ remodeling of target-SNARE protein complexes enables synaptic transmission
Authors: White K. Ian , Khan Yousuf A. , Qiu Kangqiang , Balaji Ashwin , Couoh-Cardel Sergio , Esquivies Luis , Pfuetzner Richard A. , Diao Jiajie , Brunger Axel T.
Published: October 2024

Membrane fusion is driven by SNARE complex formation across cellular contexts, including vesicle fusion during synaptic transmission. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we show that the AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) must act prior to fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations within and near these clusters using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or two binary complexes of syntaxin—SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion.
Amplification of olfactory transduction currents implements sparse stimulus encoding
Authors: Belonio Kai Clane , Haile Eyerusalem S. , Fyke Zach , Vivona Lindsay , Konanur Vaibhav , Zak Joseph D.
Published: October 2024

Sensory systems must perform the dual and opposing tasks of being sensitive to weak stimuli while also maintaining information content in dense and variable sensory landscapes. This occurs in the olfactory system, where OSNs are highly sensitive to low concentrations of odors and maintain discriminability in complex odor environments. How olfactory sensory neurons (OSNs) maintain both sensitivity and sparsity is not well understood. Here, we investigated whether the calcium-activated chloride channel, TMEM16B, may support these dual roles in OSNs. We used multiphoton microscopy to image the stimulus-response density of OSNs in the olfactory epithelium. In TMEM16B knockout mice, we found that sensory representations were denser, and the magnitude of OSN responses was increased. Behaviorally, these changes in sensory representations were associated with an increased aversion to the odorant trimethylamine, which switches perceptual valence as its concentration increases, and a decreased efficiency of olfactory-guided navigation. Together, our results indicate that the calcium-activated chloride channel TMEM16B sparsens sensory representations in the peripheral olfactory system and contributes to efficient integrative olfactory-guided behaviors.
Complex responses of soil prokaryotes, fungi and protists to prairie restoration on retired agricultural lands
Authors: Tosi Micaela , MacColl Kevin , Obregón Dasiel , MacDougall Andrew S. , Maherali Hafiz , Dunfield Kari
Published: October 2024

Restoring native ecosystems on marginal croplands has many benefits but the impacts on belowground biodiversity are less clear, in part because the limiting factors regulating soil biota are complex and poorly described. Here, we studied how grassland prairie restoration of marginal croplands affected the diversity and composition of soil microbiota on 5 conventional farms from Ontario, Canada. Soil samples (0-15 cm) were collected from annually cultivated fields and adjacent planted perennial grassland where cultivation and chemical inputs had ceased several years previously. Following DNA extraction, we estimated bacterial and fungal abundance using quantitative PCR, and microbial diversity of prokaryotes, fungi and protists using amplicon high-throughput sequencing. Under both land uses, prokaryotic communities were dominated by Proteobacteria, Actinobacteria and Acidobacteria, fungal communities by Ascomycota, and protist communities by Rhizaria (TSAR), Evosea (Amoebozoa) and Chlorophyta (Archaeplastida). Prairie restoration did not have a consistent effect on soil microbial abundance, richness or evenness, which responses varied across farms. Microbial genetic and taxonomic community composition (i.e., sequence variant and genus level) were affected by land use, farm and the interaction between these two factors. Generally, prairie soils had higher relative abundance of Latescibacterota, Desulfobacterota, Acidobacteriota and Glomeromycota, and lower of Deinococcota, Chytridiomycota and Amoebozoa_X. In terms of differentially abundant fungal genera, prairies promoted more fungal plant symbionts, less saprotrophs and no plant pathogens. Interkingdom networks revealed changes in potential microbe-microbe associations with prairie restoration, with only 8 associations in common between land uses. The relationship between soil microbial diversity and physicochemical properties varied across microbial groups, diversity metrics and land uses. Our results evidence the complexity associated with restoring soils from agricultural land to natural ecosystems, with unspecified farm-specific factors (e.g., soil type, prairie species, management history) strongly modulating the response of different microbial groups and variables.
Feature Selection Enhances Peptide Binding Predictions for TCR-Specific Interactions
Authors: Teimouri Hamid , Ghoreyshi Zahra S. , Kolomeisky Anatoly B. , George Jason T.
Published: October 2024

T-cell receptors (TCRs) play a critical role in the immune response by recognizing specific ligand peptides presented by major histocompatibility complex (MHC) molecules. Accurate prediction of peptide binding to TCRs is essential for advancing immunotherapy, vaccine design, and understanding mechanisms of autoimmune disorders. This study presents a novel theoretical method that explores the impact of feature selection techniques on enhancing the predictive accuracy of peptide binding models tailored for specific TCRs. To evaluate the universality of our approach across different TCR systems, we utilized a dataset that includes peptide libraries tested against three distinct murine TCRs. A broad range of physicochemical properties, including amino acid composition, dipeptide composition, and tripeptide features, were integrated into the machine learning-based feature selection framework to identify key features contributing to binding affinity. Our analysis reveals that leveraging optimized feature subsets not only simplifies the model complexity but also enhances predictive performance, enabling more precise identification of TCR-peptide interactions. The results of our feature selection method are consistent with findings from hybrid approaches that utilize both sequence and structural data as input as well as experimental data. Our theoretical approach highlights the role of feature selection in peptide-TCR interactions, providing a powerful tool for uncovering the molecular mechanisms of the T-cell response and assisting in the design of more advanced targeted therapeutics.
Fluorescence lifetime-based FRET biosensors for monitoring N-terminal domain interactions of TDP-43 in living cells: A novel resource for ALS and FTD drug discovery
Authors: Nathan Kochen Noah , Murray Marguerite , Vunnam Nagamani , Liao Elly E. , Chen Lihsia , Braun Anthony R. , Sachs Jonathan N.
Published: October 2024

TAR DNA-binding protein 43 (TDP-43) pathological aggregates are widely implicated in Alzheimer’s disease, frontotemporal dementia and amyotrophic lateral sclerosis. While therapeutic platforms targeting TDP-43 have predominantly targeted its aggregation, recent findings suggest that loss of functional TDP-43 dimers and multimers — essential for RNA processing — occur upstream of aggregation and is driven through disruption of N-terminal domain (NTD) interactions. Here, we demonstrate that these interactions are targetable via cellular fluorescence lifetime-based FRET biosensors which we used to screen the FDA-approved Selleck library. Our NTD-specific hit ketoconazole rescues sorbitol-induced TDP-43 mislocalization and aggregation, and ameliorates TDP-43 induced downregulation of SREBP2, a TDP-43 mRNA binding target with known implication in ALS. In addition, ketoconazole improves neurite outgrowth in a TDP-43 overexpressing neuron model and motor dysfunction in TDP-43 overexpressing C. elegans. Taken together, our platform represents a novel approach for targeting NTD-dependent TDP-43 interactions, and the identification of ketoconazole validates an exciting translational premise for TDP-43 drug discovery.
Single-molecule characterisation of soluble beta-amyloid aggregate binding by Aducanumab, Lecanemab, Gantenerumab, and Donanemab
Authors: Fertan Emre , Lam Jeff Y. L. , Albertini Giulia , Dewilde Maarten , Wu Yunzhao , Akingbade Oluwatomi E. S. , Böken Dorothea , English Elizabeth A. , De Strooper Bart , Klenerman David
Published: October 2024

Monoclonal antibodies Aducanumab, Lecanemab, Gantenerumab, and Donanemab have been developed for treatment of Alzheimer’s disease. Here, we have used single-molecule detection techniques and super-resolution imaging to characterise the binding of these antibodies to beta-amyloid aggregates including human post-mortem brain samples. Lecanemab is the best antibody in terms of binding to the small-soluble beta-amyloid aggregates, affinity, aggregate coating, and the ability to bind to post-translationally modified species, explaining its therapeutic success.
Amino acid-level differences in alpha tubulin sequences are uniquely required for meiosis
Authors: Chen Li , Chen Xi , Kashina Anna
Published: October 2024

Tubulin is the major structural constituent of the microtubule cytoskeleton. YeastSchizosaccharomyces pombecontain two α- tubulins genes,nda2andatb2, that are highly functionally distinct:nda2deletion is lethal, while lack ofatb2does not interfere with cell viability. The functional determinants underlying this distinction are unknown. Here we used CRISPR-Cas9 gene editing to generate a yeast strain expressing Atb2 amino acid sequence utilizing Nda2 codon usage in the native Nda2 locus. Such Nda2-coded Atb2 (NCA) yeast, unlike Nda2 knockout, were viable and displayed no visible abnormalities in cell morphology or vegetative life cycle. However, these NCA yeast showed strong impairments in sporulation and meiosis, including major meiotic delays and high rates of abnormal chromosome segregation. Our data indicate that the amino acid sequence of Nda2 is uniquely required for normal meiosis, and identify a novel determinant that underlies functional distinction between closely related tubulin isoforms.
Cocaine taking and craving produce distinct transcriptional profiles in dopamine neurons
Authors: Pollock Tate A. , Margetts Alexander V. , Vilca Samara J. , Tuesta Luis M.
Published: October 2024

Dopamine (DA) signaling plays an essential role in reward valence attribution and in encoding the reinforcing properties of natural and artificial rewards. The adaptive responses from midbrain dopamine neurons to artificial rewards such as drugs of abuse are therefore important for understanding the development of substance use disorders. Drug-induced changes in gene expression are one such adaptation that can determine the activity of dopamine signaling in projection regions of the brain reward system. One of the major challenges to obtaining this understanding involves the complex cellular makeup of the brain, where each neuron population can be defined by a distinct transcriptional profile. To bridge this gap, we have adapted a virus-based method for labeling and capture of dopamine nuclei, coupled with nuclear RNA-sequencing, to study the transcriptional adaptations, specifically, of dopamine neurons in the ventral tegmental area (VTA) during cocaine taking and cocaine craving, using a mouse model of cocaine intravenous self-administration (IVSA). Our results show significant changes in gene expression across non-drug operant training, cocaine taking, and cocaine craving, highlighted by an enrichment of repressive epigenetic modifying enzyme gene expression during cocaine craving. Immunohistochemical validation further revealed an increase of H3K9me3 deposition in DA neurons during cocaine craving. These results demonstrate that cocaine-induced transcriptional adaptations in dopamine neurons vary by phase of self-administration and underscore the utility of this approach for identifying relevant phase-specific molecular targets to study the behavioral course of substance use disorders.
When Rivals Are Absent: Male Aggression Towards Females in Bluefin Killifish
Authors: Karatgi Ratna , Fuller Rebecca C.
Published: October 2024

The process of obtaining mates, mating, and (potentially) caring for offspring is costly. While there are inherent costs to reproduction, behavioral interactions among individuals are often the primary drivers of reproductive costs. Males frequently compete for territories and females; females may compete for food or males; males often harass females. Here, we sought to determine whether reproductive costs were primarily due to male/male competition, female/female competition, or male/female interactions in the bluefin killifish. In this species, males guard small spawning territories where females visit them daily to spawn. To manipulate the potential for male and female competition and male/female interactions, we altered the sex ratio and density of each sex across four treatments (1 male: 1 female, 1 male : 3 females, 3 males: 1 female, 3 males: 3 females). Female mortality was higher than male mortality. Surprisingly, female mortality and male aggressive behaviors towards females (i.e., chases) were highest in treatments with a single male. Male-male aggression was present, but males often resolved these disputes via signaling by flaring their fins. There was little evidence for overt aggression among females. When males lack rivals, they turn their territorial defense towards females. These costs help explain why, in nature, females promptly leave male territories following spawning and join loose shoals with conspecific females and minnows.
miR126-mediated impaired vascular integrity in Rett syndrome
Authors: Osaki Tatsuya , Wan Zhengpeng , Haratani Koji , Jin Ylliah , Campisi Marco , Barbie David A. , Kamm Roger , Sur Mriganka
Published: October 2024

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein, and its mutation influences not only neural cells but also non-neural cells in the brain, including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis, and its alteration may be linked to the pathology of neurodegenerative disease, but a non-neurogenic effect, especially the relationship between vascular alternation and Rett syndrome pathogenesis, has not been shown. Here, we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation, doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells, we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls, indicating altered barrier function by MeCP2 mutation. Furthermore, we unveiled that hyperpermeability is involved in the upregulation of miR126-3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq, and rescue of miR126-3p level can recover their phenotype. We discover miR126-3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine.
Striatal lateral inhibition regulates action selection in a mouse model of levodopa-induced dyskinesia
Authors: Twedell Emily L. , Bair-Marshall Chloe J. , Girasole Allison E. , Scaria Lara K. , Sridhar Sadhana , Nelson Alexandra B.
Published: October 2024

Striatal medium spiny neurons (MSNs) integrate multiple external inputs to shape motor output. In addition, MSNs form local inhibitory synaptic connections with one another. The function of striatal lateral inhibition is unknown, but one possibility is in selecting an intended action while suppressing alternatives. Action selection is disrupted in several movement disorders, including levodopa-induced dyskinesia (LID), a complication of Parkinson’s disease (PD) therapy characterized by involuntary movements. Here, we identify chronic changes in the strength of striatal lateral inhibitory synapses in a mouse model of PD/LID. These synapses are also modulated by acute dopamine signaling. Chemogenetic suppression of lateral inhibition originating from dopamine D2 receptor-expressing MSNs lowers the threshold to develop involuntary movementsin vivo, supporting a role in motor control. By examining the role of lateral inhibition in basal ganglia function and dysfunction, we expand the framework surrounding the role of striatal microcircuitry in action selection.
Species-specific satellite DNA composition dictatesde novoformation of PRC1-mediated pericentric heterochromatin
Authors: Lamelza Piero , Parrado Malena , Hamlin Emma , Lampson Michael A.
Published: October 2024

Pericentromeres are heterochromatic regions adjacent to centromeres that ensure accurate chromosome segregation. Despite their conserved function, they are composed of rapidly evolving A/T-rich satellite DNA. This paradoxical observation is partially resolved by epigenetic mechanisms that maintain heterochromatin, independent of specific DNA sequences. However, it is unclear how satellite DNA sequence variation impactsde novoformation of pericentric heterochromatin, which is initially absent from paternal chromosomes in the zygote. Here we show that satellite variation has functional consequences for zygotic heterochromatin formation, recruitment of the Chromosome Passenger Complex (CPC), and interactions with spindle microtubules. InM. musculuszygotes, Polycomb Repressive Complex 1 (PRC1) is recruited to pericentric satellites by its AT-hook domain, which binds runs of A/T nucleotides, to generate H2AK119ub1 (H2Aub) heterochromatin. By fertilizingM. musculuseggs with sperm from other mouse species, we show that species-specific satellite sequences differ in their ability to recruit PRC1 and form H2Aub. This satellite-DNA mediated increase in PRC1 heterochromatin leads to reduced CPC recruitment and increased microtubule forces on kinetochores. Our results provide a direct link between satellite DNA composition and pericentromere function in the zygote, when epigenetic pathways maintaining pericentromere heterochromatin are absent.
Active RNA synthesis patterns nuclear condensates
Authors: Banani Salman F. , Goychuk Andriy , Natarajan Pradeep , Zheng Ming M. , Dall’Agnese Giuseppe , Henninger Jonathan E. , Kardar Mehran , Young Richard A. , Chakraborty Arup K.
Published: October 2024

Biomolecular condensates are membraneless compartments that organize biochemical processes in cells. In contrast to well-understood mechanisms describing how condensates form and dissolve, the principles underlying condensate patterning – including their size, number and spacing in the cell – remain largely unknown. We hypothesized that RNA, a key regulator of condensate formation and dissolution, influences condensate patterning. Using nucleolar fibrillar centers (FCs) as a model condensate, we found that inhibiting ribosomal RNA synthesis significantly alters the patterning of FCs. Physical theory and experimental observations support a model whereby active RNA synthesis generates a non-equilibrium state that arrests condensate coarsening and thus contributes to condensate patterning. Altering FC condensate patterning by expression of the FC component TCOF1 impairs ribosomal RNA processing, linking condensate patterning to biological function. These results reveal how non-equilibrium states driven by active chemical processes regulate condensate patterning, which is important for cellular biochemistry and function.
Brain Pretargeted PET – New Horizons to Image CNS Targets with Monoclonal Antibodies
Authors: Gustavsson Tobias , Kustermann Thomas , Hvass Lars , Shalgunov Vladimir , Clausen Anne Skovsbo , Stotz Sophie , Wünsche Thomas Erik , Knudsen Gitte M. , Aldana Blanca I. , Niewoehner Jens , Gobbi Luca , Honer Michael , Battisti Umberto , Kjaer Andreas , Herth Matthias M.
Published: October 2024

Antibodies are excellent targeting vectors for molecular imaging. Slow pharmacokinetics and low blood-brain barrier penetration hinder their widespread application for molecular imaging within CNS. Improved brain uptake can be achieved via transferrin-mediated transcytosis. Pretargeted imaging can increase imaging contrast and reduce radiation exposure to the patient. Here, we report for the first time that pretargeted imaging of CNS targets using intravenously administered target vectors is feasible. Specific binding to Aβ-bound antibody was achieved. We believe that this proof-of-concept study will facilitate molecular imaging of currently ‘undruggable’ targets with antibodies where small molecule PET tracer discovery has been challenging.
Chromatin Environment-Dependent Effects of DOT1L on Gene Expression in Male Germ Cells
Authors: Coulée Manon , de la Iglesia Alberto , Blanco Mélina , Gobé Clara , Lapoujade Clémentine , Ialy-Radio Côme , Alvarez-Gonzalez Lucia , Meurice Guillaume , Ruiz-Herrera Aurora , Fouchet Pierre , Cocquet Julie , El Khattabi Laïla
Published: October 2024

The H3K79 methyltransferase DOT1L is essential for multiple aspects of mammalian development where it has been shown to regulate gene expression. Here, by producing and integrating epigenomic and spike-in RNA-seq data, we deciphered the molecular role of DOT1L during mouse spermatogenesis and show that it has opposite effects on gene expression depending on chromatin environment. On the one hand, DOT1L represses autosomal genes that are devoid of H3K79me2 at their bodies and located in H3K27me3-rich/H3K27ac-poor environments. On the other hand, it activates the expression of genes enriched in H3K79me2 and located in H3K27me3-poor/H3K27ac-rich environments, predominantly X chromosome-linked genes, after meiosis I. This coincides with a significant increase in DOT1L expression at this stage and a genome-wide acquisition of H3K79me2, particularly on the sex chromosomes. Taken together, our results show that H3K79me2 positively correlates with male germ cell genetic program throughout spermatogenesis, with DOT1L predominantly inhibiting rather than activating gene expression. Interestingly, while DOT1L appears to directly regulate the (re)activation of X genes following meiotic sex chromosome inactivation, it also controls the timely expression of (autosomal) differentiation genes during spermatogenesis.
Improved optogenetic modification of the spiral ganglion neurons for future optical cochlear implants
Authors: Thirumalai Anupriya , Henseler Jana , Enayati Marzieh , Kusch Kathrin , Hessler Roland , Moser Tobias , Huet Antoine Tarquin
Published: October 2024

Optogenetic stimulation has become a promising approach for restoring lost body function. For example, partial restoration of vision has been achieved in a blind patient and proof-of-concept has been demonstrated for optogenetic hearing restoration in rodents. In order to prepare clinical translation of hearing restoration, efficient and safe optogenetic modification of spiral ganglion neurons (SGNs) in the mature cochlea remains to be developed. Here, we established microcatheter-based administration adeno-associated virus (AAV) to scala tympani of the cochlea of Mongolian gerbils and compared it to the previously developed AAV-injection into the spiral ganglion. We probed the potential AAV-PHP.S capsid to express channelrhodopsins (ChRs) under the control of the human synapsin promotor in mature SGNs in hearing and deafened gerbils. Using the microcatheter approach, but not with the AAV-modiolus injection, we achieved reliable ChR expression in SGN enabling optogenetic stimulation of the auditory pathway in 80% of the treated animals. Yet, the efficiency of SGN transduction was modest with only ∼30% ChR-expressing SGNs. Moreover, we encountered off-target expression in hair cells in hearing gerbils in both approaches, but not ChR expression in the central nervous system using microcatheter administration. Comparing optogenetic auditory brainstem responses of gerbils with and without hair cell transduction confirmed that SGNs were the primary site of optogenetic stimulation of the pathway.
Biophysical and biochemical evidence for the role of acetate kinases (AckAs) in an acetogenic pathway in pathogenic spirochetes
Authors: Deka Ranjit K. , Tso Shih-Chia , Liu Wei Z. , Brautigam Chad A.
Published: October 2024

Unraveling the metabolism ofTreponema pallidumis a key component to understanding the pathogenesis of the human disease that it causes, syphilis. For decades, it was assumed that glucose was the sole carbon/energy source for this parasitic spirochete. But the lack of citric-acid-cycle enzymes suggested that alternative sources could be utilized, especially in microaerophilic host environments where glycolysis should not be robust. Recent bioinformatic, biophysical, and biochemical evidence supports the existence of an acetogenic energy-conservation pathway inT. pallidumand related treponemal species. In this hypothetical pathway, exogenous D-lactate can be utilized by the bacterium as an alternative energy source. Herein, we examined the final enzyme in this pathway, acetate kinase (named TP0476), which ostensibly catalyzes the generation of ATP from ADP and acetyl-phosphate. We found that TP0476 was able to carry out this reaction, but the protein was not suitable for biophysical and structural characterization. We thus performed additional studies on the homologous enzyme (75% amino-acid sequence identity) from the oral pathogenTreponema vincentii, TV0924. This protein also exhibited acetate kinase activity, and it was amenable to structural and biophysical studies. We established that the enzyme exists as a dimer in solution, and then determined its crystal structure at a resolution of 1.36 Å, showing that the protein has a similar fold to other known acetate kinases. Mutation of residues in the putative active site drastically altered its enzyme activity. A second crystal structure of TV0924 in the presence of AMP (at 1.3 Å resolution) provided insight into the binding of one of the enzyme’s substrates. On balance, this evidence strongly supported the roles of TP0476 and TV0924 as acetate kinases, buttressing the existence of the acetogenic pathway in pathogenic treponemes.
High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction
Authors: Shang Xiaoyue , Bartels Nina , Weck Johann Moritz , Suppmann Sabine , Basquin Jérôme , Heuer-Jungemann Amelie , Monzel Cornelia
Published: October 2024

Background

Cluster of differentiation 95 (CD95/Fas/Apo1) as part of the Tumor-necrosis factor (TNF) receptor family is a prototypic trigger of the ‘extrinsic’ apoptotic pathway and its activation by the trimeric ligand CD95L is of high interest for anticancer therapy. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells.

Results

Here, we design a recombinant CD95L exhibiting an isoleucine zipper (IZ) motif at the N-terminus for stabilization of the trimerized CD95L and demonstrate its high apoptosis induction efficiency. A cysteine amino acid fused behind the IZ is further used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and high-yield production of CD95Lviathe HEK293T secretory expression system is presented, along with CD95L affinity purification and functionalization. We verified the biological activity of the purified protein and identified a stabilized trimeric CD95L structure as the most potent inducer of apoptosis signaling.

Conclusions

The workflow and the findings reported here will streamline a wide array of future low- or high-throughput TNF-ligand screens, and their modification towards improving apoptosis induction efficiency and anticancer therapy.
Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation
Authors: Gonzalez-Hurtado Elsie , Leveau Claire , Li Keyi , Qu Rihao , Mishra Manish , Goldberg Emily L. , Sidorov Sviatoslav , Yeung Stephen T. , Khairallah Camille , Gonzalez David , Shepard Taverlyn M. , Camell Christina , Artyomov Maxim N. , Kluger Yuval , Khanna Kamal M. , Dixit Vishwa Deep
Published: October 2024

Age-related inflammation or ‘inflammaging’ is a key mechanism that increases disease burden and may control lifespan. How adipose tissue macrophages (ATMs) control inflammaging is not well understood in part because the molecular identities of niche-specific ATMs are incompletely known. Using intravascular labeling to exclude circulating myeloid cells and subsequent single-cell sequencing with orthogonal validation, we define the diversity and alterations in niche resident ATMs through lifespan. Aging led to depletion of vessel-associated macrophages (VAMs), expansion of lipid-associated macrophages (LAMs), and emergence of a unique subset of CD38+age-associated macrophages (AAMs) in visceral white adipose tissue (VAT). Interestingly, CD169+CD11c−ATMs are enriched in a subpopulation of nerve-associated macrophages (NAMs) that declines with age. Depletion of CD169+NAMs in aged mice increases inflammaging and impairs lipolysis suggesting that they are necessary for preventing catecholamine resistance in VAT. These findings reveal specialized ATMs control adipose homeostasis and link inflammation to tissue dysfunction during aging.
Muti-omics characterization reveals brain-wide disruption of synapses and region- and age-specific changes in neurons and glia inSp4mutant mice, a genetic model of schizophrenia and bipolar disorder
Authors: Kwon Min Jee , Perzel Mandell Kira A. , Aryal Sameer , Geng Chuhan , Nicolella Ally A. , Valle-Tojeiro Antia , Natarajan Sahana , Misri Deeksha , Hall Alyssa , Song Bryan J. , Farsi Zohreh , Keshishian Hasmik , Carr Steven A. , Sheng Morgan
Published: October 2024

Schizophrenia and bipolar disorder are highly heritable mental illnesses with unclear pathophysiology. Heterozygous loss-of-function mutations ofSp4, a zinc-finger transcription factor, greatly increase risk of schizophrenia and bipolar disorder. To investigate the molecular functions ofSp4in an unbiased mannerin vivo, we performed multi-omics analyses ofSp4mutant mice. Bulk and single nucleus RNA-seq data showed prominent gene expression changes in all brain regions and most cell types, including neuronal and non-neuronal cells. Gene set enrichment analysis of transcriptomic changes revealed alterations in many molecular pathways, including synapse, oxidative phosphorylation, and ribosome. Synapse proteomics ofSp4mutants pointed to impaired glutamatergic signaling and altered presynaptic function. InSp4heterozygous mutant mice, prefrontal cortex and striatum exhibited downregulation of synapse pathways and neuronal hypoactivity at 1 month, associated with reduced sterol biosynthesis in astrocytes, whereas at 3 months, there was a shift to neuronal hyperactivity, concurrent with suppressed immune pathways in the striatal microglia. Furthermore, our study found that much of the transcriptomic changes might be accounted for by a set of transcription regulators (Nr3c1,Creb1, andKdm5b) under the control ofSp4. Overall, this study provides cellular and molecular features resulting fromSp4LoF that may explain the pathophysiology of SCZ-BD psychotic disorder spectrum.