# Impact of α-synuclein fibrillar strains and β-amyloid assemblies on mouse cortical neurons endo-lysosomal logistics

Qiao-Ling Chou, Ania Alik, François Marquier, Ronald Melki, François Treussart, Michel Simonneau

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## To cite this version:

Qiao-Ling Chou, Ania Alik, François Marquier, Ronald Melki, François Treussart, et al.. Impact of α-synuclein fibrillar strains and β-amyloid assemblies on mouse cortical neurons endo-lysosomal logistics. eNeuro, 2022, 9, pp.ENEURO.0227-21.2022. 10.1523/eneuro.0227-21.2022. cea-03683734

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Research Article: New Research | Disorders of the Nervous System

# Impact of α-synuclein fibrillar strains and β-amyloid assemblies on mouse cortical neurons endo-lysosomal logistics

https://doi.org/10.1523/ENEURO.0227-21.2022

Cite as: eNeuro 2022; 10.1523/ENEURO.0227-21.2022

Received: 20 May 2021

Revised: 19 January 2022

Accepted: 25 January 2022

This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data.

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1 **Manuscript Title:** Impact of α-synuclein fibrillar strains and β-amyloid
assemblies on mouse cortical neurons endo-lysosomal logistics

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4 **Abbreviated Title:** Protein assemblies and neurons endo-lysosomal logistics

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1 Université Paris-Saclay, ENS Paris-Saclay, CNRS, CentraleSupélec, LuMIn, 91190 Gif-sur-Yvette, France.

2 Laboratory of Neurodegenerative Diseases, Institut François Jacob (MIRCen), CNRS, CEA, Université Paris-Saclay, 92265 Fontenay-aux-Roses cedex, France.

3 Département d'Enseignement et de Recherche en Biologie, ENS Paris-Saclay, 91190 Gif-sur-Yvette, France.

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Corresponding authors: Correspondence should be addressed to François Treussart (francois.treussart@ens-paris-saclay.fr) or Michel Simonneau (michel.simonneau926@gmail.com)

Number of figures: 8 main + 8 extended figures

Number of tables: 0

Number of words:

Abstract: 257

Significant statement: 115

Introduction: 416

Discussion: 1187

Acknowledgements

This work was supported by the Joint Program on Neurodegenerative Disease Research and Agence National de la Recherche (contract TransPathND ANR-17-JPCD-0002) to M.S. and R.M.; Fondation pour la Recherche Médicale (contract ALZ201912009776) to R.M.; Q.L.C. was supported by a PhD scholarship from Taiwan Ministry for Education and University Paris-Saclay and JPND TransPathND (ANR-17-JPCD-0002).

Conflicts of Interest: Authors report no conflict of interest

Funding sources: Joint Program on Neurodegenerative Disease Research and Agence National de la Recherche (contract TransPathND ANR-17-JPCD-0002); Fondation pour la Recherche Médicale (contract ALZ201912009776); PhD scholarship from Taiwan Ministry for Education and University Paris-Saclay.
45 Abstract

46 Endosomal transport and positioning cooperate in the establishment of neuronal
47 compartment architecture, dynamics and function, contributing to neuronal
48 intracellular logistics. Furthermore, dysfunction of endo-lysosomal has been identified
49 as a common mechanism in neurodegenerative diseases. Here, we analyzed endo-
50 lysosomal transport when α-synuclein (α-syn) fibrillar polymorphs, β-amyloid (Aβ)
51 fibrils and oligomers were externally applied on primary cultures of mouse cortical
52 neurons. To measure this transport, we used a simple readout based on the
53 spontaneous endocytosis in cultured neurons of fluorescent nanodiamonds, a
54 perfectly stable nano-emitter, and the subsequent automatic extraction and
55 quantification of their directed motions at high-throughput. α-syn fibrillar polymorphs,
56 Aβ fibrils and oligomers induce a two-fold decrease of the fraction of nanodiamonds
57 transported along microtubules, while only slightly reducing their interaction with
58 cortical neurons. This important decrease in moving endosomes is expected to have
59 a huge impact on neuronal homeostasis. We next assessed lysosomes dynamics,
60 using Lysotracker. Neurons exposure to Aβ oligomers led to an increase in the
61 number of lysosomes, a decrease in the fraction of moving lysosome and an increase
62 in their size, reminiscent of that found in APP transgenic model of Alzheimer's
63 disease. We then analyzed the effect of α-syn fibrillar polymorphs, Aβ fibrils and
64 oligomers on endosomal and lysosomal transport and quantified directed transport of
65 those assemblies within cortical neurons. We report different impacts on endosomal
66 and lysosomal transport parameters and differences in the trajectory lengths of
67 cargoes loaded with pathogenic protein assemblies. Our results suggest that
68 intraneuronal pathogenic protein aggregates internalization and transport may
69 represent a target for novel neuroprotective therapeutic strategies.
## 70 Significance Statement

Neurodegenerative diseases (NDs) are characterized by the deposition of protein aggregates. These proteins exert a broad range of neuronal toxicity. Defects in endo-lysosomal traffic are increasingly viewed as key pathological features of NDs, likely contributing to synaptic dysfunction and ultimately neuronal death. Here we measured by fast fluorescence videomicroscopy the endosomal and lysosomal dynamics in the branches of primary culture of mouse cortical neurons after externally applying α-syn fibrillar polymorphs (fibrils and ribbons) and Aβ assemblies (oligomers and fibrils). We provide significant insight into the differential effects of these pathogenic protein assemblies on endosomal and lysosomal transport, and also reveal distinct transport characteristics of the compartments loaded with these protein assemblies compared to endosome ones.

## 82 Introduction

Impairment of axonal transport has recently emerged as a factor shared by several neurodegenerative disorders (Millecamps and Julien, 2013; Morfini et al., 2009). Early impact on intraneuronal transport has been thus proposed as a phenotypic trait common to neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's Disease (Stokin et al., 2005; Saudou and Humbert, 2016; Volpicelli-Daley et al., 2014). There is compelling evidence that abnormal protein accumulation in the brain is a key pathophysiological mechanism underlying the neurotoxicity observed in these age-related disorders (Golde et al., 2030; Soto et al., 2018). Selective aggregation of misfolded proteins is a hallmark of these neurodegenerative diseases (Saez-Atienzar & Masliah, 2020). An important level of complexity is due to
the fact that different species of the same molecules, such as oligomers and fibrils, contribute to a whole spectrum of toxicities (Alam et al., 2017).

Few studies have compared, within the same neurons, fibrillary and oligomeric α-synuclein (α-syn) and β-amyloid (Aβ) which are known to be involved in Parkinson and Alzheimer's diseases respectively. Brahic et al., 2016, demonstrated for instance that α-syn, Aβ₄₂ and HTTExon1 fibrils are transported anterogradely and retrogradely in mice primary neurons grown in microfluidic chambers with different efficiencies in axons. Here, we thoroughly quantified the impact of two α-syn fibrillar polymorphs namely fibrils (α-synF) and ribbons (α-synR), Aβ₄₂ fibrils (AβF) and oligomers (AβO) on endosomal and lysosomal transports in primary cultures of mouse neurons. To measure this transport and investigate finely its parameters, we relied on our previously established method (Haziza et al., 2017), in which we let perfectly stable and non-toxic fluorescent nanodiamonds (FND) being spontaneously internalized by neurons in endosomes, then follow their displacement by fast video-microscopy and finally apply to the videos our analysis pipeline to extract and analyze single particle trajectories automatically. Using fluorescently-labelled α-syn and Aβ assemblies, we conducted the same investigations on their own intraneuronal transport.

Our data allow to address three complementary questions: (i) do α-synF, α-synR, AβF and AβO influence the fraction of cargoes moving along the microtubules; (ii) do they impact the dynamics of intracellular endosomal and lysosomal transport.

We show here that all pathogenic proteins assemblies reduce the fraction of endosomes moving along microtubules and impact some of their transport parameters. Furthermore, lysosomes properties (number, fraction of lysosomes moving and transport parameters) are also affected by AβO. Finally, our data indicate that cargoes loaded with α-synF, α-synR, AβF or AβO are transported differently
118 from endosomes, considered as control cargoes, which suggests distinct molecular
119 characteristics of cargo-motor assemblies.

120 **Material and Methods**

121 **Production of α-syn fibrillar assemblies, Aβ fibrils and oligomers**

122 The expression and purification of human WT α-syn was performed as previously
described (Ghee et al., 2005). Pure WT α-syn was incubated in buffer A to obtain the
123 fibrillar polymorph “fibrils” α-synF (50 mM Tris-HCl at pH 7.5, 150 mM KCl) and in
124 buffer B for “ribbons” α-synR (5 mM Tris-HCl at pH 7.5) at 37°C under continuous
125 shaking in an Eppendorf Thermomixer (Hamburg, Germany) set at 600 rotations per
126 minute (rpm) for 4–7 days (Bousset et al., 2013). The fibrillar α-syn polymorphs were
127 centrifuged twice at 15,000 g for 10 min and resuspended twice in phosphate-
128 buffered saline (PBS) at 1,446 g/L prior to labeling with ATTO 488 NHS-ester (#AD
129 488-3, Atto-Tec, Siegen, Germany) fluorophore following the manufacturer’s
130 instructions using a protein/dye ratio of 1:2. The labeling reactions were arrested by
131 addition of 1 mM Tris (pH 7.5). The unreacted fluorophore was removed by a final
132 cycle of two centrifugations at 15,000 g for 10 min and resuspensions of the pellets in
133 PBS. This labeling protocol typically yields ≥ 1 ATTO molecule incorporated per α-
134 syn monomer on average as previously demonstrated (Shrivastava et al., 2015). The
135 assemblies were examined by transmission electron microscopy after adsorption on
136 200 mesh carbon-coated electron microscopy grids and negative stained with 1%
137 uranyl acetate before and after fragmentation using a JEOL 1400 electron
138 microscope (JEOL, Tokyo, Japan).

140 The expression and purification of Met-Aß 1-42 was performed as described (Walsh
141 et al., 2009). Aß was assembled in PBS, at 4°C or 37°C without shaking for 2 or 24 h
142 to obtain oligomers AßO or fibrils AßF, respectively. The two kinds of assemblies
143 were labeled with ATTO 488 NHS-ester at a protein/dye ratio of 1:2. The labeling
144 reactions were arrested by addition of 1 mM Tris at pH 7.5. For fibrillar Aß, the
145 unreacted fluorophore was removed by two cycles of centrifugation and resuspension
146 of the pelleted fibrils in PBS as described for α-syn. For oligomeric Aβ, the oligomers
147 were separated from the monomeric and fibrillar forms of the protein by size
148 exclusion chromatography on a Superose 6 HR10/300 column (GE Healthcare, Life
149 Sciences, Wauwatosa, WI, USA) equilibrated in PBS pH 7.4 at a flow rate of
150 0.5 mL/min. Elution was monitored by measuring absorbance at 280 nm wavelength.
151 The Superose 6 column was calibrated with Dextran blue (over 2200 kDa), (670 kDa),
152 β-amylase (200 kDa), BSA (66 kDa), and carbonic anhydrase (29 kDa) standards
153 (Sigma-Aldrich).

154 Primary mouse cortical neuron cultures

155 We used commercial primary mouse cortical neurons (ref. Gibco A15586,
156 ThermoFisher) because the provider quality check guarantees a purity of 98% of
157 neurons. The cells were grown on high optical quality glass coverslips (high-precision
158 170±5 µm thick, 18 mm diameter, ref. 0117580, Marienfeld GmbH, Germany). The
159 coverslips are first cleaned with 70% ethanol, rinsed with water for injection (ref.
160 A128730, ThermoFisher Inc., USA) and exposed during 1 h to UV light. They were
161 then coated with 0.1 mg/ml poly-L-ornithine (ref. P3655, Sigma-Aldrich Merck KGaA,
162 Germany) and placed for 2 h in an incubator set at 37°C, then rinsed twice with water
163 and let dry at biological hood for one hour. We plated an amount of 6x10⁵ primary
164 mouse cortical neurons (ref. Gibco A15586, ThermoFisher) on each coated coverslip,
165 which was then put at the bottom of a 6-wells plate, each well-being finally filled with
166 3 mL of neurobasal phenol red-free medium (ref. 12348017, ThermoFisher)
167 containing 0.5 mM GlutaMax (ref. 35050061 ThermoFisher), 2% B-27 (ref. 17504044,
ThermoFisher) and 1% PenStrep (ref. 15070063, ThermoFisher). The 6-well plate was then placed in an incubator at 37 °C and 5% CO₂. Half of the volume of the medium was replaced with fresh medium 24 h after plating. We made the subsequent medium changes every 3 days to reduce glutamate toxicity. Neurons were grown until 21 days in culture.

### Exposure of mouse cortical neurons to α-syn fibrillar assemblies, Aβ fibrils or oligomers

In all the measurements dealing with (1) the impact of pathogenic protein assemblies on endosomal-lysosomal transport, (2) their colocalization with FND-labelled compartment or lysosomes, or (3) the tracking of their intraneuronal transport by fluorescence videomicroscopy, cortical neurons were incubated with either 0.2 µM ATTO 488-labeled α-synF or R, or 1 µM ATTO 488-labeled AβF or AβO.

α-synF or R were added at 24 h, 48 h, or 72 h before observations, while the addition time was either 24 h or 48 h for ATTO 488-labeled AβF or AβO. The video acquisitions of all the experiments were performed at DIC21.

### Washing protocol to test the protein assemblies interaction with the neuron membrane

The coverslip with the culture attached to them, were extracted from the well and flushed with PBS first and then twice with culture medium, before FND internalization was carried out following the procedure described in the next paragraph.

### Intraneuronal transport cargo labeling

To evaluate the endosomal transport parameters, we relied on our fluorescent nanodiamond assay (Haziza et al. 2017). We used commercially available sized 35 nm FND (brFND-35, FND Biotech, Taiwan). Each NP contains an average of 15
nitrogen-vacancy emitters displaying a peak emission wavelength around 700 nm and a full-width at half-maximum of ≈100 nm. This far-red emission allows also to investigate the colocalization of green-emitting ATTO 488-labelled neurodegenerative-disease related species with FND-labeled cargos. FND were internalized in cortical neurons just before the transport analysis, at DIC21. Each culture coverslip was removed from the 6-well plate containing maintaining medium and put in contact with 400 µL of fresh culture medium to which we added 2 µl of stock solution of FNDs (1 mg/mL), reaching a final FND concentration of 5 µg/mL. After 10 mins incubation, the extra FND-containing medium was absorbed by a wiper sheet and the coverslip was placed back to the dish containing the old maintaining medium. The culture was then placed back during 20 mins in the incubator before the video acquisition.

To measure lysosomal transport or investigate the colocalization of neurodegenerative disease-related species with lysosomes, cortical neurons were stained at DIC21, just before the observation, with LysoTracker Deep Red (ref. L12492, ThermoFisher) or Magic Red Cathepsin B substrate (ref. ICT937, Bio-Rad). These dye molecules have an emission spectrum within the similar range than the one of FND. The coverslip was removed from maintaining medium and incubated with prewarmed (37°C) culture medium containing 50 nM LysoTracker or Magic Red (1:20 dilution) for 1 h. The probe-containing medium was replaced with the old maintaining medium and followed by video acquisition.

Pseudo-total internal reflection (TIRF) live-cell videomicroscopy

Pseudo-TIRF illumination was implemented on an inverted microscope (Eclipse Ti-E, Nikon, Japan) as described in details in (Haziza et al. 2017). The whole microscope is enclosed in a cage incubator (Okolab, Italy) to maintain temperature at 37°C. For
the intraneuronal transport recording, each coverslip supporting the neuron culture is mounted at the bottom a Ludin chamber (type 1, Life Imaging Service, Switzerland), installed inside the environmental chamber (in which 5% partial CO₂ pressure and 100% hydrometry is maintained) having a hole at its bottom allowing direct optical access of the microscope objective to the coverslip. We used a ×100 magnification and 1.49 numerical aperture immersion oil objective (CFI Apo TIRF ×100 Oil, Nikon), compatible with differential interference contrast (DIC) mode. Field of views of interest of the neuron cultures were selected in white-light illumination DIC mode. Two continuous-wave lasers are coupled to the microscope and fluorescence was recorded on a cooled EMCCD array detector (DU-885K-CS0, Andor Technologies, UK) of 1004×1002 pixels, with 80 nm pixel size in the sample plane. Two-minutes duration videos were acquired at 20 full frame/s rate large enough to be able to detect short pausing duration in cargoes displacements. EMCCD parameters were selected to provide the largest signal-to-background ratio for FND label tracking at the selected frame rate, leading to EM gain of 90, preamplification gain ×3.8, and digitalization speed of 35 MHz. FNDs and Lysotracker fluorophore were excited with a diode-pumped solid-state laser at a wavelength of 561 nm (SLIM-561-100, Oxxius S.A., France), while ATTO 488 dye was excited with a laser diode emitting at a wavelength of 488 nm (LBX-488-200-CSB-PP, Oxxius S.A). Each excitation laser power was adjusted so that the detection dynamic range of all channels was identical for the above mentioned fixed EMCCD settings. This leads to 561 nm laser excitation power of 60 mW for FND, 200 µW for LysoTracker and 1 mW for Magic Red, and to 488 nm laser excitation power of 200 µW for ATTO 488 dye.

To perform two-color acquisitions and record simultaneously FND (or LysoTracker) and ATTO 488 we combined the two laser beams with a dual-band dichroic filter (ref.
Di01-R488/561, Semrock, USA), and placed a dual imaging system (W-VIEW GEMINI, Hamamatsu, Japan) in front of the EMCCD array detector (DU-885K-CS0, Andor Technologies, UK). This system splits half of the detection field of view (FoV) in two color channels with a dichroic beamsplitter (FF560-FDi01, Semrock) and projects each color on half of the array detector, further preceded by bandpass detection filters (red channel: HC697/75, Semrock; green channel: ET525/50, Chroma Corporation, USA). The result is that each frame of the video contains the same rectangular FoV (1004×501 pixels) in green (ATTO 488) and red (FND and LysoTracker) emission range, allowing to identify spots that colocalize dynamically.

## Video processing and intraneuronal transport quantification

Two programs written in python were developed to extract quantitative parameters from videos automatically. The first one relies on Trackpy 0.4.2 package (Trackpy 2019), from which it uses two functions: locate to identify isolated spots in each fluorescence frame and fit them with gaussians, and link which connects the spots between frames to form trajectories using Crocker-Grier algorithm (Crocker & Grier 1996). Transport parameters are then calculated with a second program which first parses each trajectory into "go" and "stop" phases based on the confinement ratio calculation as described in (Haziza et al., 2017). Four main transport parameters are extracted for each trajectory: velocity, which is the average speed of all go phases; run length: average distance traveled during all go phases; pausing time: average duration of the stop phases, and pausing frequency (events/min). In addition to these four main parameters, we also calculated the total length of the trajectory as the sum of all run lengths during go phases.

## Lysosomes size estimation
To get an estimate of the lysosome size from the diffraction limited fluorescence images, we considered the LysoTracker spots as the result of the convolution of the microscope point spread function (assimilated to a gaussian of standard deviation, SD, σ<sub>PSF</sub>) and the lysosome assimilated to a symmetrical gaussian of SD σ<sub>L</sub>. The result of this convolution is also a gaussian of SD σ<sub>T</sub>, related to σ<sub>L</sub> and σ<sub>PSF</sub> by σ<sub>T</sub><sup>2</sup> = σ<sub>PSF</sub><sup>2</sup> + σ<sub>L</sub><sup>2</sup>. σ<sub>T</sub> is the so-called “size” output of the Trackpy locate function. The knowledge of σ<sub>T</sub> and σ<sub>PSF</sub> allows the derivation of σ<sub>L</sub>. We then defined the lysosome “diameter” d<sub>L</sub> as the full width at half maximum of its gaussian approximation, inferred by d<sub>L</sub> = 2√2 ln 2 σ<sub>L</sub>. For the PSF size σ<sub>PSF</sub> we took the measured value of the smallest FND spot observed in several trajectories with our microscope. This value was σ<sub>PSF</sub>=112 nm, consistent with the theoretical Airy radius ρ<sub>A</sub>=286 nm (diffraction limit at 700 nm maximum emission wavelength for the 1.49 numerical aperture objective used), and the empirical relation σ<sub>PSF</sub> = ρ<sub>A</sub>/3, giving here an experimental PSF SD of 286/3=95 nm.

Quantification of fluorescence intensity of ATTO 488-labeled α-synF in cortical neuron branches

We quantify the fluorescence intensity from the first frame of the green channel videos, using Fiji Image J software (Schindelin et al, 2012). We first identify from the DIC image some well-separated and mainly straight neuronal branches, that we surround with the region-of-interest (ROI) polygonal selection tool as close as possible to the branch to include all the fluorescence signal, over a length of 30 µm. We then used the Analyze function to measure the average intensity counts per pixel in the defined ROI, to which we subtract the average background counts, measured after having moved the ROI in a region without branches.

Data representation and statistical analysis
All bar plots display the ± standard error on the mean of the distribution. Box plots display the median value as the horizontal line within the box whose limits are 25% and 75% percentiles; bottom and top horizontal lines correspond to 10% and 90% percentiles. As all the data compared between two conditions were random and normally distributed but with unequal variance (as tested with a *F*-test), we performed the relevant comparison test which is the non-parametric Wilcoxon Mann-Whitney two-tailed (implemented in Igor Pro 8, Wavemetrics Inc., USA). Stars referred to the following *p*-value significance level: \* *p*<0.05; \*\* *p*<0.01; \*\*\* *p*<0.001.

## 299 Results

### 300 Quantification of intraneuronal transport using fluorescent nanodiamonds

We quantified the intraneuronal transport with our FND tracking assay (Haziza et al., 2017). We first used a simple readout consisting in counting the number of FND detected in field-of-views (FoV) of size 40×80 µm during 2 minutes. We selected each FoV based on the criteria that it contains approximately the same density of neuronal branches, as estimated from differential interference contrast images (Fig. 1). Our incubation protocol was designed to strongly limit any non-specific interactions of FND, like their attachment to the coverslip supporting the culture, and favor their interaction with neuron membranes and their subsequent internalization in endosomes (Haziza et al., 2017). The perfectly stable fluorescence of FND allows to reconstruct the endosome trajectories accurately and identify “go” and “stop” (none or very slow motion) phases (Fig. 1B) in the transport of FND-labelled endosomes along neuronal branches as observed in differential interference contrast microscopy (Fig. 1C).
314 α-synF and R affect the number of cargos transported along microtubules
315 without major changes in trajectory length

316 Primary cultures of mouse cortical neurons were incubated at day in culture (DIC) 20
317 with α-synF or R at a concentration of 0.2 µM for 24 hours. The intraneuronal
318 transport in these cultures was investigated at DIC21.

319 Both exposures to α-synF or R led to a small decrease (26% for α-synF and 13% for
320 α-synR) in the number of FND (moving or not) present in each FoV, indicating that
321 both fibrillar polymorphs impact the FND binding to neuronal membrane and their
322 transport dynamics within neurons (Fig. 2A). Indeed, if we consider the fraction of
323 these FND having a directed motion, corresponding to those being first internalized in
324 endosomes and then taken in charge by molecular motors, we observed that it
325 decreases by 49%, upon exposure of neurons to α-synF and 45% in the case of α-
326 synR (Fig. 2B). The unknown mechanism involved in such a large decrease in the
327 number of cargoes transported along microtubules is expected to impair the functions
328 of cortical neurons.

329 In order to determine if this important reduction of FND moving fraction is related to
330 the binding of the protein assemblies to the neuronal branches membrane, possibly
331 leading to a reduced endocytosis, we tried in the case of α-synF to wash away the
332 aggregates just before the addition of FND (Materials and Methods), but we found
333 that the interaction of the nanodiamonds with neuron did not change in washed-
334 neuron condition compared to unwashed (Fig. 2-1A-C); in particular, we did not
335 observe differences in the fraction of moving FND. We concomitantly measured the
336 amount of ATTO 488-labelled assemblies along the neuronal branches (as quantified
337 by the dye fluorescence intensity) and could not see any differences before and after
338 washing (Fig. 2-1D-E), which is in agreement with FND-neuron interaction results,
339 and indicate a strong binding of α-synF to the neuronal membrane.

340 Using our established FND-based intraneuronal transport assay (Haziza et al., 2017)
341 we detected and quantitatively analyzed the alternation of movement and pause
342 phases of intraneuronal cargoes motion. We first measured the length of trajectories
343 (see Material & Methods) for control FND, FND in the presence of either α-synF or α-
344 synR and we did not evidence any major changes (Fig. 2C, Fig. 2D; decrease of 4%
345 for α-synF and 5% for α-synR).

346 We then measured four parameters: the curvilinear velocity of each moving phase, its
347 run length, the duration of the pauses and the pausing frequency. The velocity
348 (Fig. 2E) and run length (Fig. 2F) increases (velocity: 31% and 38%; run length: 80%
349 and 100%, for α-synF and R respectively), the pausing time decreases (Fig. 2G; 40%
350 decrease for both fibrillar assemblies) while the pausing frequency increases (Fig. 2H;
351 19% and 25%, for α-synF and R respectively). These results are summarized in
352 Fig. 2-2A.

353 We next analyzed the same parameters for lysosomes labelled with Lysotracker red,
354 an established marker of lysosomes, with the difference, compared to FND, that all
355 the fluorescent spots, including the static ones, correspond to lysosomes because
356 Lysotracker only become fluorescent once inside lysosomes. α-synR treatment
357 induces a slight but significant decrease (15%) in the number of lysosomes per field-
358 of-view, while α-synF does not (Fig. 3A). This result suggests that α-synR reduce the
359 endocytosis. It is consistent with the observed decrease of FND interacting with
360 neuron (Fig. 2A) that possibly reveals their reduced uptake. Furthermore, like for
361 endosomes (Fig. 2B), α-synF and R induce a 46% and 32% decrease respectively
362 (resp.) in the fraction of lysosomes having a directed motion (Fig. 3B). Analysis of
lysosome trajectory lengths indicates a slight decrease (Fig. 3C; 9% and 3% for α-synF and R resp.). Example of FoV showing lysosome trajectories in the different conditions are shown in Fig. 3D, where the large decrease in the fraction of lysosomes having a directed motion can be clearly seen.

We quantified the same transport parameters for lysosomes than for FND, using the same experimental paradigm. In contrast to what we observed for endosomes transport, exposure of neurons to α-synF did not lead to any changes in lysosomes transport parameters (Fig. 3E-H). Interestingly however, in cortical neurons exposed to α-synR, we measured a slight increase of 6% in lysosomes velocity (Fig. 3E) and larger one of 26% in run length (Fig. 3F), no significant change in pausing time (Fig. 3G) and a slight increase of 4% in pausing frequency (Fig. 3H). These results are summarized in Fig. 2-2B.

**Aβ assemblies affect the number of cargos transported along microtubules without major changes in trajectory length**

We also analyzed the same parameters after DIC20 mouse cortical neurons exposure to either AβF or AβO, for 24 hours, followed by intracellular transport measurement at DIC21. We used the common 1 µM concentration that has been reported to have a biological impact (Marshall et al., 2020). Figure 4A shows a slight decrease (3% and 7% for AβF and AβO resp.) of FND interacting with neurons exposed to AβF or AβO, accompanied by a much larger decrease (56% and 29% for AβF and AβO resp.) of the fraction having directed motions (Fig. 4B). FND trajectory lengths stay almost the same for AβF (3% decrease) but are reduced by AβO (Fig. 4C, Fig. 4D; 13% decrease). We also investigated the effect of Aβ assemblies at the smaller concentration of 0.2 µM, identical to the one of α-syn assemblies. Even at this lower concentration, we could detect for both AβF and AβO small decreases
388 (19% and 18% for AßF and AßO resp.) in the number of FND per FoV (Fig. 4-1A)
389 and in the fraction of FND having a directed motion (Fig. 4-1B; 11% and 18% for AßF
390 and AßO respectively). To summarize, as for α-syn assemblies (Fig. 3), the exposure
391 of cortical neurons to Aß assemblies induce important and significant decreases of
392 the endosomal transport.

393 We then measured more precisely the impact of AßF and AßO on endosomal
394 transports parameters. We observed an increase of FND velocity (Fig. 4E; 20% and
395 15% for AßF and AßO resp.) and run length (Fig. 4F; 5% and 7% for AßF and AßO
396 resp.), a decrease in their pausing time (Fig. 4G; 36% and 12% for AßF and AßO
397 resp.) and an increase of the pausing frequency (Fig. 4H; 28% and 15% for AßF and
398 AßO resp.), with effects more pronounced for AßF than for AßO. Interestingly, the
399 same trends of changes were also observed at the lower AßF and AßO concentration
400 of 0.2 µM (Fig. 4-1C-F). Let us finally point out that for AßF, the important changes of
401 some transport parameters overall combine in an only very slight decrease in
402 trajectory length as shown in Fig 4C, which makes the detailed quantitative analysis
403 performed all the more useful. These results are summarized in Fig. 2-2A.

404 Regarding the impact of Aß on lysosomal transport, we noticed large differences
405 between the two types of assemblies AßF and AßO. In neurons exposed to AßO, the
406 total number of lysosomes detected in a FoV as compared to controls increased by
407 50% (Fig. 5A) while it stayed unchanged in case of exposure to AßF. The intracellular
408 transport measurements we performed showed that the fraction of moving lysosomes
409 decreased by 1.6-fold and 5.7-fold in neurons exposed to AßF and AßO, respectively
410 (Fig. 5B; from 23% for control to 14% for AßF and 4.5% for AßO). Lysosome
411 trajectory lengths were only slightly decreased for AßF (5%) and more significantly
412 reduced for AßO (Fig. 5C and Fig. 5D; 17% decrease) exposure.
We also measured lysosome transport parameters in the presence of 1 µM AßF or AßO (Fig. 5E-H). For AßF, we observed almost no change in velocity (Fig. 5E), pausing time (Fig. 5G), and pausing frequency (Fig. 5H). In contrast, exposure to AßO, led to a 1.7-fold increase of the pausing time. The run length decreased significantly for both assemblies (Fig. 5F; with 9% and 13% decrease for AßF and AßO exposure resp.). These results are summarized in Fig. 2-2B.

Furthermore, as changes in lysosome size was described in APP mouse transgenic model of Alzheimer's disease (Gowrishankar et al., 2015), we asked if detectable changes in lysosome diameter can be quantified upon 24 or 48 h exposure to 1 µM AßF and AßO. We detected a slight increase (7%) of lysosome diameter upon exposure of neurons to AßF at 24 h that disappears at 48 h. This contrasts with the increase we observed at both time points (11% and 7% at 24 h and 48 h resp.) in neurons exposed to 1 µM AßO (Fig. 5I-J and Fig. 5-1A-C). The finding that AßO addition triggers an increase in lysosomes number and size and a decrease in lysosome movements is in agreement with previous reports (Gowrishankar et al., 2015; Marshall et al., 2020).

Finally, as LysoTracker can also label other acidic compartments than lysosomes, in particular late endosomes, we repeated the transport experiment with Magic Red substrate that reveals by fluorescence the Cathepsin B protease activity, taking place more specifically in lysosomes. We observed that for both α-synF (Fig. 5-2) and AßF (Fig. 5-3) Magic Red-labelled compartments (lysosomes) behaved the same as LysoTracker-labelled ones for all parameters, with in particular a ≈40% decrease of the mobile fraction of Magic Red and LysoTracker-labelled compartment in the presence of the fibrillar assemblies, and similar colocalization data at 24 h, i.e. small colocalization of 4-7% for α-synF compared to 40% for AßF. These results indicate
that, in our case, LysoTracker and Magic Red labeling largely overlap and that we
can rely on LysoTracker puncta density to quantify endocytic activity as we did.
These results are summarized in Fig. 2-2C.

441 Transport of α-syn and Aβ assemblies within cortical neurons.

442 We also assessed α-syn and Aβ assemblies transport within cortical neurons while
443 documenting their impact on endosomes and lysosomes dynamics. As ATTO 488
444 dye used to label α-syn and Aβ assemblies exhibit no emission spectrum overlap
445 with neither FND nor Lysotracker deep red, we were able to measure simultaneously
446 the transport properties of endosome or lysosome and the assemblies on two-color
447 channels.

448 We first studied α-syn fibrillar assemblies transport (Fig. 6). We found that α-synF
449 and R display directed movements as shown by examples of trajectories in Fig. 6A-B.

450 We compared these motions to the endosomal transport in the presence of α-syn
451 fibrillar polymorphs. Interestingly, α-syn F and R trajectories are about 29% shorter
452 than those of FND (Fig. 6C). We also compared the transport parameters and found
453 smaller velocity (Fig. 6D; 5% and 10% for α-synF and α-synR resp.), run length
454 (Fig. 6E; 39% and 46% for α-synF and α-synR resp.) and pausing time (Fig. 6F; 19%
455 and 21% for α-synF and α-synR resp.) for α-synF and α-synR-loaded cargoes
456 compared to those of FND-containing endosomes. On the contrary, α-synF and R
457 pausing frequencies were larger than those of FND (Fig. 6G; 19% and 8% for α-synF
458 and α-synR resp.). The shorter trajectories length and run-length together with the
459 larger pausing frequency, suggest that cargoes loaded with α-synF- and R are
460 transported less efficiently than those containing FND.

461 Similarly, we also investigated ATTO 488-labelled AβF and AβO intraneuronal
462 transport (Fig. 7) at the concentration of 1 µM, as we could not detect their
fluorescence signal at 0.2 µM. Both species exhibit directed transport as shown by examples of trajectories in Fig. 7A-B. These trajectories are shorter than the ones of FND in the same conditions (Fig. 7C), as for α-syn fibrillar assemblies. Regarding the transport parameters, compared to FND, AßF and AßO have slightly larger velocity (Fig. 7D; 8% and 12% for AßF and AßO resp.), and a trend towards a shorter run-length (Fig. 7E; 7% and 28% for AßF and AßO resp.). As α-syn fibrillar assemblies, AßF and AßO exhibit a much shorter pausing time (Fig. 7F; 49% and 53% for AßF and AßO resp.) and a much larger pausing frequency (Fig. 7G; 41 and 36% for AßF and AßO resp.). As for α-syn fibrillar assemblies the shorter trajectories length and run-length together with the larger pausing frequency suggest that cargoes loaded with AßF and AßO are transported less efficiently than those containing FND.

Moreover, since differences in AßO and AßF uptake in cultured neurons were recently reported (Vadukul et al., 2020), we also investigated the related aspect of the number of Aß assembly trajectories per field-of-view at 24 h and 48 h time points (Fig. 7-1). We did not observe differences in the number of trajectories 24 h after addition of the assemblies (Fig. 7-1A) in agreement with Fig. 4B of (Vadukul et al. 2020). However, at 48 h time point, we measured a ≈2.5 lower number of AßO trajectories compared to AßF ones (Fig. 7-1B), which differs to uptake results of (Vadukul et al. 2020) at 72 h time point, who reported a ≈1.5 times larger amount of internalized AßO compared to AßF(sonicated). Our observations differ from (Vadukul et al. 2020), but several reasons may explain this discrepancy: (i) we quantify only the moving fraction of Aß assemblies; (ii) we do not have the 72 h point, and finally (iii) we do not use the exact same AßF.

Finally, we assessed in a quantitative manner the co-localization of α-syn and Aß assemblies with lysosomes, as a function of neuron exposure time at 24 and
488 48 hours (Fig. 8). While the fraction of α-syn fibrillar assemblies moving within
489 lysosomes increases from ≈4% at 24 h to 12-14% at 72 h (Fig. 8A), it reaches
490 already ≈41% at 24 h for Aβ fibrils continuing its increase up to ≈51% at 48 h
491 (Fig. 8B). AβO moving in or with lysosomes have slightly lower colocalization
492 proportions, however much larger than for α-syn fibrillar assemblies. We repeated
493 these analyses with Magic Red labeling instead of LysoTracker for α-synF (Fig. 8-1A)
494 and AβF (Fig. 8-1B) at 24 h and achieved similar results, with a ≈40% colocalization
495 fraction of AβF with Magic Red-labelled lysosomes, and only ≈7% for α-synF.

496 Discussion

497 In this work, we investigated the generic effects of α-syn fibrillar polymorphs (fibrils
498 and ribbons) and Aβ assemblies (oligomers and fibrils) on endosomal and lysosomal
499 movements in mouse cortical neurons. In neurons, early endosomes and lysosomes
500 move using different types of machinery. Also, the former are compartmentalized
501 (dendrites vs. axon) while the latter are not (Winckler et al., 2018). However, due to
502 the high density of the cultures it was not possible to identify unambiguously the
503 compartment (dendrite or axon) in which the traced vesicles moved, and therefore we
504 could not study separately the impact of the protein assemblies on axonal and
505 dendritic endolysosomal transports.

506 Potential consequences of a decrease in the number of cargos transported at a
given time within cortical neuron

508 The exposure of cortical neurons to α-syn and Aβ assemblies led to important
509 reductions (between 32% and 56%) of moving endosomes and lysosomes along
510 neuronal branches (Figs. 1-2). We previously showed that pathogenic α-syn and Aβ
511 assemblies bind the plasma membrane with, as a consequence, a redistribution of
512 essential membrane proteins (Renner et al. 2010; Shrivastava et al., 2013;
513 Shrivastava et al., 2015). We further reviewed the physico-pathogenic mechanisms
514 at the origin and resulting from pathogenic proteins assemblies-plasma membrane
515 components interactions (Shrivastava et al., 2017). The reduction we report here
516 might be due to changes in membrane dynamics and endocytosis rate.

517 Exposure of cortical neurons to α-syn and Aβ assemblies affected moving FND-
518 containing endosome properties. α-syn fibrillar assemblies increased their velocity by
519 31-38% and run length by 80-100% (Fig. 2E-F) and decreased their pausing time by
520 40% (Fig. 2G) while increasing their pausing frequency to a lesser extend (19-25%,
521 Fig. 2H). We observed similar effects but less pronounced for moving FND-
522 containing endosomes when neurons are exposed to AβF or AβO (Fig. 4E-H). These
523 changes reflect an increase in mobility of moving FND-containing endosomes. Hence,
524 while decreasing the fraction of moving FND-labeled endosomes, α-synF and R or
525 AβF and AβO, increase the overall mobility of the moving ones (Fig. 2-2A).

526 A decrease in the number of moving endosomes or lysosomes can affect protein
527 quality control, accompanied by limited elimination of damaged membrane and
528 cytosolic proteins, protein aggregates, and membranous organelles (Winckler et al.,
529 2018). Furthermore, considering that lysosomes and late endosomes act as mRNA
530 translation platforms (Cioni et al., 2019; Liao et al., 2019; Fernandopulle et al., 2021),
531 changes in the number of cargos transported at a given time within a cortical neuron
532 is expected to dramatically impact mRNA translation platform either in dendrites or in
533 axons. In particular, the regulation of protein synthesis and degradation at the
534 neuronal synapse is local and dynamic and modify the synaptic proteome
535 autonomously during plasticity (Giandomenico et al., 2021). Hence, the synaptic
536 function can be impacted if the number of moving lysosomes is affected.
537 **Effect of α-syn and Aβ assemblies on lysosomes transport**

538 We observed that α-synF (Fig. 3E-H) and AβF (Fig. 5E-H) barely impact the
539 lysosomal transport parameters as compared to the control. In contrast, α-synR
540 (Fig. 3E-H) and AβO (Fig. 5E-H) induce significant changes of lysosomes transport
541 parameters (Fig. 2-2B). Furthermore, the size of lysosomes significantly increased in
542 the presence of AβO (Fig. 5I-J). This set of impairments of lysosomal transport in
543 Alzheimer-related context are in full agreement with previous reports (Gowrishankar
544 et al., 2015; Marshall et al., 2020). Indeed, using a mouse model of Alzheimer's
545 disease, (Gowrishankar et al., 2015) evidenced axonal lysosome accumulations with
546 local impairment in the retrograde axonal transport of lysosome precursors. Similarly,
547 (Marshall et al., 2020) found that misfolded Aβ₄₂ impacts the endo-lysosomal
548 pathway. They reported impairments in the uptake of proteins that use a dynamin-
549 dependent endosomal mechanism and accumulation of lysosomes.

550 **Intraneuronal transport of neurodegenerative-related molecular species**

551 We were able to quantify the intraneuronal transport parameters for α-syn and Aβ
552 pathogenic species (Figs 6-7). We found that cargoes loaded with α-synF and R
553 exhibit a more dynamical transport compared to those containing FND that are
554 characterized by a larger pausing frequency and shorter run-length and pausing time
555 (Fig. 6D-G). AβF and AβO-containing endosomes exhibit similar characteristics
556 (Fig. 7D-G) with in addition an increase in velocity, not observed for α-synF and R.
557 These results suggest that similar molecular mechanisms are at play in the transport
558 of the two α-syn fibrillar polymorphs, the AβF and AβO. However, the ≈7-fold larger
559 fraction of AβF and AβO found in moving lysosomes compared to α-syn assemblies
560 (Fig. 8), also indicate differences in the molecular interactions of Aβ assemblies with
lysosomes. These results suggest either a cellular triage leading to differential transport of α-syn and Aβ assemblies.

## Potential application in drug discovery assay

We report here the use of a model based on primary mouse cortical neurons where it is possible to identify a robust decrease in the number of vesicles moving intracellularly (Figs. 2B, 3B, 4B and 5B), in the order of 30-50%, and up to 5-fold for lysosomes in neurons exposed to AβO (Fig. 5B). Such a large decrease is likely to impact the physiology of neurons and it is reasonable to consider that the transport of other cargoes, such as mitochondria and RNA granules, is also affected. This endolysosomal transport impairment endophenotype can be instrumental in generating large-scale drug-discovery campaigns (i.e., >10⁵ compounds) as used in more complex human cellular models (Park et al., 2021).

The cargoes transport blockade within cortical neurons we report could either result from a direct interaction between pathogenic aggregates and the intraneuronal transport machinery or a pathogenic aggregates-mediated transcriptional changes in transport proteins expression (Encalada and Goldstein, 2014, Lee et al., 2014; Guo et al., 2020). Analysis of neuronal immuno-precipitates of α-syn fibrillar assemblies and Aβ polymorphs may be instrumental to identify the molecular partners that directly interact with these protein assemblies. A recent postmortem proteomics study identified proteins whose abundance changed at different stages of Alzheimer's disease (Li et al., 2021). At its early stage, differentially expressed proteins of "clathrin-coated endocytic vesicle membrane" (GO: 0030669) and the secretory pathway (R-HSA-432720: "Lysosome Vesicle Biogenesis" and R-HSA-432722: "Golgi Associated Vesicle Biogenesis") classes were over-represented. Comparison of proteome profile changes in our neuronal model with (Li et al., 2021) profiles can be
instrumental to identify druggable targets in order to enhance the number of transported cargoes.

Finally, we quantified intraneuronal transport of neurodegenerative-linked molecular species (α-syn fibrillar polymorphs, AβF and oligomers) whose transport characteristics are distinct from those of endosomes but for which no molecular characterization is yet available. Furthermore, we observed that all these protein assemblies are transported intracellularly in cortical neurons with very similar quantitative characteristics. We could not detect differences in their transport parameters. Further work will be required to identify a possible common transport mechanism and the identification of specific molecules involved in this transport can allow to selectively inhibit it.

These results also need to be considered from the standpoint of the prion-like spread of pathogenic protein particles between neurons (Brundin et al., 2010; Hardy and Revesz, 2012; Jucker and Walker, 2018). Selective inhibition may avoid the spread of these neurotoxic species. Thus, advances in the identification of targets involved in cargoes loaded with pathogenic protein aggregates transport may lead to novel neuroprotective therapeutic avenues.

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733 **Figure Legends**

734 **Figure 1.** Recording fluorescent nanodiamond trajectories in mouse cortical neurons
735 at DIC21. **A**, Schematic representation of the cargoes that are tracked thanks to
736 FND, at different stages after their endocytosis. We previously showed (Haziza et al.,
737 2017) that FND are present in cargos at different stage of their lifetime after
738 endocytosis, as shown by colocalization measurements with specific membrane
739 protein markers: Rab5 for early endosome; Rab7 for late endosome; Rab11 for
740 recycling endosome; Lysotracker for the lysosome. **B**, Illustration of FND trajectories
741 with go (green) and stop (red) phases. **C**, Differential interference contrast images of
742 cortical neurons overlapped with 10 representative trajectories. Scale bar: 10 µm.

743

744 **Figure 2.** Effect of α-synF or R on the mobility of endosomes and their transport as
745 measured by tracking FND-containing cargoes in mouse cortical neurons at DIC21.
746 24 h exposure to α-synF or R at 0.2 µM concentration, compared to nothing added
747 control (Ctrl). **A**, Number of FNDs detected per field-of-view of 40 µm x 80 µm size
748 during 2 mins of observation. **B**, Fraction of FNDs-containing cargoes having a
749 directed motion. **C**, Length of FND trajectories. **D**, Examples of FND trajectories.
750 Scale bar: 10 µm. **E-H**, Comparison of four transport parameters: **E**, curvilinear
751 velocity, **F**, run length, **G**, pausing time and **H**, pausing frequency. The number within
752 each bar represents the total number of FoV (**A**, **B**) or trajectories (**C**, **E-H**) analyzed
753 from *n*=8 coverslips (four independent cultures). Inset: box-plots representation of the
754 same dataset. See also Figure 2-1 and Fig. 2-2.

755

756 **Figure 3.** Effect of α-synF or α-synR on the mobility of LysoTracker-labelled
757 lysosomes and their transport in mouse cortical neurons at DIC21. 24 h exposure to
758 α-synF or α-synR at 0.2 µM concentration, compared to nothing added control (Ctrl).
759 A, Number of lysosomes detected per field-of-view of 40 x 80 µm size during 2 mins
760 of observation. B, Fraction of lysosomes having a directed motion. C, Length of
761 lysosome trajectories. D, Examples of lysosome trajectories. Scale bar: 10 µm. E-H,
762 Comparison of four transport parameters: curvilinear velocity (E), run length (F),
763 pausing time (G) and H, pausing frequency (H). The number within each bar
764 represents the total number of FoV (A, B) or trajectories (C, E-H) analyzed from n=2
765 coverslips (from one culture). Inset: box-plots representation of the same dataset.
766

767 **Figure 4.** Effect of AßF and AßO on the mobility of endosomes and their transport as
768 measured by tracking FND-containing cargoes in mouse cortical neurons at DIC21.
769 24 h exposure to AßF and AßO at 1 µM concentration, compared to nothing added
770 control (Ctrl). A, Number of FNDs detected per field-of-view of 40 x 80 µm size during
771 2 mins of observation. B, Fraction of FNDs-containing cargoes having a directed
772 motion. C, Length of FND trajectories. D, Examples of FND trajectories. Scale bar:
773 10 µm. E-H, Comparison of four transport parameters: curvilinear velocity (E), run
774 length (F), pausing time (G) and pausing frequency (H). The number inside the bar
775 represents the total number of FoV (A, B) or trajectories (C, E-H) analyzed from n=6
776 coverslips (three independent cultures). Inset: box-plots representation of the same
777 dataset. See also Figure 4-1.
778

779 **Figure 5.** Effect of AßF and AßO on the mobility of LysoTracker-labelled lysosomes
780 and their transport in mouse cortical neurons at DIC21. 24 h exposure to AßF and
781 AßO at 1 µM concentration, compared to nothing added control. **A**, Number of
782 lysosomes detected per field-of-view of 40 x 80 µm size during 2 mins of observation.
B, Fraction of lysosomes having a directed motion. C, Length of lysosome trajectories. D, Examples of lysosome trajectories. Scale bar: 10 µm. *E-H*, Comparison of four transport parameters: curvilinear velocity (*E*), run length (*F*), pausing time (*G*) and pausing frequency (*H*). I-J) Comparison of Lysosome size. The number inside the bar represents the total number of FoV (*A*, *B*), trajectories (*E-H*) and lysosomes (*I*, *J*) analyzed from *n*=2 coverslips (from one culture). Inset: box-plots representation of the same dataset. See also Figure 5-1, 5-2 and 5-3.

790

**Figure 6.** Intraneuronal transport of ATTO 488-labeled α-synF and R in mouse cortical neurons at DIC21. DIC20 cortical neurons were exposed to α-synF and R during 24 h, at concentration of 0.2 µM. **A-B**, Examples of α-synF and R, and FND trajectories (in the presence of α-synF and R). Scale bar: 10 µm. **C**, Length of α-synF, α-synR and FND trajectories. **D-G**, Comparison of four transport parameters: curvilinear velocity (**D**), run length (**E**), pausing time (**F**) and pausing frequency (**G**). The number inside the bar represents the total number of trajectories analyzed from *n*=8 coverslips (four independent cultures) is indicated in each bar. Inset: box-plots representation of the same dataset.

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**Figure 7.** Intraneuronal transport of ATTO 488-labeled AβF and AβO in mouse cortical neurons at DIC21. DIC20 cortical neurons were exposed to AβF and AβO (1 µM) during 24 h. **A-B**, Examples of trajectories. Scale bar: 10 µm. **C**, Length of AβF, AβO and FND trajectories. **D-G**, Comparison of four transport parameters: curvilinear velocity (**D**), run length (**E**), pausing time (**F**) and pausing frequency (**G**). The number inside the bar represents the total number of trajectories analyzed from
n=6 coverslips (from three independent cultures) is indicated in each bar. Insets: box
plots representation of the same dataset. See also Figure 7-1.

**Figure 8.** Colocalized events between moving neurodegenerative disease-related molecular species and moving lysosomes at different time points. A, α-synF and α-synR were incubated for 24 and 48 h, at concentration of 0.2 µM. B, AβF and AβO were incubated for 24 and 48 h, at concentration of 1 µM. The number inside the donut plot represents the percentage of moving α-syn or Aβ assemblies colocalized with lysosomes (LysoTracker labelled). The table on the right panel indicates the number of neurodegenerative-related molecular species trajectories colocalized with lysosomes. n represents the total number of trajectories. The percentage and number of trajectories in each time point were analyzed from 2 coverslips (from one culture). See also Figure 8-1.

818
819 **Figures**

820 **Figure 1**

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

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A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>

D

<figure><img src="image_9.png" /></figure>

FNDs Trajectories_Ctrl

<figure><img src="image_10.png" /></figure>

FNDs Trajectories_α-synF

<figure><img src="image_11.png" /></figure>

FNDs Trajectories_α-synR

E

<figure><img src="image_12.png" /></figure>

F

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G

<figure><img src="image_14.png" /></figure>

H

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<figure><img src="image_16.png" /><figcaption>826 **Figure 3**</figcaption></figure>
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<figure><img src="image_19.png" /><figcaption>835 **Figure 6**</figcaption></figure>
A

<figure><img src="image_20.png" /></figure>

FNDs Trajectories_AβF

<figure><img src="image_21.png" /></figure>

AβF Trajectories

B

<figure><img src="image_22.png" /></figure>

FNDs Trajectories_AβO

<figure><img src="image_23.png" /></figure>

AβO Trajectories

C

<figure><img src="image_24.png" /></figure>

D

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<caption>841 **Figure 8**</caption>

A

<figure><img src="image_29.png" /></figure>

<table><thead><tr><th>α-syn</th><th>fibrils</th><th>ribbons</th></tr></thead><tbody><tr><td>24h</td><td>12 (n=320)</td><td>6 (n=135)</td></tr><tr><td>48h</td><td>46 (n=616)</td><td>19 (n=171)</td></tr></tbody></table>

B

<figure><img src="image_30.png" /></figure>

<table><thead><tr><th>AB</th><th>fibrils</th><th>oligomers</th></tr></thead><tbody><tr><td>24h</td><td>102 (n=252)</td><td>22 (n=80)</td></tr><tr><td>48h</td><td>326 (n=646)</td><td>64 (n=141)</td></tr></tbody></table>

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Extended Figure Legends

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**Figure 2-1.** FNDs interaction with neurons and fluorescence intensity of ATTO 488-labeled α-synF with and without (w/o) washing. 24 h exposure of neurons to α-synF at concentration of 0.2 µM, compared to nothing added control. **A**, Number of FNDs detected per field-of-view of 40 µm x 80 µm during 2 mins of observation for the different conditions. **Ctrl**: no addition of α-synF; α-synF Wash: addition of α-synF during 24 h and washing of the culture just before addition of FND tracers. **Inset**: box-plots representation of the same dataset. **B**, Fraction of FNDs-containing cargoes having a directed motion. The numbers inside the bar in A and B represent the total number of FOV analyzed from *n*=2 coverslips (from one culture). **C**, Example of FND trajectories in the different conditions. **Scale bar**: 10 µm. **D**, Average fluorescence intensity of ATTO 488-labeled α-synF evaluated for 30 µm-long branches, with or w/o washing (*n*= 25 branches from 25 fields-of-view). **E**, Examples of first frames of TIRF videomicroscopy of ATTO 488-labeled α-synF decoration of DIC21 cortical neurons with (top) or w/o (bottom) washing. **Scale bar**: 10 µm.

860

**Figure 2-2.** Summary of the effect of different protein assemblies on the transport parameters of endosomes (A), LysoTracker-labeled lysosomes (B), and Magic Red-labeled lysosomes (C).

864

**Figure 4-1.** Impact of Aβ fibrils and oligomers at the concentration of 0.2 µM on the number of nanodiamonds interacting with cells (A), on the fraction of moving nanodiamonds (B) and on the FND-labelled endosome transport parameters (C-F).

868

**Figure 5-1.** Lysotracker-labelled compartments size in control (A) and 24 h exposure of Aβ fibrils (B) and oligomers (C) at concentration of 1 µM. **Scale bar**: 2 µm.

871

**Figure 5-2.** Effect of α-synF on the mobility of Magic Red-labelled lysosomes and their transport in mouse cortical neurons at DIC21, after 24 h exposure to α-synF at concentration of 0.2 µM, compared to nothing added control. **A**, Number of lysosomes detected per field-of-view of 40 µm x 80 µm size during 2 mins of observation. **B**, Fraction of lysosomes having a directed motion. **C**, Trajectory length. **D**, Examples of lysosome trajectories. **Scale bar**: 10 µm. **E-H**, Comparison of four
transport parameters: **E**) curvilinear velocity (**E**), run length (**F**), pausing time (**G**) and pausing frequency (**H**). **I**, Diameter of Magic Red-labelled lysosomes. General remarks: the number inside the bar represents the total number of FoV (**A**, **B**), trajectories (**E-I**). Inset: box-plots representations of the same datasets (**C-I**).

**Figure 5-3.** Effect of AßF on the mobility of Magic Red-labelled lysosomes and their transport in mouse cortical neurons at DIC21 after 24 h exposure to AßF at concentration of 1 µM, compared to nothing added control. **A**, Number of lysosomes detected per field-of-view of 40 µm x 80 µm size during 2 mins of observation. **B**, Fraction of lysosomes having a directed motion. **C**, Trajectory length. **D**, Examples of lysosome trajectories. Scale bar: 10 µm. (**E-H**) Comparison of four transport parameters: curvilinear velocity (**E**), run length (**F**), pausing time (**G**) and pausing frequency (**H**). **I**, Comparison of Magic Red-labelled lysosome size. General remarks: The number inside the bar represents the total number of FoV (**A**, **B**) and trajectories (**E-I**). Inset: box-plots representation of the same dataset.

**Figure 7-1.** Number of AßF and AßO trajectories detected per field-of-view of 40 x 80 µm during 2 mins of observation. Cortical neurons were exposed to AßF and AßO (1µM) during 24 h (**A**) and 48 h (**B**). The number inside the bar represents the total number of FOV analyzed from *n*= 6 coverslips (from three cultures) for 24 h and *n*=2 coverslips (from one culture).

**Figure 8-1.** Fraction of protein assemblies, α-synF (**A**) or AßF (**B**) trajectories colocalizing with Magic Red puncta trajectories, as inferred from the tables indicating the number of protein assembly trajectories colocalizing with Magic Red puncta and the total number of assembly trajectories in parenthesis. Data from 2 coverslips and one culture in both α-synF and Aß cases.