Qiao-Ling Chou

Ania Alik

François Marquier

Ronald Melki

François Treussart
email: francois.treussart@ens-paris-saclay.fr

Michel Simonneau
email: michel.simonneau926@gmail.com

assemblies on mouse cortical neurons endo-lysosomal logistics

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

Significance Statement

Neurodegenerative diseases (NDs) are characterized by the deposition of protein aggregates. These proteins exert a broad range of neuronal toxicity. Defects in endolysosomal 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 -syn fibrillar polymorphs (fibrils and ribbons) and Aß 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.

Introduction

Impairment of axonal transport has recently emerged as a factor shared by several neurodegenerative disorders [START_REF] Millecamps | Axonal transport deficits and neurodegenerative diseases[END_REF][START_REF] Morfini | Axonal Transport Defects in Neurodegenerative Diseases[END_REF].

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 [START_REF] Stokin | Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease[END_REF][START_REF] Saudou | The Biology of Huntingtin[END_REF][START_REF] Volpicelli-Daley | Parkinson's disease Formation of α-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes[END_REF]. 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., 203;[START_REF] Soto | Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases[END_REF].

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 ⍺synuclein (⍺-syn) and ß-amyloid (Aß) which are known to be involved in Parkinson and Alzheimer's diseases respectively. Brahic et al., 2016, demonstrated for instance that ⍺-syn, Aß 42 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 ⍺-syn fibrillar polymorphs namely fibrils (⍺-synF) and ribbons (⍺-synR), Aß 42 fibrils (AßF) and oligomers (AßO) 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 [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF], 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 ⍺-syn and Aß assemblies, we conducted the same investigations on their own intraneuronal transport.

Our data allow to address three complementary questions: (i) do ⍺-synF, ⍺-synR, AßF and AßO 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 AßO. Finally, our data indicate that cargoes loaded with ⍺-synF, ⍺-synR, AßF or AßO are transported differently from endosomes, considered as control cargoes, which suggests distinct molecular characteristics of cargo-motor assemblies.

Material and Methods

Production of -syn fibrillar assemblies, Aß fibrils and oligomers

The expression and purification of human WT ⍺-syn was performed as previously described [START_REF] Ghee | PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly[END_REF]. Pure WT ⍺-syn was incubated in buffer A to obtain the fibrillar polymorph ''fibrils'' ⍺-synF (50 mM Tris-HCl at pH 7.5, 150 mM KCl) and in buffer B for ''ribbons'' ⍺-synR (5 mM Tris-HCl at pH 7.5) at 37°C under continuous shaking in an Eppendorf Thermomixer (Hamburg, Germany) set at 600 rotations per minute (rpm) for 4-7 days [START_REF] Shrivastava | ß-amyloid and ATP-induced diffusional trapping of astrocyte and neuronal metabotropic glutamate type-5 receptors[END_REF]. The fibrillar ⍺-syn polymorphs were centrifuged twice at 15,000 g for 10 min and resuspended twice in phosphatebuffered saline (PBS) at 1,446 g/L prior to labeling with ATTO 488 NHS-ester (#AD 488-3, Atto-Tec, Siegen, Germany) fluorophore following the manufacturer's instructions using a protein/dye ratio of 1:2. The labeling reactions were arrested by addition of 1 mM Tris (pH 7.5). The unreacted fluorophore was removed by a final cycle of two centrifugations at 15,000 g for 10 min and resuspensions of the pellets in PBS. This labeling protocol typically yields ≥ 1 ATTO molecule incorporated per ⍺syn monomer on average as previously demonstrated [START_REF] Shrivastava | alpha-synuclein assemblies sequester neuronal alpha3-Na+/K+-ATPase and impair Na+ gradient[END_REF]. The assemblies were examined by transmission electron microscopy after adsorption on 200 mesh carbon-coated electron microscopy grids and negative stained with 1% uranyl acetate before and after fragmentation using a JEOL 1400 electron microscope (JEOL, Tokyo, Japan).

The expression and purification of Met-Aß 1-42 was performed as described [START_REF] Walsh | A facile method for expression and purification of the Alzheimer's diseaseassociated amyloid beta-peptide[END_REF]. Aß was assembled in PBS, at 4°C or 37°C without shaking for 2 or 24 h to obtain oligomers AßO or fibrils AßF, respectively. The two kinds of assemblies were labeled with ATTO 488 NHS-ester at a protein/dye ratio of 1:2. The labeling reactions were arrested by addition of 1 mM Tris at pH 7.5. For fibrillar Aß, the unreacted fluorophore was removed by two cycles of centrifugation and resuspension of the pelleted fibrils in PBS as described for ⍺-syn. For oligomeric Aß, the oligomers were separated from the monomeric and fibrillar forms of the protein by size exclusion chromatography on a Superose 6 HR10/300 column (GE Healthcare, Life Sciences, Wauwatosa, WI, USA) equilibrated in PBS pH 7.4 at a flow rate of 0.5 mL/min. Elution was monitored by measuring absorbance at 280 nm wavelength.

The Superose 6 column was calibrated with Dextran blue (over 2200 kDa), (670 kDa), β -amylase (200 kDa), BSA (66 kDa), and carbonic anhydrase (29 kDa) standards (Sigma-Aldrich).

Primary mouse cortical neuron cultures

We used commercial primary mouse cortical neurons (ref. Gibco A15586, ThermoFisher) because the provider quality check guarantees a purity of 98% of neurons. The cells were grown on high optical quality glass coverslips (high-precision 170±5 µm thick, 18 mm diameter, ref. 0117580, Marienfeld GmbH, Germany). The coverslips are first cleaned with 70% ethanol, rinsed with water for injection (ref.

A128730, ThermoFisher Inc., USA) and exposed during 1 h to UV light. They were then coated with 0.1 mg/ml poly-L-ornithine (ref. P3655, Sigma-Aldrich Merck KGaA, Germany) and placed for 2 h in an incubator set at 37°C, then rinsed twice with water and let dry at biological hood for one hour. We plated an amount of 6x10 5 primary mouse cortical neurons (ref. Gibco A15586, ThermoFisher) on each coated coverslip, which was then put at the bottom of a 6-wells plate, each well-being finally filled with 3 mL of neurobasal phenol red-free medium (ref. 12348017, ThermoFisher) 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% CO 2 . 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 -syn fibrillar assemblies, Aß 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 M ATTO 488-labeled -synF or R, or 1 M ATTO 488-labeled AßF or AßO.

-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 AßF or AßO. 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 [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF]. We used commercially available sized 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 l of stock solution of FNDs (1 mg/mL), reaching a final FND concentration of 5 g/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 [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF] 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 [START_REF] Crocker | Methods of Digital Video Microscopy for Colloidal Studies[END_REF]. 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 [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF]. 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, PSF ) and the lysosome assimilated to a symmetrical gaussian of SD L . The result of this convolution is also a gaussian of SD T , related to L and PSF by T = PSF + L . T is the so-called "size" output of the Trackpy locate function.

The knowledge of T and PSF allows the derivation of L . We then defined the lysosome "diameter" L as the full width at half maximum of its gaussian approximation, inferred by L = 2√2 ln 2 L . For the PSF size PSF we took the measured value of the smallest FND spot observed in several trajectories with our microscope. This value was PSF =112 nm, consistent with the theoretical Airy radius A =286 nm (diffraction limit at 700 nm maximum emission wavelength for the 1.49 numerical aperture objective used), and the empirical relation PSF = A , giving here an experimental PSF SD of 286/3=95 nm.

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

We quantify the fluorescence intensity from the first frame of the green channel videos, using Fiji Image J software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. 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.

Results

Quantification of intraneuronal transport using fluorescent nanodiamonds

We quantified the intraneuronal transport with our FND tracking assay [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF]. 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 [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF]. 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).

⍺ ⍺-synF and R affect the number of cargos transported along microtubules without major changes in trajectory length

Primary cultures of mouse cortical neurons were incubated at day in culture (DIC) 20

with ⍺-synF or R at a concentration of 0.2 μM for 24 hours. The intraneuronal transport in these cultures was investigated at DIC21.

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

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

Using our established FND-based intraneuronal transport assay [START_REF] Haziza | Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors[END_REF] we detected and quantitatively analyzed the alternation of movement and pause phases of intraneuronal cargoes motion. We first measured the length of trajectories (see Material & Methods) for control FND, FND in the presence of either ⍺-synF or ⍺-synR and we did not evidence any major changes (Fig. 2C, Fig. 2D; decrease of 4% for ⍺-synF and 5% for ⍺-synR).

We then measured four parameters: the curvilinear velocity of each moving phase, its run length, the duration of the pauses and the pausing frequency. The velocity (Fig. 2E) and run length (Fig. 2F) increases (velocity: 31% and 38%; run length: 80% and 100%, for ⍺-synF and R respectively), the pausing time decreases (Fig. 2G; 40% decrease for both fibrillar assemblies) while the pausing frequency increases (Fig. 2H;

19% and 25%, for ⍺-synF and R respectively). These results are summarized in Fig. 2-2A.

We next analyzed the same parameters for lysosomes labelled with Lysotracker red, an established marker of lysosomes, with the difference, compared to FND, that all the fluorescent spots, including the static ones, correspond to lysosomes because Lysotracker only become fluorescent once inside lysosomes. α-synR treatment induces a slight but significant decrease (15%) in the number of lysosomes per fieldof-view, while α-synF does not (Fig. 3A). This result suggests that α-synR reduce the endocytosis. It is consistent with the observed decrease of FND interacting with neuron (Fig. 2A) that possibly reveals their reduced uptake. Furthermore, like for endosomes (Fig. 2B), α-synF and R induce a 46% and 32% decrease respectively (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 ⍺-synF did not lead to any changes in lysosomes transport parameters (Fig. 3E-H). Interestingly however, in cortical neurons exposed to ⍺-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.

Aß 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 AßF or AßO, 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 [START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF]. Figure 4A shows a slight decrease (3% and 7% for AßF and AßO resp.) of FND interacting with neurons exposed to AßF or AßO, accompanied by a much larger decrease (56% and 29% for AßF and AßO resp.) of the fraction having directed motions (Fig. 4B). FND trajectory lengths stay almost the same for AßF (3% decrease) but are reduced by AßO (Fig. 4C, Fig. 4D; 13% decrease). We also investigated the effect of Aß assemblies at the smaller concentration of 0.2 μM, identical to the one of ⍺-syn assemblies. Even at this lower concentration, we could detect for both AßF and AßO small decreases (19% and 18% for AßF and AßO resp.) in the number of FND per FoV (Fig. 4-1A)

and in the fraction of FND having a directed motion (Fig. 4-1B; 11% and 18% for AßF and AßO respectively). To summarize, as for α-syn assemblies (Fig. 3), the exposure of cortical neurons to Aß assemblies induce important and significant decreases of the endosomal transport.

We then measured more precisely the impact of AßF and AßO on endosomal transports parameters. We observed an increase of FND velocity (Fig. 4E; 20% and 15% for AßF and AßO resp.) and run length (Fig. 4F; 5% and 7% for AßF and AßO resp.), a decrease in their pausing time (Fig. 4G; 36% and 12% for AßF and AßO Regarding the impact of Aß on lysosomal transport, we noticed large differences between the two types of assemblies AßF and AßO. In neurons exposed to AßO, the total number of lysosomes detected in a FoV as compared to controls increased by 50% (Fig. 5A) while it stayed unchanged in case of exposure to AßF. The intracellular transport measurements we performed showed that the fraction of moving lysosomes decreased by 1.6-fold and 5.7-fold in neurons exposed to AßF and AßO, respectively (Fig. 5B; from 23% for control to 14% for AßF and 4.5% for AßO). Lysosome trajectory lengths were only slightly decreased for AßF (5%) and more significantly 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 [START_REF] Gowrishankar | Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques[END_REF], 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 [START_REF] Gowrishankar | Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques[END_REF][START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF].

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 ⍺-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 ⍺-synF compared to 40% for AßF. These results indicates 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.

Transport of -syn and Aß assemblies within cortical neurons.

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

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

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

Similarly, we also investigated ATTO 488-labelled AßF and AßO intraneuronal 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 ⍺-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 runlength (Fig. 7E; 7% and 28% for AßF and AßO resp.). As ⍺-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 ⍺-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 [START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF], 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 we do not use the exact same AßF.

Finally, we assessed in a quantitative manner the co-localization of ⍺-syn and Aß assemblies with lysosomes, as a function of neuron exposure time at 24 and 48 hours (Fig. 8). While the fraction of ⍺-syn fibrillar assemblies moving within lysosomes increases from ≈4% at 24 h to 12-14% at 72 h (Fig. 8A), it reaches already ≈41% at 24 h for Aß fibrils continuing its increase up to ≈51% at 48 h (Fig. 8B). AßO moving in or with lysosomes have slightly lower colocalization proportions, however much larger than for ⍺-syn fibrillar assemblies. We repeated these analyses with Magic Red labeling instead of LysoTracker for ⍺-synF (Fig. 8-1A)

and AßF (Fig. 8-1B) at 24 h and achieved similar results, with a ≈40% colocalization fraction of AßF with Magic Red-labelled lysosomes, and only ≈7% for ⍺-synF.

Discussion

In this work, we investigated the generic effects of ⍺-syn fibrillar polymorphs (fibrils and ribbons) and Aß assemblies (oligomers and fibrils) on endosomal and lysosomal movements in mouse cortical neurons. In neurons, early endosomes and lysosomes move using different types of machinery. Also, the former are compartmentalized (dendrites vs. axon) while the latter are not [START_REF] Winckler | The Endolysosomal System and Proteostasis: From Development to Degeneration[END_REF]. However, due to the high density of the cultures it was not possible to identify unambiguously the compartment (dendrite or axon) in which the traced vesicles moved, and therefore we could not study separately the impact of the protein assemblies on axonal and dendritic endolysosomal transports.

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

The exposure of cortical neurons to -syn and Aß assemblies led to important reductions (between 32% and 56%) of moving endosomes and lysosomes along neuronal branches (Figs. 12). We previously showed that pathogenic ⍺-syn and Aß assemblies bind the plasma membrane with, as a consequence, a redistribution of essential membrane proteins [START_REF] Renner | Deleterious effects of amyloid b oligomers acting as an extracellular scaffold for mGluR5[END_REF][START_REF] Shrivastava | ß-amyloid and ATP-induced diffusional trapping of astrocyte and neuronal metabotropic glutamate type-5 receptors[END_REF][START_REF] Shrivastava | alpha-synuclein assemblies sequester neuronal alpha3-Na+/K+-ATPase and impair Na+ gradient[END_REF]. We further reviewed the physico-pathogenic mechanisms at the origin and resulting from pathogenic proteins assemblies-plasma membrane components interactions [START_REF] Shrivastava | Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions[END_REF]. The reduction we report here might be due to changes in membrane dynamics and endocytosis rate.

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

A decrease in the number of moving endosomes or lysosomes can affect protein quality control, accompanied by limited elimination of damaged membrane and cytosolic proteins, protein aggregates, and membranous organelles [START_REF] Winckler | The Endolysosomal System and Proteostasis: From Development to Degeneration[END_REF]. Furthermore, considering that lysosomes and late endosomes act as mRNA translation platforms [START_REF] Cioni | Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons[END_REF][START_REF] Liao | RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether[END_REF][START_REF] Fernandopulle | RNA transport and local translation in neurodevelopmental and neurodegenerative disease[END_REF], changes in the number of cargos transported at a given time within a cortical neuron is expected to dramatically impact mRNA translation platform either in dendrites or in axons. In particular, the regulation of protein synthesis and degradation at the neuronal synapse is local and dynamic and modify the synaptic proteome autonomously during plasticity [START_REF] Giandomenico | Proteostatic regulation in neuronal compartments[END_REF]. Hence, the synaptic function can be impacted if the number of moving lysosomes is affected.

Effect of ⍺ ⍺-syn and Aß assemblies on lysosomes transport

We observed that ⍺-synF (Fig. 3E-H) and AßF (Fig. 5E-H) barely impact the lysosomal transport parameters as compared to the control. In contrast, ⍺-synR (Fig. 3E-H) and AßO (Fig. 5E-H) induce significant changes of lysosomes transport parameters (Fig. 2-2B). Furthermore, the size of lysosomes significantly increased in the presence of AßO (Fig. 5I-J). This set of impairments of lysosomal transport in Alzheimer-related context are in full agreement with previous reports [START_REF] Gowrishankar | Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques[END_REF][START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF]. Indeed, using a mouse model of Alzheimer's disease, [START_REF] Gowrishankar | Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques[END_REF] evidenced axonal lysosome accumulations with local impairment in the retrograde axonal transport of lysosome precursors. Similarly, [START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF] found that misfolded Aß 42 impacts the endo-lysosomal pathway. They reported impairments in the uptake of proteins that use a dynamindependent endosomal mechanism and accumulation of lysosomes.

Intraneuronal transport of neurodegenerative-related molecular species

We were able to quantity the intraneuronal transport parameters for ⍺-syn and Aß pathogenic species (Figs 6-7). We found that cargoes loaded with ⍺-synF and R exhibit a more dynamical transport compared to those containing FND that are characterized by a larger pausing frequency and shorter run-length and pausing time (Fig. 6D-G). AßF and AßO-containing endosomes exhibit similar characteristics (Fig. 7D-G) with in addition an increase in velocity, not observed for ⍺-synF and R.

These results suggest that similar molecular mechanisms are at play in the transport of the two ⍺-syn fibrillar polymorphs, the AßF and AßO. However, the ≈7-fold larger fraction of AßF and AßO found in moving lysosomes compared to ⍺-syn assemblies (Fig. 8), also indicate differences in the molecular interactions of Aß assemblies with lysosomes. These results suggest either a cellular triage leading to differential transport of ⍺-syn and Aß 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 AßO (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., >10 5 compounds) as used in more complex human cellular models [START_REF] Park | A logical network-based drug-screening platform for Alzheimer's disease representing pathological features of human brain organoids[END_REF].

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 [START_REF] Encalada | Biophysical challenges to axonal transport: motorcargo deficiencies and neurodegeneration[END_REF]Goldstein, 2014, Lee et al., 2014;[START_REF] Guo | Axonal transport defects and neurodegeneration: Molecular mechanisms and therapeutic implications[END_REF]. Analysis of neuronal immuno-precipitates of -syn fibrillar assemblies and Aß 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 [START_REF] Li | Sequence of proteome profiles in preclinical and symptomatic Alzheimer's disease[END_REF]. 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 [START_REF] Li | Sequence of proteome profiles in preclinical and symptomatic Alzheimer's disease[END_REF] 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 ( -syn fibrillar polymorphs, A F 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 [START_REF] Brundin | Prion-like transmission of protein aggregates in neurodegenerative diseases[END_REF][START_REF] Hardy | The spread of neurodegenerative disease[END_REF][START_REF] Jucker | Propagation and spread of pathogenic protein assemblies in neurodegenerative diseases[END_REF]. 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.

  resp.) and an increase of the pausing frequency (Fig.4H; 28% and 15% for AßF and AßO resp.), with effects more pronounced for AßF than for AßO. Interestingly, the same trends of changes were also observed at the lower AßF and AßO concentration of 0.2 μM (Fig.4-1C-F). Let us finally point out that for AßF, the important changes of some transport parameters overall combine in an only very slight decrease in trajectory length as shown in Fig 4C, which makes the detailed quantitative analysis performed all the more useful. These results are summarized in Fig. 2-2A.

  -1A) in agreement with Fig.4Bof[START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF]. 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[START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF]) at 72 h time point, who reported a ≈1.5 times larger amount of internalized AßO compared to AßF(sonicated). Our observations differ from[START_REF] Marshall | Misfolded amyloid-beta-42 impairs the endosomal-lysosomal pathway[END_REF]), 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)

Figure 1 .

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

Figure 2 .

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

Figure 3 .

 3 Figure 3. Effect of ⍺-synF or ⍺-synR on the mobility of LysoTracker-labelled lysosomes and their transport in mouse cortical neurons at DIC21. 24 h exposure to

Figure 4 .

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

Figure 5 .

 5 Figure 5. Effect of AßF and AßO on the mobility of LysoTracker-labelled lysosomes and their transport in mouse cortical neurons at DIC21. 24 h exposure to AßF and AßO at 1 μM concentration, compared to nothing added control. A, Number of lysosomes detected per field-of-view of 40 x 80 μm size during 2 mins of observation.

Figure 6 .

 6 Figure 6. Intraneuronal transport of ATTO 488-labeled ⍺-synF and R in mouse cortical neurons at DIC21. DIC20 cortical neurons were exposed to ⍺-synF and R during 24 h, at concentration of 0.2 M. A-B, Examples of ⍺-synF and R, and FND trajectories (in the presence of ⍺-synF and R). Scale bar: 10 m. C, Length of ⍺-synF, ⍺-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.

Figure 7 .

 7 Figure 7. Intraneuronal transport of ATTO 488-labeled AßF and AßO in mouse cortical neurons at DIC21. DIC20 cortical neurons were exposed to AßF and AßO (1 M) during 24 h. A-B, Examples of trajectories. Scale bar: 10 m. C, Length of AßF, AßO 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

Figure 8 .

 8 Figure 8. Colocalized events between moving neurodegenerative disease-related molecular species and moving lysosomes at different time points. A, -synF and -synR were incubated for 24 and 48 h, at concentration of 0.2 μM. B, AßF and AßO were incubated for 24 and 48 h, at concentration of 1 μM. The number inside the donut plot represents the percentage of moving -syn or Aß 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.

  Figures

  Figure 2

Figure 2 - 1 .

 21 Figure 8

Figure 2 - 2 .

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

Figure 4 - 1 .

 41 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).

Figure 5 - 1 .

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

Figure 5 - 2 .

 52 Figure 5-2. Effect of ⍺-synF on the mobility of Magic Red-labelled lysosomes and their transport in mouse cortical neurons at DIC21, after 24 h exposure to ⍺-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 m. E-H, Comparison of four

Figure 5 - 3 .

 53 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 m. (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 .

 71 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 exposure 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 .

 81 Figure 8-1. Fraction of protein assemblies, ⍺-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 ⍺-synF and Aß cases.

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.