Rania Znaidi

Olivia Massiani-Beaudoin

Philippe Mailly

Héloïse Monnet

Rajiv L Joshi
email: rajiv.joshi@college-de-france.fr

Julia Fuchs
email: julia.fuchs@college-de-france.fr

Nuclear translocation of LINE-1 encoded ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in neurons

Keywords: Retrotransposons, LINE-1, ORF1p, nuclear envelope, nuclear pore complex, nucleo-cytoplasmic transport, neurodegeneration

Introduction

Repetitive sequences derived from transposable elements (TEs) represent 45% of the human genome, including non-LTR and LTR retroelements. Non-LTR retrotransposons of the Long Interspersed Element-1 (LINE-1) family have been the most active TEs in mammalian genomes and LINE-1-derived sequences represent almost 21% of the human genome which carries nearly 500000 LINE-1 copies [START_REF] Lander | Initial sequencing and analysis of the human genome[END_REF] . Although most of these LINE-1 copies are remnants of past retrotransposition events and are truncated or mutated, the reference human genome still carries 146 full-length LINE-1 copies which can be autonomously active and competent for retrotransposition [START_REF] Penzkofer | L1Base 2: more retrotransposition-active LINE-1s, more mammalian genomes[END_REF] . Full-length LINE-1 sense strand codes for two proteins, namely ORF1p and ORF2p, which are required for retrotransposition. ORF1p is an RNA binding protein and

Drug treatment

For LUHMES treatment with Remodelin drug (a gift from Dr. Raphaël Rodriguez), differentiated neurons at day 4 of differentiation, were incubated in fresh culture medium containing Remodelin (10 µM) or DMSO for 24 h and then subjected to oxidative stress using Arsenite (50 µM for 1 h at 37°C). For Stavudine treatment differentiated neurons (at day 4 of differentiation) were incubated in fresh culture medium containing Stavudine (10 µM) or NaCl for 24 h. The next day, cells were subjected to oxidative stress using Arsenite (50 µM for 1 h at 37°C).

Transfection methods

Adherent electroporation (Nucleofection): LUHMES cells were seeded at a density of 150 000 cells/well into 24-well plates already containing cover-slips pre-coated with poly-L-ornithine and fibronectine and were differentiated as described in Cell culture section. After 3 days of differentiation, cell culture medium was replaced by pre-warmed fresh medium and incubated for 1 h at 37°C prior to transfection. LUHMES cells were transfected by Nucleofection using the AMAXA 4D-Nucleofector® Y Unit (Lonza) which enables the direct Nucleofection (by electroporation) of cells in adherence in 24-well culture plates using the AD1 4D-Nucleofector™ Y Kit (Lonza, V4YP-1A24) for neuronal cells electroporation. The ER-100 program was the most appropriate to LUHMES cells in terms of transfection efficiency and viability and was used for all subsequent transfections. One Nucleofection sample contained 150 000 cells/well, 10 µg plasmid DNA (pcDNA3-ORF1-HA, a gift from Dr. John and Dr. Y. Ariumi) or 10 µg pmaxGFP vector and 350 µl AD1 4D-Nucleofector Y solution. The 24-well dipping electrode array (provided in the kit) was inserted into the plate and cells were transfected by using the ER-100 Nucleofector program. After nucleofection, AD1 solution was removed and the cells were immediately washed once with fresh medium and incubated in humidified 37°C / 5% CO2 incubator. Media was not changed until analysis at day 5 of differentiation (48 h after transfection). Transfection efficiency (48 h after transfection) was monitored by fluorescence microscopy.

Protein extraction

After aspirating media, cells were washed one time with 1X PBS. 200 to 500 µl of 1X RIPA lysis buffer (50 mM Tris-HCL pH 8, 150 mM Nacl, 0,5% Sodium deoxycholate, 1% NP-40, 0,1% SDS) with 1X protease inhibitor (Sigma, 11836153001) was added to each well in the plate. Cells were scraped and lysates were transferred to a sterile microtube. The lysates were incubated on ice for 30 min and sonicated using the Bioruptor UCD-200 for 15 min (30s on/30s off) on ice at high power to shear contaminating DNA. After collecting the supernatant (avoiding the pellet) into new microtubes, protein concentration was determined using Pierce TM BCA Protein Assay Kit. Aliquot of lysates were stored at -20ºC avoiding multiple freeze/thaw cycles. Proteins were taken in 1X Laemmli buffer (250 mM Tris pH 6,8, 10% sodium dodecyl sulfate, 1,25% bromophenol blue, 5% β-mercaptoethanol, 50% glycerol) containing DTT, boiled 10 minutes at 95°C and stored at -20°C until analyzed.

Western blot analysis

A 1.5 mm NuPAGE 4-12% Bis-Tris-Gel (Invitrogen™M) polyacrylamide gel was used for Western blots. 10 ug of protein were loaded per well. Gel migrated in NuPAGE™ MES SDS Running Buffer (1X) (Invitrogen™M) for 55 minutes at 200 V. Gels were transferred into a methanol activated PVDF membrane (Immobilon) in a transfer buffer (Tris 25 mM, pH 8,3 and glycine 192 mM) during 1 h at 400 mA at 4°C. For blocking, 5% milk in TBST (0,2% Tween 20, 150 mM NaCI, 10 mM Tris pH:8) was used during 1 h at room temperature. The membranes were then incubated overnight with primary antibody anti-ORF1p 1:500 (Abcam, ab245249).

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The membranes were incubated with anti-actin peroxidase antibody 1:20000 (Sigma, A3854) diluted in 5% milk in TBST, 1 h at room temperature on a shaker. The membranes were washed in TBST (1X) and incubated with secondary antibody including anti-rabbit HRP 1:2000 (Cell Signaling, 7074S) and anti-mouse HRP 1:2000 (Cell Signaling, 7076S) for 1 h at room temperature. After washing in TBST (1X), membranes were revealed using Clarity Western ECL Substrate (Bio Rad, #1705060) or Maxi Clarity Western ECL Substrate (BioRad, #1705062) in a LAS-4000 Fujifilm system.

Immunoprecipitation

For immunoprecipitation (IP) we used ORF1p (Millipore, MABC 1152) and IgG mouse (Thermofisher, #31903) antibodies. The antibodies were coupled to magnetic beads using the Dynabeads® Antibody Coupling Kit (Invitogen, 14311D) according to the manufacturer's recommendations. We used 8 µg of antibody for 1 mg of beads. The appropriate volume of buffer was added to the coupled beads to achieve a final concentration of 10 mg/ml. LUHMES cells were plated at a density of 20x10 6 cells in 2 T75 flasks and grown in differentiation medium for 5 days. One flask was treated with 50 µM Arsenite for 1 h. Control and treated cells were washed with 1X PBS and harvested using 1 ml of lysis buffer (10 mM Tris HCl pH 8, 150 mM NaCl, NP40 0.5% v/v, protease inhibitor 10 µl/ml) per T75 flask.

Samples were sonicated for 15 min at 4°C, then centrifuged at 1200 rpm for 15 min at 4°C. The supernatants obtained were transferred to a new microcentrifuge tube and then separated into 3 tubes. One tube containing 50 µl of supernatant (1/10th of the test samples) was stored at 4°C until the samples were analyzed. The other two tubes containing 445 µl of the remaining supernatants were used for IP. Mouse IgG beads (75 µl) were added to one tube and ORF1p antibody beads (75 µl) to the other tube. Each of these two tubes was then diluted to 1.5 ml with buffer (10 mM Tris HCl pH8, 150 mM NaCl, 10 µl/ml protease inhibitor) to dilute the NP40. The samples were then incubated overnight on a wheel at 4°C. Samples were then washed 3 times with 1 ml buffer (10 mM Tris HCl pH 8, 150 mM NaCl, 10 µl/ml protease inhibitor) using a magnet and then resuspended in 50 µl of the same buffer. The samples were boiled in Laemmli buffer (95°C, 10 min) and 20 µl of each sample were deposited on a 4-12% Nupage gel (Invitrogen, NP0336). Migration was carried out in MES SDS running buffer (Invitrogen, NP0002) for 45 min at 200 V. The gel was then transferred onto a PVDF membrane activated with methanol (Sigma, Immobilon-P 2659478) in glycine buffer (25 mM Tris, 200 mM glycine pH 8,3) for 1 h at 400 mA. The membranes were revealed using the primary antibodies anti-ORF1p rabbit 1:500 (Abcam, ab245249), anti-lamin B1 rabbit 1:1000 (Abcam, ab16048) or anti-KPNB1 rabbit 1:1000 (Cell Signaling, #51186), the secondary anti-rabbit HRP 1:2000 (Ozyme, 7074S) with the Maxi Clarity Western ECL Substrate kit (BioRad, #1705062). Analysis of immunoblotting images was performed manually using Fiji software.

RT-qPCR

Total RNA from 5x10 5 lysed cells was extracted using RNeasy Plus Micro Kit (Qiagen, 74034) according to the manufacturer's instructions. The removal of contaminating genomic DNA (gDNA) was assessed by using the ezDNase kit (Invitrogen TM , 11766051). After gDNA elimination, RNA (300 ng) was reverse transcribed using the All-In-One 5X RT MasterMix kit (Abcam, G592) for first-strand cDNA synthesis. The newly synthesized first-strand cDNA is ready for immediate real-time PCR analysis.

Proximity Ligation Assay (PLA)

The detection of the interactions between ORF1p and Lamin B1, NUP153 and KPNB1 were analyzed using proximity ligation Assay (PLA) method using Duolink® In Situ Red Starter Kit Mouse/Rabbit (Sigma, DUO92101). Cells were treated according to the manufacturer's instructions. After PLA, cells are mounted with a Fluoromount (Invitrogen™, 15586276).

Fluorescence images were acquired using a Spinning Disk W1 Yokogawa imaging system.

Immunofluorescence

Immunostaining experiments were assessed at 1 h after Arsenite treatment. Stressed and control differentiated LUHMES cells cultured in 24-well plates (150 000 cells/well) were fixed with 4% paraformaldehyde (PFA) for 10 min. Immunofluorescence were performed overnight at 4°C after a 20 min permeabilization in 0,2% Triton X100 and incubation in blocking buffer (10 % bovine serum albumin BSA) for 1 h with the following antibodies: ORF1p 1:500 (Millipore, MABC1152 clone 4H1), TH 1:500 (Millipore, AB9702), Tuj1 1:1000 (Abcam, ab18207), Lamin B1 1:1000 (Abcam, ab16048), KPNB1 1:500 (GTX, 133733), NUP153 1:1000 (Novus biological, NB100-93329), H4K20me3 1:1000 (Abcam, ab9053), HP1 1:1000 (Cell Signaling, #2623S), TDP-43 1:600 (Proteintech, 107-82-2AP), HA 1:1000 (Roche, 11867423001), RANGAP1 1:500 (Santa Cruz, sc-28322), ssDNA 1:500 (ENZO, (F7-26) ALX 804-192) diluted in the blocking buffer, overnight at 4°C. For ssDNA immunostaining, cells were fixed with PFA, followed by 24 h methanol incubation at -20°C, then incubated at 37°C for 4 h with RNaseA. Fluorescence secondary antibodies: Alexa Fluor 555, Alexa Fluor 488, Alexa Fluor 642, 1:2000 (ThermoFisher Scientific) were added for 1 h at room temperature. Nuclei were stained with Hoechst 1:2000 (Invitrogen, H3570). We used Fluoromount (Invitrogen™, 15586276). Cells were imaged by Spinning Disk W1 Yokogawa and STED Abberior Facility line imaging systems.

Quantification and image analysis

The quantification of ORF1p fluorescence was performed with a custom-written plugin (https://github.com/orion-cirb/Julia_Nucleus_Analysis) developed for the Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] , using Bio-Formats [START_REF] Linkert | Metadata matters: access to image data in the real world[END_REF] and 3D Image Suite 26 libraries. Hoechst channel is downscaled by a factor 2, before detecting nuclei with the 2D-stitched version of Cellpose 27 (model = 'cyto2', diameter = 100, flow threshold = 0.4, cell probability threshold = 0.0, stitching threshold = 0.5).

Segmented image is rescaled to original size and obtained 3D nuclei are filtered by volume to avoid false positive detections. Cells in ORF1p channel are detected in the same manner as nuclei. Then, each cell is associated with a nucleus having at least half of its volume in contact with it. Cells without any associated nucleus are filtered out. For each cell, ORF1p signal is analyzed in the following cellular compartments: cytoplasm, nucleus, outer and inner nuclear membranes and nucleoplasm (Fig. 2C,E). Compartmentalization is obtained using combinations of morphological operations (dilation, erosion and subtraction) of the cell and nucleus masks.

In each compartment, the integrated intensity of ORF1p is measured and normalized to ensure comparability across images. Normalization is done by subtracting the image background noise, estimated as the median intensity value of the minimum intensity Zprojection of the ORF1p channel. ORF1p foci are detected with Stardist 2D [START_REF] Schmidt | Cell Detection with Star-Convex Polygons[END_REF] To quantify nuclear deformation, another custom plugin (https://github.com/orioncirb/HA_ORF1P_Prot/) was developed for the Fiji software, that uses the same libraries listed above, CLIJ [START_REF] Haase | CLIJ: GPU-accelerated image processing for everyone[END_REF] and Find Focused Slices (https://sites.google.com/site/qingzongtseng/findfocus?authuser=0). In brief, best focussed slice in the stack is selected. Nuclei are detected on this slice with Cellpose 2D (model = 'cyto2', diameter = 120, flow threshold = 0.4, cell probability threshold = 0.0), filtered by area and their circularity is computed. For each nucleus, ORF1p integrated intensity is analyzed in the following compartments: nucleus, outer and inner nuclear membranes and nucleoplasm. Compartments are obtained with the same combinations of morphological operations than previously described.

In case a HA-ORF1p channel is provided, cells are detected on the best focussed slice with Cellpose 2D (model = 'cyto2', diameter = 200, flow threshold = 0.4, cell probability threshold = 0.0) and filtered by area. Nuclei having at least one pixel in contact with a HA-ORF1p cell are considered as HA-ORF1p+, other nuclei as HA-ORF1p-. The intensity quantification of other markers than ORF1p was also performed with this plugin.

Statistical Analysis

Data were obtained from independent experiments. The number of cells analyzed in each experiment is described in corresponding figure legends. Data analysis was performed using GraphPad Prism 8. Shapiro Wilk normality tests were performed prior to the statistical test. Two-tailed Student's t-test or One-way ANOVA with Tukey's multiple comparisons test were used to determine statistical significance. Data are expressed as mean ± standard error of the mean (SEM). The significant threshold was defined as p<0.5 and depicted as follows on all graphs: *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Results

Stress-induced activation of LINE-1 in human post-mitotic dopaminergic neurons

LUHMES cells represent a robust model to investigate the molecular and cellular mechanisms leading to neurodegeneration [START_REF] Zhang | Cell-based assays for Parkinson's disease using differentiated human LUHMES cells[END_REF][START_REF] Harris | 3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects[END_REF][START_REF] Harischandra | Enhanced differentiation of human dopaminergic neuronal cell model for preclinical translational research in Parkinson's disease[END_REF] . LUHMES cells were differentiated into post-mitotic neurons according to the experimental paradigm [START_REF] Lotharius | Effect of Mutant α-Synuclein on Dopamine Homeostasis in a New Human Mesencephalic Cell Line[END_REF][START_REF] Lotharius | Progressive Degeneration of Human Mesencephalic Neuron-Derived Cells Triggered by Dopamine-Dependent Oxidative Stress Is Dependent on the Mixed-Lineage Kinase Pathway[END_REF][START_REF] Scholz | Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line: LUHMES as widely applicable neuronal model system[END_REF] displayed in Fig. 1A and Suppl. Fig. 1A. These cells acquire a mature dopaminergic neuronal phenotype as revealed by immunostaining for the neuronal marker Tuj1 (β-III tubulin) and the dopaminergic marker TH (Tyrosine hydroxylase) at day 5 of differentiation as shown in Figure 1B and quantified in Supplementary Figure 1B. The dopaminergic phenotype of these cells was further confirmed by analyzing TH protein expression by Western blot at different days of differentiation (Suppl. Fig. 1C).

Surprisingly, but as previously observed in mouse dopaminergic neurons [START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF] , full-length LINE-1 elements are expressed at steady-state in human dopaminergic neurons as revealed by immunostaining for the LINE-1 ORF1p protein (Fig. 1B). Human ORF1p, as mouse ORF1p, shows a predominant cytoplasmic expression pattern. Oxidative stress is a common feature of aging [START_REF] Junqueira | Aging and oxidative stress[END_REF][START_REF] Romano | Oxidative stress and aging[END_REF][START_REF] Liguori | Oxidative stress, aging, and diseases[END_REF] , cancer [START_REF] Hayes | Oxidative Stress in Cancer[END_REF] and NDs [START_REF] Emerit | Neurodegenerative diseases and oxidative stress[END_REF][START_REF] Chen | Oxidative stress in neurodegenerative diseases[END_REF][START_REF] Kim | The Role of Oxidative Stress in Neurodegenerative Diseases[END_REF][START_REF] Fischer | Interrelation of Oxidative Stress and Inflammation in Neurodegenerative Disease: Role of TNF[END_REF] and has been associated with LINE-1 activation [START_REF] Giorgi | LINE-1 retrotransposition in human neuroblastoma cells is affected by oxidative stress[END_REF][START_REF] Wongpaiboonwattana | Oxidative Stress Induces Hypomethylation of LINE-1 and Hypermethylation of the RUNX3 Promoter in a Bladder Cancer Cell Line[END_REF][START_REF] Whongsiri | Oxidative stress and LINE-1 reactivation in bladder cancer are epigenetically linked through active chromatin formation[END_REF] . To generate a human neuronal model of endogenous LINE-1 activation, we induced oxidative stress in differentiated dopaminergic neurons with low-dose arsenite (NaAsO2 ; 50 µM) for 1 hour at day 5 of differentiation as described in Figure 1A. This treatment did not affect the neuronal and dopaminergic phenotypes as shown by Tuj1 and TH immunofluorescence quantification (Suppl. Fig. 1D,E) and triggered no apparent cell death. Oxidative stress increased the expression of LINE-1 in human neurons as revealed by Western blot and immunofluorescence analysis of LINE-1 ORF1p protein (Fig. 1C,D). LINE-1 RNA quantification by RT-qPCR using several primers targeting different regions of the LINE-1 sequence also showed an up-regulation of LINE-1 transcripts (Suppl. Fig. 1F). In order to test LINE-1 encoded protein activity, we analyzed two known consequences of ORF2p activity (for which no effective antibodies are available). Increased LINE-1 ORF2p has been previously reported to induce DNA damage due to the endonuclease activity [START_REF] Gasior | The Human LINE-1 Retrotransposon Creates DNA Double-strand Breaks[END_REF][START_REF] Belgnaoui | Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells[END_REF][START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF] . Upon oxidative stress, differentiated human dopaminergic neurons displayed increased DNA damage as quantified by an increase in γH2AX (Phosphorylated histone H2AX)-positive foci, which decreased in the presence of Stavudine, an inhibitor of the reverse transcriptase of LINE-1 ORF2p (Fig. 1E).

The reverse transcriptase activity of LINE-1 ORF2p has also been reported to generate the accumulation of single-stranded DNA (ssDNA) in the cytosol leading to inflammation [START_REF] Simon | LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation[END_REF][START_REF] Thomas | Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation[END_REF] . We also observed an increase of ssDNA foci volume and intensity in the cytosol of stressed neurons (Suppl. Fig. 2A). Full-length LINE-1 and its encoded proteins are thus expressed at steady-state in human dopaminergic neurons, are inducible by oxidative stress and functionally active. This in vitro model therefore represents a valuable tool to investigate cellular effects of LINE-1 activation mediated by the encoded proteins.

ORF1p localizes to the nuclear envelope and translocates into the nucleus upon oxidative stress

We next investigated the precise cellular localization and quantity of ORF1p upon stress using confocal imaging (Fig. 2A). ORF1p was predominantly cytoplasmic both in steady-state as well as in stress conditions and showed both, diffuse and dot-like patterns. Surprisingly and against the current assumptions 45 , we observed the presence of ORF1p in the nuclear compartment and an accumulation of ORF1p at the nuclear periphery both at the outer and the inner NE as distinguishable by Hoechst DNA staining (Fig. 2A,B). We further quantified ORF1p intensities using an automated deep-learning based quantification script developed in-house as schematized (Fig. 2C). This script allows for the quantification of immunofluorescence intensities, dot numbers and volumes in pre-defined cellular compartments including the nucleus and the cytosol. The increase in staining intensities was accompanied by a rearrangement of staining patterns as revealed by an increase in ORF1p-positive dot numbers and volumes, both in the cytoplasm and the nucleus (Fig. 2D), indicating a possible condensation of LINE-1 ORF1p in stress conditions. As ORF1p appeared to accumulate along the NE, the script was further applied to analyze ORF1p intensity in more detailed compartments including the outer NE, the inner NE and the nucleoplasm (not including the inner NE) (Fig. 2E). The quantification showed a significant increase of ORF1p in both the inner and outer NE and in the nucleoplasm (Fig. 2F).

In summary, ORF1p immunostainings using an unbiased image quantification method revealed increased LINE-1 ORF1p levels in the cytoplasm, in the nucleus, and in the inner and outer NE, indicating a general increase of ORF1p, the accumulation of ORF1p at the NE, a rearrangement of ORF1p in a dot-like manner particularly in the cytoplasm and a significant nuclear translocation upon stress.

ORF1p targets nuclear envelope components upon oxidative stress

To confirm ORF1p localization to the inner NE, we performed co-immunostainings for ORF1p and Lamin B1, a nuclear lamina protein which decorates the inner NE and plays a key role in the maintenance of nuclear architecture. These experiments revealed the colocalization of ORF1p and Lamin B1 at the inner NE which increased upon stress. This colocalization is particularly visible at the level of lamina invaginations (Fig. 3A). Co-immunoprecipitation experiments confirmed the interaction between ORF1p and Lamin B1 under stress conditions (Fig. 3B). Direct binding of ORF1p to Lamin B1 was further examined by proximity ligation assay (PLA). The results obtained indicate a direct interaction between ORF1p and Lamin B1 at the nuclear periphery upon stress (Fig. 3C) in line with the colocalization signals observed by co-immunostainings (Fig. 1A). However, direct interaction of ORF1p and Lamin B1 is also detected in the nucleoplasm of neurons and PLA signal quantification confirmed an increase of the number of dots (= direct interaction events) at the inner NE but also in the nucleoplasmic compartment (Fig. 3D). This suggests a possible scenario in which the stress-induced nuclear translocation of ORF1p and direct ORF1p binding to Lamin B1 induces Lamin B1 mislocalization from the inner NE to the nucleoplasm.

Previous studies have shown that ORF1p interacts with importin beta family members (i.e, KPNB1) [START_REF] Freeman | Identification of charged amino acids required for nuclear localization of human L1 ORF1 protein[END_REF] , which could mediate its nuclear translocation using nuclear pore complexes (NPCs). To examine whether ORF1p nuclear translocation observed in human neurons upon oxidative stress (Fig. 2B) might depend on KPNB1 mediated import, we performed coimmunostaining of ORF1p with the importin KPNB1 or NUP153 (a nucleoporin of the nuclear basket), a core NPC protein shown to interact with KPNB1 [START_REF] Kapinos | Karyopherins regulate nuclear pore complex barrier and transport function[END_REF] . The results shown in Figure 3E indicate the colocalization of ORF1p with KPNB1 and NUP153 at the NE suggesting that nuclear translocation of ORF1p in human neurons upon oxidative stress might indeed be mediated by these proteins. Our results are in line with previous reports describing that KPNB1 may be involved in active nuclear import of LINE-1 ORF1p [START_REF] Idica | MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by down-regulating the nuclear import factor TNPO1[END_REF] . In addition, several cytoplasmic ORF1p-positive foci colocalize with KPNB1 (Fig. 3E) suggesting possible sequestration of this importin with ORF1p. Direct interaction of ORF1p with KPNB1 and NUP153 was further confirmed by PLA (Fig. 3F). PLA signal of ORF1p and KPNB1 interaction is not affected by oxidative stress (Fig. 3G), whereas the direct interaction between ORF1p and NUP153 is clearly increased in stress condition (Fig. 3H). These results suggest that ORF1p directly interacts with components of the inner NE and the nucleo-cytoplasmic transport (NCT) machinery and might mislocalize these proteins interfering with their physiological functions in nuclear architecture and NCT function. This let us to investigate whether ORF1p localization to the NE and direct protein-protein interaction with Lamin B1, KPNB1 and NUP153 might affect NE morphology and/or NCT efficiency.

Nuclear ORF1p accumulation correlates with nuclear envelope alterations

Lamin B1-based staining of the inner nuclear lamina of human dopaminergic neurons revealed various NE dysmorphologies including nuclear invaginations and blebbing which appeared upon oxidative stress (exemplified in Fig. 4A). It is noteworthy that nearly 70% of stressed cells presented such deformations (Fig. 4B). Co-immunostaining for ORF1p and Lamin B1 revealed the accumulation of ORF1p at the level of NE invaginations and blubs, which is clearly observable in STED acquired 3D images and 3D reconstituted images (Fig. 4A). Nuclear dysmorphology was accompanied by increased Lamin B1 levels at the inner NE but even more so in the nucleoplasm of stressed cells (Fig. 4C), again suggesting a mislocalization or relocalization of Lamin B1 possibly as a result of direct protein interactions with ORF1p. The degree of NE distortion (expressed as a circularity index) was analyzed using an automated script (detailed in the Materiel and Methods section) which measures nuclear circularity using Lamin B1 staining as a mask. This analysis showed a significant decrease of nuclear circularity in stressed cells (Fig. 4D). Importantly, the degree of nuclear distortion (the lower the index, the higher the distortion) was significantly correlated with the intensity of nuclear ORF1p staining (thus nuclear ORF1p quantity) both in the nucleoplasm and in the inner NE (Fig. 4E). This correlation strongly suggests that nuclear ORF1p translocation and accumulation in the nucleoplasm and at the inner NE, at least in part, is functionally involved in the occurrence of nuclear dysmorphologies observed in neurons upon oxidative stress. It is noteworthy that ORF2p activity inhibitor (Stavudine) which prevented ORF2p induced DNA strand breaks (Fig. 1E) did not have any effect on nuclear circularity, or Lamin B1 delocalization, nor on ORF1p nuclear and cytoplasmic levels (Suppl. Fig. 3A-C). This suggests that the effect observed is not due to ORF2p activity.

Since nuclear lamina plays an important role in anchoring heterochromatin at the inner NE (= nuclear lamina), we examined whether the observed alteration of the nuclear lamina is accompanied by heterochromatin disorganization by immunostaining for the heterochromatin markers HP1 (heterochromatin marker protein-1) and the repressive histone mark H4K20me3 (histone H4, trimethylated on lysine 20) in control and stressed cells. HP1 was significantly depleted from the inner NE (Fig. 4F) and H4K20me3 levels decreased in stress conditions (Fig. 4G) suggesting possible decondensation or detachment of heterochromatin from the inner NE.

Nuclear envelope targeting by ORF1p is accompanied by nucleo-cytoplasmic transport defects

In view of the interaction of ORF1p with KPNB1 and NUP153 reported above, we examined whether the accumulation of ORF1p at the NE could lead to NCT deficiencies.

Immunostainings and subsequent quantification of intensity, distribution and localization of KPNB1 showed an accumulation of KPNB1 in the cytoplasm, as revealed by an increase in the intensity of KPNB1. We also observed a change in KPNB1 distribution, as revealed by an increase in the number of counted dots and their volume in the cytoplasm (Fig. 5A) indicating a possible alteration of its function. To examine whether these changes in KPNB1 upon stress were associated with NCT defects, we first analyzed the localization of a major nucleocytoplasmic transport protein, RANgap1, by immunostaining. The GTPase activity of this protein is involved in maintaining the RAN gradient required for NCT and its delocalization is associated with NCT defects [START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF] to a pathological gain-of-function in the cytoplasm and loss-of-function in the nucleus [START_REF] Neumann | Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis[END_REF][START_REF] Aizawa | Impaired Nucleoporins Are Present in Sporadic Amyotrophic Lateral Sclerosis Motor Neurons that Exhibit Mislocalization of the 43-kDa TAR DNA-Binding Protein[END_REF] contributing to cellular dysregulation culminating in neurodegeneration. We therefore performed immunostainings for TDP-43 which revealed the presence of TDP-43-positive foci in the cytoplasm under stress conditions which were absent in non-stressed controls.

Quantification showed that the intensity as well as the number and volume of TDP-43 positive dots were increased in the cytoplasm in stressed neurons (Fig. 5C). The quantification of the nucleoporin NUP153 showed an abnormal intranuclear and cytoplasmic accumulation of NUP153 in stress conditions (Fig. 5D).

Attempts to establish a causal link between the increase of LINE-1 ORF1p and NE alterations and NCT defects using siRNA were technically not successful. We therefore performed LINE-1 ORF1p gain of function experiments to examine whether increased ORF1p (and only ORF1p) could lead to the above-described effects in the absence of stress.

ORF1p overexpression leads to nuclear envelope dysmorphology and nucleo-cytoplasmic transport defects in the absence of oxidative stress

Dopaminergic neurons were nucleofected at day 3 of differentiation with a plasmid encoding either ORF1-HA or GFP control and analyzed 48 h later. Co-immunostainings for HA and Lamin B1 showed the presence of HA-ORF1 in the nucleus and at the inner NE, colocalizing with Lamin B1 (Fig. 6A). HA-ORF1 transfected cells displayed pronounced NE deformations as revealed by Lamin B1 staining (Fig. 6A), decreased circularity and increased nucleoplasmic Lamin B1 levels compared to GFP expressing cells (Fig. 6B). Interestingly, nuclear circularity was highly correlated with the nuclear intensity of HA-ORF1 in transfected cells (Fig. 6B), strongly suggesting a causal link between ORF1p nuclear localization and NE dysmorphology.

Neurons transfected with HA-ORF1 also presented increased levels of RANgap1 in the cytoplasm and TDP-43 in both the cytoplasm and nucleus compared to GFP expressing cells (Fig. 6C-F). These results indicate that overexpression of ORF1p, in the absence of stress, is sufficient to induce NCT defects and NE dysmorphology in human dopaminergic neurons.

Rescue of nuclear envelope dysmorphology under stress conditions by Remodelin

NE morphology is disrupted during aging [START_REF] Scaffidi | Lamin A-Dependent Nuclear Defects in Human Aging[END_REF] and in premature aging syndromes caused by laminopathies [START_REF] Worman | Nuclear lamins and laminopathies[END_REF][START_REF] Stiekema | Structural and Mechanical Aberrations of the Nuclear Lamina in Disease[END_REF] including progeria syndromes [START_REF] Sandre-Giovannoli | Lamin A Truncation in Hutchinson-Gilford Progeria[END_REF] . In Hutchinson-Gilford progeria syndrome (HGPS), nuclear integrity was shown to be restored by the small molecule "Remodelin", an inhibitor of the acetylase-transferase protein NAT10 [START_REF] Larrieu | Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway[END_REF] but whether this molecule is efficient in restoring misshapen nuclear morphology in cell types other than fibroblasts or epithelial cells was not known. We therefore examined whether NE dysmorphology induced by ORF1p under oxidative stress in human neurons could be restored by Remodelin. NE deformations (i.e invaginations) were clearly restored in cells pre-treated with Remodelin for 24 h (Fig. 7A,B).

Quantification showed that the decrease of nuclear circularity following arsenite treatment was restored by Remodelin (Fig. 7B), which was paralleled by a reduction of nucleoplasmic Lamin B1 levels quantified in Figure 7B. In order to investigate whether Remodelin treatment had an influence on ORF1p levels, we performed ORF1p immunostainings on cells treated or not with Remodelin. Arsenite-induced nuclear ORF1p increase was completely normalized in the presence of Remodelin, both at the inner NE and in the nucleoplasm (Fig. 7C) Interestingly, in these conditions cytoplasmic ORF1p levels remained unchanged at an expected higher level than non-stressed controls. Since Remodelin has been reported to restore a "juvenile" chromatin state in Progeria models [START_REF] Larrieu | Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway[END_REF][START_REF] Larrieu | Chemical Inhibition of NAT10 Corrects Defects of Laminopathic Cells[END_REF] , we examined H4K20me3, known to repress LINE-1 elements 58,59 . As shown in Figure 7A, H4K20me3 immunostaining was significantly decreased following stress and restored with Remodelin treatment as confirmed by quantification of H4K20me3 intensity in the nucleus (Fig. 7D). This suggests that the decrease of nuclear ORF1p upon Remodelin treatment in stressed cells might induce indirect blockage of nuclear ORF1p translocation. Alternatively, this decrease could also be due to a transcriptional inhibition of LINE-1 or a consequence of deposition of H4K20me3 at LINE-1 loci.

Discussion

In recent years, TEs have emerged as possible new pathogenic players in NDs [START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF][START_REF] Li | Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders[END_REF][START_REF] Sun | Pathogenic tauinduced piRNA depletion promotes neuronal death through transposable element dysregulation in neurodegenerative tauopathies[END_REF][START_REF] Rekaik | Engrailed Homeoprotein Protects Mesencephalic Dopaminergic Neurons from Oxidative Stress[END_REF][START_REF] Peze-Heidsieck | Retrotransposons as a Source of DNA Damage in Neurodegeneration[END_REF][START_REF] Casale | Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease[END_REF][START_REF] Tam | Postmortem Cortex Samples Identify Distinct Molecular Subtypes of ALS: Retrotransposon Activation, Oxidative Stress, and Activated Glia[END_REF][START_REF] Ravel-Godreuil | Transposable elements as new players in neurodegenerative diseases[END_REF] . However, the mechanisms through which LINE-1 might contribute to the pathogenesis of NDs still remain poorly understood potentially implicating cis-genetic or epigenetic, LINE-1 RNA or LINE-1 protein-mediated effects. Concerning the latter, the study of potential pathogenic effects of LINE-1 ORF2p has received most attention due to its enzymatic activities as an endonuclease and reverse transcriptase while ORF1p has widely been reduced to an RNA chaperone with cis-preference for its own RNA and a necessary component of the LINE-1 ribonucleoprotein particle (RNP) for retrotransposition. Previous work suggests that ORF2p is not only a source of genomic instability [START_REF] Gasior | The Human LINE-1 Retrotransposon Creates DNA Double-strand Breaks[END_REF][START_REF] Belgnaoui | Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells[END_REF][START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF][START_REF] Simon | LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation[END_REF][START_REF] Peze-Heidsieck | Retrotransposons as a Source of DNA Damage in Neurodegeneration[END_REF] , but can also trigger the innate immune response through reverse transcription of RNA generating cytoplasmic ssDNA [START_REF] Simon | LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation[END_REF][START_REF] De Cecco | Author Correction: L1 drives IFN in senescent cells and promotes age-associated inflammation[END_REF][START_REF] Thomas | Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation[END_REF] . While ORF2p reverse transcriptase activity can be targeted by inhibitors (nucleoside reverse transcriptase inhibitors, NRTIs, i.e. Stavudine), the analysis of ORF2p protein expression and localization remains challenging due to a lack of efficient antibodies and/or its lesser abundance compared to ORF1p (1 ORF2p for 240 ORF1p molecules per LINE-1 RNP [START_REF] Dai | Expression and detection of LINE-1 ORF-encoded proteins[END_REF] . In contrast, no inhibitor has been identified to target ORF1p activity, but effective antibodies are available to explore its expression and subcellular localization in pathological situations. However, a better understanding of the implication of ORF1p in the LINE-1 life cycle and beyond is interesting as accumulating data suggests that full-length LINE-1 are expressed, predominantly in epithelial cancers [START_REF] Rangasamy | Activation of LINE-1 Retrotransposon Increases the Risk of Epithelial-Mesenchymal Transition and Metastasis in Epithelial Cancer[END_REF] but also at steady-state in mouse neurons [START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF] , human neurons [START_REF] Macia | Engineered LINE-1 retrotransposition in nondividing human neurons[END_REF] and postmortem brain tissues [START_REF] Sur | Detection of the LINE-1 retrotransposon RNA-binding protein ORF1p in different anatomical regions of the human brain[END_REF] .

Using a human neuronal model of oxidative stress which displays increased LINE-1 activity as monitored by an increase in ORF1p expression and ORF2p-dependent genomic instability, we observed not only an increase of ORF1p in the cytoplasm, but also a striking translocation of ORF1p to the nucleus. Current consensus postulates that LINE-1 encoded proteins enter the nucleus only during cell cycle [START_REF] Mita | LINE-1 protein localization and functional dynamics during the cell cycle[END_REF] which thus would exclude the presence of LINE-1 proteins in the nucleus of non-dividing cells including post-mitotic neurons. However, others have reported the presence of ORF1p in the nucleus in human cells in neurodegenerative context [START_REF] Pereira | Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis[END_REF] . We also observed ORF1p staining in the nucleus of human dopaminergic neurons in vitro and in mouse dopaminergic neurons in vivo even at steady-state [START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF] , and oxidative stress dramatically increases the nuclear translocation of ORF1p. The most probable mode of import for ORF1p is the importin-beta pathway. Indeed, ORF1p, as we show here in neurons and others have shown in HeLa cells [START_REF] Freeman | Identification of charged amino acids required for nuclear localization of human L1 ORF1 protein[END_REF] binds directly to the importin beta family member KPNB1, which is a transport mediator carrying cargo proteins through the NPC. This importin-assisted mode of nuclear entry is widely used by several viral proteins [START_REF] Twyffels | Transportin-1 and Transportin-2: Protein nuclear import and beyond[END_REF][START_REF] Kimura | Identification of Cargo Proteins Specific for Importin-β with Importin-α Applying a Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based in Vitro Transport System *[END_REF] , certain retrotransposons (i.e Ty1 [START_REF] Manhas | The yeast Ty1 retrotransposon requires components of the nuclear pore complex for transcription and genomic integration[END_REF] , LINE-1 ORF1p itself [START_REF] Idica | MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by down-regulating the nuclear import factor TNPO1[END_REF] ) but also a pathological form of Tau which aggregates in neurodegeneration [START_REF] Candia | Importin-Mediated Pathological Tau Nuclear Translocation Causes Disruption of the Nuclear Lamina, TDP-43 Mislocalization and Cell Death[END_REF] . The binding of ORF1p to KPNB1 does not increase under stress condition indicating that the stress-induced increase of ORF1p nuclear translocation probably requires other protein partners. It is noteworthy that ORF1p, in a stress-dependent manner, accumulates both at the outer NE and the inner NE which corresponds to the nuclear lamina and co-localizes with and binds directly to the NPC protein NUP153. This suggests a NUP153dependent nuclear import of ORF1p under stress. Indeed, NUP153 binding of cytoplasmic proteins can mediate nuclear import of proteins [START_REF] Ando | Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells[END_REF] . ORF1p and NUP153 interaction might be favored by KPNB1, which also binds NUP153 [START_REF] Ando | Nuclear Pore Complex Protein Mediated Nuclear Localization of Dicer Protein in Human Cells[END_REF] . However, both pathways also interact [START_REF] Walther | The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins[END_REF] making it difficult to dissect the precise mechanisms.

The small transcriptional increase in LINE-1 RNA following arsenite treatment suggests that the overall increase in ORF1p could be partially transcriptional and partially posttranscriptional, possibly by releasing LINE-1 RNA stored in P-bodies for translation [START_REF] Arora | Sequestration of LINE -1 in cytosolic aggregates by MOV10 restricts retrotransposition[END_REF][START_REF] Briggs | RIP-seq reveals LINE-1 ORF1p association with p-body enriched mRNAs[END_REF] . Once ORF1p translocates to the nucleus, it accumulates at the inner NE by directly binding to Lamin B1 in a stress-specific manner. This targeting of the inner NE by ORF1p is paralleled by NE dysmorphology including invaginations and blebs which significantly correlated with ORF1p intensity, both in the nucleus and the inner NE, suggesting that it is the stress-dependent nuclear translocation of ORF1p and the direct binding to Lamin B1 that participate in NE dysmorphology. A direct functional role of ORF1p in NE dysmorphology independent of oxidative stress is further supported by direct ORF1p gain of function experiments showing that neurons over-expressing ORF1p (and not ORF2p) display a significant decrease of nuclear circularity. These experiments also suggest that NE dysmorphology is mediated by the nuclear presence of ORF1p alone and does not rely on the presence of the entire RNP or ORF2p. NE dysmorphology is associated with NCT deficiencies [START_REF] Paonessa | Microtubules Deform the Nuclear Membrane and Disrupt Nucleocytoplasmic Transport in Tau-Mediated Frontotemporal Dementia[END_REF] and both have been described to be disturbed in the context of NDs [START_REF] Aizawa | Impaired Nucleoporins Are Present in Sporadic Amyotrophic Lateral Sclerosis Motor Neurons that Exhibit Mislocalization of the 43-kDa TAR DNA-Binding Protein[END_REF][START_REF] Kim | Lost in Transportation: Nucleocytoplasmic Transport Defects in ALS and Other Neurodegenerative Diseases[END_REF][START_REF] Kinoshita | Nuclear Contour Irregularity and Abnormal Transporter Protein Distribution in Anterior Horn Cells in Amyotrophic Lateral Sclerosis[END_REF][START_REF] Liu | Parkinson's Disease-Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria[END_REF][START_REF] Sheffield | Nuclear Pore Complex Proteins in Alzheimer Disease[END_REF][START_REF] Gasset-Rosa | Polyglutamine-Expanded Huntingtin Exacerbates Age-Related Disruption of Nuclear Integrity and Nucleocytoplasmic Transport[END_REF][START_REF] Chou | TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD[END_REF][START_REF] Grima | Mutant Huntingtin Disrupts the Nuclear Pore Complex[END_REF] . Indeed, targeting of the NE by ORF1p in neurons was accompanied by NCT deficiencies as revealed by the mislocalization and abnormal and compartmentalized accumulation of several proteins involved in NCT function, some of which interact with ORF1p directly (KPNB1 and NUP153) or do not interact (i.e. RANgap1). KPNB1 sequestration by binding to ORF1p might alter nuclear import but could also inhibit its important disaggregase function [START_REF] Guo | Therapeutic Dissolution of Aberrant Phases by Nuclear-Import Receptors[END_REF] . Analogous, the abnormal localization of NUP153 in the nucleoplasm suggests an alteration of its physiological function, potentially mediated by the interaction with ORF1p as illustrated by the accumulation of nucleoplasmic PLA foci.

Nucleoporins are long-lived proteins [START_REF] Savas | Extremely Long-Lived Nuclear Pore Proteins in the Rat Brain[END_REF][START_REF] Toyama | Identification of Long-Lived Proteins Reveals Exceptional Stability of Essential Cellular Structures[END_REF] and rely on cell division for their renewal. Postmitotic neurons do not divide and nucleoporins therefore are not renewed [START_REF] D'angelo | Age-Dependent Deterioration of Nuclear Pore Complexes Causes a Loss of Nuclear Integrity in Postmitotic Cells[END_REF][START_REF] Hetzer | The Nuclear Envelope[END_REF] . Their continuous functioning in non-dividing cells throughout the life time of an individual is thus of crucial importance and any perturbations will have potentially irreversible consequences [START_REF] D'angelo | Age-Dependent Deterioration of Nuclear Pore Complexes Causes a Loss of Nuclear Integrity in Postmitotic Cells[END_REF] . Indeed abnormal intranuclear and/or cytoplasmic accumulation of nucleoporins (such as NUP62, NUP88) have been described in the context of NDs (i.e, HD) [START_REF] Grima | Mutant Huntingtin Disrupts the Nuclear Pore Complex[END_REF] . Similarly, delocalization and abnormal cytoplasmic accumulation of RANgap1, a protein with GTPase activity involved in maintaining a RAN gradient required for NCT, have been described in ND-affected tissues [START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF] .

NCT deficiencies have been consistently linked to the abnormal subcellular distribution of TDP-43, a nuclear protein involved in RNA metabolism [START_REF] Buratti | The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation[END_REF][START_REF] Colombrita | TDP-43 and FUS RNA-binding Proteins Bind Distinct Sets of Cytoplasmic Messenger RNAs and Differently Regulate Their Post-transcriptional Fate in Motoneuron-like Cells[END_REF] , mutated in ALS/FTD [START_REF] Barmada | Cytoplasmic Mislocalization of TDP-43 Is Toxic to Neurons and Enhanced by a Mutation Associated with Familial Amyotrophic Lateral Sclerosis[END_REF][START_REF] Benajiba | TARDBP mutations in motoneuron disease with frontotemporal lobar degeneration[END_REF][START_REF] Kabashi | TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis[END_REF][START_REF] Sreedharan | TDP-43 Mutations in Familial and Sporadic Amyotrophic Lateral Sclerosis[END_REF] and mislocalized to the cytoplasm in numerous NDs [START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF][START_REF] Neumann | Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis[END_REF][START_REF] Aizawa | Impaired Nucleoporins Are Present in Sporadic Amyotrophic Lateral Sclerosis Motor Neurons that Exhibit Mislocalization of the 43-kDa TAR DNA-Binding Protein[END_REF] not linked to TDP-43 mutations. Indeed, in our model, NCT deficiencies as a consequence of mislocalized NCT proteins resulted in the cytoplasmic accumulation of TDP-43. TDP-43 exits the nucleus through passive [START_REF] Duan | Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export[END_REF] or active nuclear export [START_REF] Winton | Disturbance of Nuclear and Cytoplasmic TAR DNA-binding Protein (TDP-43) Induces Disease-like Redistribution, Sequestration, and Aggregate Formation *[END_REF] through nuclear pores. We also observed an increase in the nuclear pool which could be indicative of a perturbed export mechanism of this shuttling protein [START_REF] Ederle | TDP-43 and FUS en route from the nucleus to the cytoplasm[END_REF] . Together, our results suggest a possible scenario in which ORF1p might sequester and/or mislocalize NE components such as Lamin B1 or NUP153 (see below). NE alteration and NCT defects are emerging pathogenic features in several NDs [START_REF] Kim | Lost in Transportation: Nucleocytoplasmic Transport Defects in ALS and Other Neurodegenerative Diseases[END_REF][START_REF] Eftekharzadeh | Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease[END_REF][START_REF] Frost | Alzheimer's disease: An acquired neurodegenerative laminopathy[END_REF] including HD [START_REF] Gasset-Rosa | Polyglutamine-Expanded Huntingtin Exacerbates Age-Related Disruption of Nuclear Integrity and Nucleocytoplasmic Transport[END_REF] , ALS/FTD 82 , PD [START_REF] Liu | Parkinson's Disease-Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria[END_REF] , AD [START_REF] Eftekharzadeh | Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease[END_REF][START_REF] Frost | Alzheimer's disease: An acquired neurodegenerative laminopathy[END_REF] and more generally in the context of tauopathies 100 . For example, aggregated Tau directly interacts with the nuclear pore complex [START_REF] Eftekharzadeh | Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease[END_REF] and oligomeric Tau with lamin proteins 101 resulting in loss of nuclear integrity including NCT failure [START_REF] Paonessa | Microtubules Deform the Nuclear Membrane and Disrupt Nucleocytoplasmic Transport in Tau-Mediated Frontotemporal Dementia[END_REF] , similar to what we describe here. Loss of nuclear integrity in the form of NE deformation and loss of NE function has been extensively described in the context of aging 102 including in the brain 103 with associated changes in chromatin organization and NCT dysfunction [START_REF] Frost | Alzheimer's disease: An acquired neurodegenerative laminopathy[END_REF][103][104][105] . Progerin, a mutant form of lamin A, is the cause of the premature aging syndrome Hutchinson-Gilford progeria. Similar to mutant Tau, it accumulates at the NE and disrupts NE morphology, NCT and chromatin organization [START_REF] Larrieu | Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway[END_REF] . In cellular and mouse models of accelerated aging, these alterations can be successfully corrected by treatment with the small molecule Remodelin [START_REF] Larrieu | Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway[END_REF][START_REF] Larrieu | Chemical Inhibition of NAT10 Corrects Defects of Laminopathic Cells[END_REF] . Remodelin acts by inhibiting the acetyltransferase NAT10 [START_REF] Larrieu | Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway[END_REF] . In our model of oxidative stress, pre-treatment of neurons with Remodelin 24 h prior to arsenite application restored, just as in progeria cells, NE abnormalities. In parallel, Remodelin decreased nuclear ORF1p while not affecting cytoplasmic ORF1p content. Remodelin might thus, through the restoration of the nuclear architecture, prevent ORF1p nuclear translocation and therefore further ORF1p-induced NE dysmorphology. Nuclear lamina-alterations do not only affect NCT but alter global chromatin organization 106 and potentially cell type specific gene expression 107 . Chromatin organization in cells is spatial and specific heterochromatin domains are directly linked to the nuclear lamina, called lamina-associated domains (LADs).

One marker associated with LADs is H4K20me3, a repressive heterochromatin mark known to silence LINE-1 elements [START_REF] Ren | DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation[END_REF] . Indeed, oxidative stress induced a global loss of H4K20me3 which is rescued by Remodelin pre-treatment. Together, these data indicate that Remodelin can preserve and/or restore nuclear integrity of neurons including chromatin disorganization induced by oxidative stress possibly or at least partly through an inhibitory effect on ORF1p nuclear translocation. The destructuration of nuclear lamina-anchored heterochromatin as indicated by the delocalization of H4K20me3 from the nuclear periphery could lead to the expression of genes normally repressed in post-mitotic neurons leading to neuronal fate loss, an emerging feature of aging and NDs 108 . Indeed, mutated Tau can lead to chromatin relaxation, re-expression of certain fetal genes in the brain in a fly model [START_REF] Frost | Tau promotes neurodegeneration through global chromatin relaxation[END_REF] and activation of TEs [START_REF] Guo | Tau Activates Transposable Elements in Alzheimer's Disease[END_REF][START_REF] Frost | Tau promotes neurodegeneration through global chromatin relaxation[END_REF] .

Further, the loss of H4K20me3 at the nuclear periphery could either be the source of LINE-1 activation (as H4K20me3 is a repressive mark known to decorate repeat DNA 109,110 ) or the consequence of nuclear ORF1p translocation leading to a decrease in H4K20me3 through a yet to be identified mechanism. This would indeed lead to the amplification of the initial stressinduced increase in LINE-1 via a positive feedback loop. Another possibility is an adaptive response aimed at protecting the genome from damage as a short-term response to deformation 111-113 .

Recent studies have shown that aggregated proteins such as Tau [START_REF] Candia | Importin-Mediated Pathological Tau Nuclear Translocation Causes Disruption of the Nuclear Lamina, TDP-43 Mislocalization and Cell Death[END_REF]114 , mutant Huntingtin [START_REF] Gasset-Rosa | Polyglutamine-Expanded Huntingtin Exacerbates Age-Related Disruption of Nuclear Integrity and Nucleocytoplasmic Transport[END_REF] and C9ORF72 poly(GA) [START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF] can target the NE and alter its integrity and function.

Our data suggests that ORF1p might have a pathogenic action similar to ND-linked proteins by perturbing nuclear integrity. In addition, stress-granule independent cytoplasmic TDP-43 droplets can drive nuclear import defects and cell death 115 . Whether nuclear import defects are driven by a direct ORF1p dependent mechanism or via ORF1p-dependent TDP-43 cytoplasmic accumulation remains to be determined.

Condensation is a feature of ORF1p which is intrinsic to its role in LINE-1 mobilization 116 . We observed large ORF1p foci at the level of NE invaginations and an increase in the size of cytoplasmic and nuclear ORF1p foci (dots) indicative of ORF1p condensation. Whether ORF1p itself might be aggregation-prone or participate in the organization of LLPS (Liquid liquid phase separation)-related cytoplasmic or nuclear membraneless organelles requires further investigation.

In conclusion, we provide evidence that the LINE-1 encoded protein ORF1p, independent on the LINE-1 RNP or ORF2p, translocates to the nucleus upon cellular stress in an importin-beta and NUP153 involving process. Upon translocation, ORF1p binds to Lamin B1 and sequesters and/or mislocalizes this protein, which is essential to NE integrity, leading to further deformation of the NE. In parallel, while entering the nucleus through nuclear pores, ORF1p binds to NUP153, leading to its mislocalization to the nucleoplasm and thereby contributing to NCT deficiencies including the cytoplasmic accumulation of TDP-43. NE deformation is associated with chromatin disorganization as illustrated by HP1 loss at the inner NE and global H4K20me3 depletion in the nucleus. Nuclear deformation and chromatin disorganization upon cell stress were rescued by the small molecule Remodelin which might indirectly act as an inhibitor of ORF1p nuclear translocation. This suggests that that LINE-1 encoded protein ORF1p, for which no pathogenic function had yet been assigned, might play a pathogenic role by targeting the NE. As LINE-1 activation is a recurring observation in several neurological diseases [117][118][119][120][121][122][123] , in the aging process [START_REF] Simon | LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation[END_REF] and more generally upon oxidative stress [START_REF] Blaudin De Thé | Engrailed homeoprotein blocks degeneration in adult dopaminergic neurons through LINE-1 repression[END_REF] and if this activation includes full-length LINE-1 elements with coding potential, the result could be an increase in ORF1p, in addition to ORF2p and LINE-1 RNA. Passing a certain threshold and in conjunction with a fragilization of the NE either through aging, ND-linked mutated proteins known to affect the NE or oxidative stress, this then might trigger the nuclear translocation of ORF1p contributing to NE deformation and NCT dysfunction. One consequence we identified is the cytoplasmic accumulation of TDP-43 which could favor its aggregation 124 , one of the hallmarks of several NDs [START_REF] Zhang | The C9orf72 repeat expansion disrupts nucleocytoplasmic transport[END_REF][START_REF] Neumann | Ubiquitinated TDP-43 in Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis[END_REF][START_REF] Aizawa | Impaired Nucleoporins Are Present in Sporadic Amyotrophic Lateral Sclerosis Motor Neurons that Exhibit Mislocalization of the 43-kDa TAR DNA-Binding Protein[END_REF] .

Activation of LINE-1 elements in neurons, through ORF2p-induced genomic instability 5 , ORF2p-induced inflammation [START_REF] Simon | LINE1 Derepression in Aged Wild-Type and SIRT6-Deficient Mice Drives Inflammation[END_REF][START_REF] De Cecco | Author Correction: L1 drives IFN in senescent cells and promotes age-associated inflammation[END_REF][START_REF] Thomas | Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation[END_REF] or LINE-1 RNA mediated heterochromatin erosion 125 and, as we show here, through the ORF1-dependent fragilization of nuclear integrity could contribute in multiple ways to pathological features common to several NDs. This reinforces the idea that LINE-1 elements might represent a novel therapeutic target for neuroprotection.

  Quantitative PCR reactions were carried out in duplicates with SsoAdvanced Universal SYBR® Green Supermix (Biorad, 1725274) on a CFX Opus 384 (Bio-Rad, #12011452) with the following program: enzyme activation at 95°C 2 min, primer denaturation at 95°C, annealing at 57°C and primer extension at 72°C for 42 cycles. The following primers were used: HPRT (F: CAGCCCTGGCGTCGTGATTAGT; R: CCAGCAGGTCAGCAAAGA AT); TBP (F: CAGCATCACTGTTTCTTGGCGT; R: AGATAGGGATTCCGGGAGTCAT) ; LINE-1 5'UTR (F: GTACCGGGTTCATCTCACTAGG R: TGTGGGATATAGTCTCGTG GTG), ORF1 (F: AGGAAATACAGAGAACGCCACAA) R: GCTGGATATGAAATTCTG GGTTGA), LINE-1 ORF2 (F: AAATGGTGCTGGGAAAACTG ; R: GCCATTGCTTTTGG TGTTTT ), 3'UTR (F : GGGAATTGAACAATGAGATCAC; R: TATACATGTGCCATGC TGGTG). Data were analyzed using the ddCt method and values normalized to hypoxanthine-guanine phosphoribosyl transferase (HPRT) or to glyceraldehyde-3-phosphate dehydrogenase (TBP) TATA-binding protein.

  , applying a model trained on a dataset of 1950 images of foci. The following settings are used: percentile normalization = [0.2 -99.8], probability threshold = 0.2, overlap threshold = 0.25. ORF1p foci are then labeled in 3D and filtered by volume. The number of foci, their volume and their normalized integrated intensity are computed in each cellular compartment. Results are provided as an Excel file.

  . RANgap1 accumulated in the cytoplasm, shown by increased intensity, number and dot volume in the cytoplasm (Fig. 5B) which is indicative of an alteration of NCT. Disruption of NCT can lead to the abnormal localization of the RNA binding protein TDP-43, a predominantly nuclear protein. TARDBP mutations are causal in familial forms of FTD and ALS and a change from its predominantly nuclear to cytoplasmic localization has been observed in several NDs. Current consensus suggests that TDP-43 misdistribution leads

Figure 1 .

 1 Figure 1. LINE-1 are upregulated in differentiated LUHMES cells following stress.

Figure 2 .

 2 Figure 2. ORF1p increases, changes its conformational pattern and translocates to the

Figure 3 .

 3 Figure 3. ORF1p association with nuclear envelope components and nuclear transport

Figure 4 .

 4 Figure 4. Stress induced association of ORF1p to the nuclear envelope leads to nuclear

Figure 5 .

 5 Figure 5. Stress induced association of ORF1p with the nuclear envelope is accompanied

Figure 6 .

 6 Figure 6. LINE-1 over-expression following transfection leads to nuclear envelope

Figure 7 .

 7 Figure 7. Restauration of nuclear alterations by the small molecule Remodelin

  Fig.2

  Fig.3

  Fig.4

  Fig.6

  Fig.7

Acknowledgements

RZ acknowledges the Fondation Recherche Alzheimer (FRA) for a Ph.D. fellowship and is enrolled with the Ecole Doctorale ED3C at Sorbonne University. This work was supported by grants to JF from the Fondation de France (00086320), the Fondation du Collège de France, the Fondation NRJ/Institut de France and the National French Agency for Research (ANR-20-CE16-0022 NEURAGE). We gratefully acknowledge Julien Dumont and the Collège de France Orion imaging facility (IMACHEM-IBiSA), member of the French National Research Infrastructure France-BioImaging (ANR-10-INBS-04), which received support from the program «Investissements d'Avenir» ANR-10-LABX-54 MEMOLIFE. We also thank the Fondation Bettencourt Schueller for their support.

Author contributions

RZ performed most of the experiments. OMB contributed experimentally, PM and HM wrote the scripts for the automated image analysis workflow, RLJ and JF supervised the study and wrote the paper with RZ. JF received the funding.

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