Nuclear translocation of the LINE-1 encoded ORF1
protein alters nuclear envelope integrity in human
neurons

Rania Znaidi, Olivia Massiani-Beaudoin, Philippe Mailly, Héloïse Monnet,
Tom Bonnifet, Rajiv L Joshi, Julia Fuchs

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To cite this version:

Rania Znaidi, Olivia Massiani-Beaudoin, Philippe Mailly, Héloïse Monnet, Tom Bonnifet, et al. Nuclear translocation of the LINE-1 encoded ORF1 protein alters nuclear envelope integrity in human neurons. Brain Research, 2025, 1857, pp.149579. 10.1016/j.brainres.2025.149579. hal-05339194

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# Nuclear translocation of the LINE-1 encoded ORF1 protein alters nuclear envelope integrity in human neurons

Rania Znaidi<sup>a</sup>, Olivia Massiani-Beaudoin<sup>a</sup>, Philippe Mailly<sup>b</sup>, Héloïse Monnet<sup>b,†</sup>, Tom Bonnifet<sup>a</sup>, The Brainbank Neuro- CEB Neuropathology Network<sup>1</sup>, Rajiv L. Joshi<sup>a, *, ‡</sup>, Julia Fuchs<sup>a, *, ‡</sup>

## ARTICLE INFO

### Keywords:

Retrotransposons

LINE-1

ORF1p

Nuclear envelope

Nuclear pore complex

Nucleocytoplasmic transport

Neurodegeneration

## ABSTRACT

LINE-1 retrotransposons are increasingly implicated in aging and neurodegenerative diseases, yet the precise pathogenic mechanisms remain elusive. While the endonuclease and reverse transcriptase activities of LINE-1-encoded ORF2p can induce DNA damage and inflammation, a role of LINE-1 ORF1p in cellular dysfunctions stays unassigned. Here we demonstrate, using a neuronal cellular model, that ORF1p translocates into the nucleus upon arsenite-induced stress, directly interacting with nuclear import (KPNB1), nuclear pore complex (NUP153), and nuclear lamina (Lamin B1) proteins. Nuclear translocation of ORF1p disrupts nuclear integrity, nucleocytoplasmic transport, and heterochromatin structure, features linked to neurodegeneration and aging. Elevated nuclear ORF1p levels induced either by arsenite-induced stress, ORF1p overexpression, or as observed in Parkinson's disease post-mortem brain tissues correlate with impaired nuclear envelope (NE) morphology. Stress-induced nuclear alterations are mitigated by blocking ORF1p nuclear import or with the anti-aging drug remodelin. This study thus reveals a pathogenic action of nuclear ORF1p in human neurons driving NE alterations and thereby contributing to LINE-1-mediated cell toxicity.

## 1. Introduction

Repetitive sequences derived from transposable elements (TEs) represent 45 % of the human genome, including LTR (Long Terminal Repeat) and non-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 500,000 LINE-1 copies (International Human Genome Sequencing Consortium et al., 2001). 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 (according to L1Base database (http://l1base.charite.de/) which can be autonomously active and competent for retrotransposition (Penzkofer et al.,

2017). The 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 ORF2p encompasses reverse transcriptase and endonuclease activities. Since LINE-1 activation can lead to deleterious consequences such as DNA damage and genomic instability (Gasior et al., 2006; Belgnaoui et al., 2006; Blaudin de Thé et al., 2018; Simon et al., 2019), senescence and inflammation (Thomas et al., 2017; De Cecco et al., 2019), these TEs are repressed in most somatic cells at the transcriptional and post-transcriptional level. Failure of these repressive mechanisms can lead to the activation of LINE-1 and other TEs in the context of aging or human diseases like cancer.

Several recent studies have reported that TE activation might be linked to neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), non-AD tauopathies, Parkinson's disease (PD), Amyotrophic

<footnote><sup>a</sup> CIRB, Collège de France, Université PSL, CNRS, INSERM, 75005 Paris, France</footnote>

<footnote><sup>b</sup> Orion Imaging Facility, CIRB, Collège de France, Université PSL, CNRS, INSERM, Labex Memolife, 75005 Paris, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: rajiv.joshi@college-de-france.fr (R.L. Joshi), Julia.fuchs@college-de-france.fr (J. Fuchs).</footnote>

<footnote n="1">¹ The Neuro-CEB Neuropathology network includes: Franck Letournel (CHU Angers), Marie-Laure Martin-Négrier (CHU Bordeaux), Maxime Faisant (CHU Caen), Catherine Godfraind (CHU Clermont-Ferrand), Claude-Alain Maurage (CHU Lille), Vincent Deramecourt (CHU Lille), Mathilde Duchesne (CHU Limoges), David Meyronnet (CHU Lyon), Clémence Delteil (CHU Marseille), Valérie Rigau (CHU Montpellier), Danielle Seilhean (CHU PS, Paris), Susana Boluda (CHU PS, Paris), Isabelle Plu (CHU PS, Paris), Dan Christian Chiforeanu (CHU Rennes), Florent Marguet (CHU Rouen), Béatrice Lannes (CHU Strasbourg).</footnote>
Lateral Sclerosis (ALS) and Huntington's disease (HD) (Blaudin de Thé et al., 2018; Douville et al., 2011; Li et al., 2012; Krug et al., 2017; Prudencio et al., 2017; Sun et al., 2018; Guo et al., 2018). Although NDs present many common hallmarks such as pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, oxidative stress and neuronal cell death (Gan et al., 2018; Fu et al., 2018), the genetic basis and the molecular and cellular mechanisms underlying neurodegeneration still remain elusive. In this regard, recent work from our group (Blaudin de Thé et al., 2018; Ravel-Godreuil et al., 2021) and others (Sun et al., 2018; Guo et al., 2018; Frost et al., 2014) suggests that TE activation could directly contribute to neuronal dysfunctions associated with NDs. Indeed, several pathogenic features of NDs might actually be linked to TE de-repression (Rekaik et al., 2015; Peze-Heidsieck et al., 2022). While the deleterious consequences attributed to the endonuclease and reverse transcriptase activities of LINE-1 ORF2p (i.e. DNA strand breaks, cytosolic single-stranded DNA inducing innate immune activation and inflammation) are beginning to be well unraveled, no particular pathogenic function has yet been assigned to the RNA chaperone ORF1p.

To directly address the consequences of endogenous full-length LINE-1 activation and increased LINE-1-encoded proteins in adult neurons, we used (i) a neuronal model of acute stress displaying an increase in LINE-1 encoded ORF1p expression, (ii) direct overexpression of tagged ORF1p and (iii) post-mortem human brain tissues of PD-affected or non-PD-affected individuals. We found that ORF1p translocation to the nucleus alters nuclear envelope (NE) integrity including NE morphology, nucleocytoplasmic transport and heterochromatin structure and that blocking ORF1p nuclear translocation by either nuclear import inhibition or the small molecule remodelin prevented NE deformations. This suggests that neurons are susceptible to ORF1p mediated NE perturbations potentially leading to the amplification of NE dysfunctions which are an established feature of aging and an emerging feature of many NDs.

# 2. Materiel and methods

## 2.1. Cell culture

The Lund Human Mesencephalic (LUHMES) cell line is a conditionally immortalized cell line. These cells are derived from the tetracycline-controlled, v-myc-overexpressing human mesencephalic cell line MESC2.10 (Lotharius et al., 2002; Lotharius et al., 2005). The culturing and handling procedure of LUHMES cells were as described previously (Lotharius et al., 2002). LUHMES cells can be differentiated into mature dopaminergic neurons. The differentiation was performed following the two-step protocol previously established (Scholz et al., 2011). Cell culture flasks, multi well plates and glass coverslips used for LUHMES culture were pre-coated by adding 50 µg/ml poly-L-ornithine (Sigma) diluted in 1X PBS (Phosphate buffered saline) for 1 h at 37 °C, washed 3 times in 1X PBS, and then incubated with 1 µg/ml fibronectin (Sigma) diluted in 1X PBS for 3 h minimum at 37 °C to promote cell attachment.

Cell culture medium consisted of Advanced DMEM/F12 (ThermoFisher Scientific), N2-Supplement (ThermoFisher Scientific) and 2 mM GlutaMAX (ThermoFisher Scientific, 35050061) which was supplemented by 40 ng/ml recombinant human basic fibroblast growth factor FGF (Peprotech) during proliferation. Cells were kept at 37 °C in 100 % humidified air with 5 % CO2 until 70 %–80 % of cellular confluence is reached.

The differentiation process was initiated by the shut-down of v-myc by adding 1 µg/ml doxycycline (Sigma) in the base medium supplemented with 2 ng/ml recombinant human glial-derived neurotrophic factor GDNF (Peprotech) and 1 mM cAMP (Sigma). The pre-differentiated neurons were replated 48 h later at a defined density of 150 000 cells/well in 24-well plates or 10 (Simon et al., 2019) cells/well in 6-well plates. To confirm successful generation of post-mitotic

LUHMES, expression of neuronal and dopaminergic markers was assessed throughout the differentiation protocol, which lasts a total of 5 days (Fig. 1A; Suppl. Fig. 1A). All experiments were performed on day 5 of differentiation, at which point LUHMES cells were considered to be completely mature post-mitotic neurons.

## 2.2. Arsenite-induced stress

Sodium arsenite solution (NaAsO₂) (Sigma) is stored at room temperature. Dilutions were carried out shortly before each experiment. Fully differentiated LUHMES cells (at day 5 of differentiation) were subjected to arsenite during 1 h at 37 °C at a concentration of 50 µM.

## 2.3. Cell viability

Cytotoxicity was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780) following the manufacturer's instructions. This colorimetric assay quantifies the release of lactate dehydrogenase (LDH) from damaged cells as an indicator of cytotoxicity. LDH released in culture supernatants is measured using a 30-minute coupled enzymatic assay that results in the conversion of a tetrazolium salt (INT) to a red formazan product. The amount of colour formed is proportional to the number of damaged cells. Differentiated cells on day 5 were changed to a new medium and treated with 50 µM or 500 µM sodium arsenite, left untreated as a control for the number of cells that die naturally or completely lysed with a 1 % triton treatment to quantify the maximum amount of LDH that could be released. Supernatants were collected 1 h, 4 h, 17 h and 22 h after treatment. At each time point, 50 µl of the supernatant was transferred to a new 96-well plate, and 50 µl of CytoTox 96® reagent was added to each well. After incubation for 30 min at room temperature, the reaction was stopped by adding 50 µl of stop solution. Absorbance was measured at 490 nm using a microplate reader. The background absorbance of the medium was subtracted from all readings and cytotoxicity was expressed as a percentage of the maximum LDH released from lysed cells.

## 2.4. Drug treatment

For treatment with remodelin (a generous gift from Drs. R. Rodriguez and L. Colombeau), 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 arsenite-induced stress at a concentration of 50 µM for 1 h (in the presence of remodelin). 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, and then subjected to arsenite-induced stress at a concentration of 50 µM for 1 h (in the presence of stavudine). For treatment using nuclear import inhibitors, differentiated neurons at day 5 of differentiation, were incubated in fresh culture medium containing ivermectin (5 µM; CliniSciences) and importazole (10 µM; Scientific Laboratory Supplies) or DMSO for 2 h, and were then treated with arsenite (50 µM) for 1 h. In all cases, neurons were then fixed and processed for immunocytochemistry.

## 2.5. 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 above. 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, expressing ORF1p with an N-Ter HA tag; a gift from Dr. J. Moran 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 was monitored by fluorescence microscopy.

## 2.6. 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) containing 1X protease inhibitor (Sigma) 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 (30 s on/30 s off) on ice at high power to shear genomic DNA. After collecting the supernatant into new microtubes, protein concentration was determined using Pierce™ BCA Protein Assay Kit. Aliquot of lysates were stored at -20 °C avoiding multiple freeze/ thaw cycles. Laemmli buffer (250 mM Tris pH 6.8, 10 % sodium dodecyl sulfate, 1,25 % bromophenol blue, 5 % β-mercaptoethanol, 50 % glycerol) containing DTT was added to protein lysates (1X final), boiled 10 min at 95 °C and stored at -20 °C until analyzed.

## 2.7. Western blot analysis

A 1.5 mm NuPAGE 4–12 % Bis-Tris-Gel (Invitrogen™) polyacrylamide gel was used for Western blots. 10 µg of protein was loaded per well. Gel migrated in NuPAGE™ MES SDS running buffer (1X) (Invitrogen™) for 55 min 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 NaCl, 10 mM Tris-HCl pH 8) was used during 1 h at room temperature. The membranes were then incubated overnight with the primary antibodies: anti-ORF1p 1:500 (Abcam, ab245249), TH 1:500 (Millipore, AB9702). The membranes were incubated with anti-actin peroxidase antibody 1:20000 (Sigma) 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) and anti-mouse HRP 1:2000 (Cell Signaling) for 1 h at room temperature. After washing in TBST (1X), membranes were revealed using Clarity Western ECL Substrate (BioRad) or Maxi Clarity Western ECL Substrate (BioRad) in a LAS-4000 Fujifilm system. Analysis of immunoblotting images was performed manually using Fiji software.

## 2.8. Immunoprecipitation

For immunoprecipitation (IP) we used ORF1p (Millipore, MABC 1152) and IgG mouse (Thermofisher) antibodies. The antibodies were coupled to magnetic beads using the Dynabeads® Antibody Coupling Kit (Invitogen) 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 20×10⁶ cells in 2 T75 flasks and grown in differentiation medium for 5 days. 50 µM arsenite for 1 h was added to treatment groups. 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 pH 8, 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). Migration was carried out in MES SDS running buffer (Invitrogen) for 45 min at 200 V. The gels were then transferred onto a PVDF membrane activated with methanol (Immobilon, Sigma) 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), the secondary anti-rabbit HRP 1:2000 (Cell signaling) with the Maxi Clarity Western ECL Substrate kit (BioRad). Analysis of immunoblotting images was performed manually using Fiji software.

## 2.9. RT-qPCR

Total RNA from 5×10⁵ lysed cells was extracted using RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's instructions. The removal of contaminating genomic DNA (gDNA) was assured by using the ezDNase kit (Invitrogen™). After gDNA elimination, RNA (300 ng) was reverse transcribed using the All-In-One 5X RT MasterMix kit (Abm) for first-strand cDNA synthesis ready for immediate real-time PCR analysis.

Quantitative PCR reactions were carried out in duplicates with Advanced Universal SYBR® Green Supermix (Biorad) on a CFX Opus 384 (Bio-Rad,) 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: AGTAGGGATTCCGGGAGTCAT); LINE-1 5'UTR (F: GTACCGGGTTCATCTCACTAGG R: TGTGGGATATAGTCTCGTG GTG), ORF1 (F: AGGAAATACAGAGAACGCCACAA) R: GCTGGATATGAAATTCTGGGTTGA), LINE-1 ORF2 (F: AAATGGTGCTGGGAAAAACTG; R: GCCATTGCTTTTTGG TGTTTT), 3'UTR (F: GGGAAATTGAACAATGAGATCAC; 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.

## 2.10. Proximity ligation assay (PLA)

Proximity Ligation Assay (PLA) is a method that allows in situ detection of endogenous proteins, and protein interactions with high specificity and sensitivity. It utilizes a pair of oligonucleotide-labeled secondary antibodies (PLA probes) which binds to the primary antibodies raised in different species specific to detect unique protein targets. Then, connector oligos hybridize to join the PLA probes only when they are in close proximity, forming a closed circular DNA template necessary for rolling-circle amplification. The PLA probe serves as a primer for a DNA polymerase, initiating the production of concatemeric sequences through rolling-circle amplification producing detectable signals visualized and quantified as discrete spots by microscopy image,
indicating the presence and localization of the interacting proteins. The detection of the interactions between ORF1p and Lamin B1, NUP153 and KPNB1 were analyzed using 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 were mounted with a Fluoromount (Invitrogen™, 15586276). Fluorescence images were acquired using a Spinning Disk W1 Yokogawa imaging system. The quantification was again conducted with the custom-written plugin (https://github.com/orion-cirb/Nuclei_ORF1P) described previously. In short, nuclei and cytoplasms are segmented in 3D using Cellpose. Within each cell, PLA foci are detected using Stardist 2D on each slice of the cell mask and then reconstructed in 3D. The foci parameters are ultimately computed in each cellular compartment (cytoplasm, nucleus, outer and inner nuclear membranes, and nucleoplasm).

## 2.11. Immunofluorescence

Immunostaining experiments on cells 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) in PBS for 20 min. Immunofluorescence were performed overnight at 4 °C after a 20 min permeabilization in 0,2% Triton X100 in PBS, followed by incubation in 100 mM glycine in PBS for 20 min at room temperature and incubation in blocking buffer (10 % Normal goat serum (NGS), 0,1% Triton X-100 in PBS) 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), γH2AX 1:1000 (Abcam, ab2893), ssDNA 1:500 (ENZO, (F7-26) ALX 804–192) diluted in 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 647, 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 (stimulated-emission-depletion) Abberior Facility line imaging systems.

For immunostaining of post-mortem human brain tissues, sections were fixed with 4 % paraformaldehyde (PFA) for 10 min. Sections were permeabilized in 0,5% Triton X-100 in PBS for 20 min followed by incubation in 100 mM Glycine in PBS for 20 min at room temperature and incubated at 100 °C for 20 min in citrate buffer (10 mM Sodium citrate buffer pH 6.0, 0.05 % Tween 20). After blocking for 1 h (10 % Normal goat serum (NGS), 0,1% Triton X-100, in PBS), tissues were incubated overnight at 4 °C with the following primary antibodies: ORF1p 1:500 (Millipore, MABC1152 clone 4H1), Lamin A/C 1:800 (Abcam, ab108595), NeuN 1:1000 (GENTEX, GTX00837) diluted in blocking buffer. The following secondary antibodies were added for 1 h at room temperature: Alexa Fluor 555, Alexa Fluor 488, Alexa Fluor 647 1:2000 (ThermoFisher Scientific). Nuclei were stained with Hoechst 1:2000 (Invitrogen, H3570). Sections were treated with TrueBlack lipofuscin autofluorescence quencher (OZYME, BTM23014) diluted in PBS for 10 min in the absence of light. The slides were rinsed 3 × 10 min in PBS to remove excess quencher and mounted with Fluoromount (Invitrogen™, 15586276). Sections were imaged by Spinning Disk W1 Yokogawa imaging system.

The quantification of ORF1p fluorescence was performed with a custom-written plugin (https://github.com/orion-cirb/Nuclei_ORF1P) developed for the Fiji software (Schindelin et al., 2012), using BioFormats (Linkert et al., 2010) and 3D Image Suite (Ollion et al., 2013)

libraries. The Hoechst channel was downscaled by a factor 2, before detecting nuclei with the 2D-stitched version of Cellpose (Stringer et al., 2021) (model = 'cyto2', diameter = 100, flow threshold = 0.4, cell probability threshold = 0.0, stitching threshold = 0.5). Segmented images were rescaled to original size and the obtained 3D nuclei were filtered by volume to avoid false positive detections. Cells in the ORF1p channel were detected in the same manner as nuclei. Then, each cell was associated with a nucleus having at least half of its volume in contact with it. Cells without any associated nucleus were filtered out. For each cell, ORF1p signal was analyzed in the following cellular compartments: cytoplasm, nucleus, outer and inner nuclear membranes and nucleoplasm (Fig. 2C, E). Compartmentalization was obtained using combinations of morphological operations (dilation, erosion and subtraction) of the cell and nucleus masks.

In each compartment, the integrated intensity of ORF1p was measured and normalized to ensure comparability across images. Normalization was done by subtracting the image background noise, estimated as the median intensity value of the minimum intensity Z-projection of the ORF1p channel. ORF1p foci were detected with Stardist 2D (Schmidt et al., 2018), applying a model trained on a dataset of 1950 images of foci. The following settings were used: percentile normalization = [0.2–99.8], probability threshold = 0.2, overlap threshold = 0.25. ORF1p foci were then labeled in 3D and filtered by volume. The number of foci, their volume and their normalized integrated intensity were computed in each cellular compartment. Results were provided as an Excel file.

To quantify nuclear deformation, another custom plugin (https://github.com/orion-cirb/HA_ORF1P_Prot/) was developed for the Fiji software, that uses the same libraries listed above, CLIJ (Haase et al., 2020) and Find Focused Slices (https://sites.google.com/site/qingzongtseng/find-focus?authuser = 0). In brief, the best focused slice in a stack is selected. Nuclei were 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 was computed. For each nucleus, ORF1p integrated intensity was analyzed in the following compartments: nucleus, outer and inner nuclear membranes and nucleoplasm. Compartments were obtained with the same combinations of morphological operations as described above.

In case a HA-ORF1p channel was provided, cells were detected on the best focused 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 were considered as HA-ORF1p+, other nuclei as HA-ORF1p-. The quantification of the intensity of other markers than ORF1p was also performed with this plugin.

The analysis of human sample images was conducted using a third custom-written plugin (https://github.com/orion-cirb/Hoechst_ORF1p_pTAU/) developed for the Fiji software, incorporating BioFormats and 3D Image Suite libraries. Nuclei detection in the Hoechst channel was performed using the 2D-stitched version of Cellpose (model = 'cyto', diameter = 100, flow threshold = 0.4, cell probability threshold = 0.0, stitching thresh-old = 0.5). The resulting 3D nuclei were filtered based on volume, and measurements of nuclei volume, circularity, as well as ORF1p intensity mean and standard deviation were conducted. Circularity was assessed on the middle plane of the nucleus, and intensity measurements were normalized by subtracting the image background noise, as described above.

If a NeuN channel was provided, NeuN-positive cells detection followed the same process as nuclei detection, with the exception that the model used was trained through the human-in-the-loop procedure implemented in the Cellpose GUI (Pachitariu and Stringer, 2022). Specifically, the model was initialized from the 'cyto2' pretrained Cellpose model and trained on 20 images (approximately 50 cells annotated). During the inference phase, the following parameters were employed: diameter = 140, flow threshold = 0.4, cell probability threshold = 0.0, stitching threshold = 0.5. Subsequently, each cell was linked to a
nucleus with at least a quarter of its volume in contact with it. Nuclei were thus classified as either NeuN + or NeuN-.

## 2.12. Statistical analysis

Data were obtained from independent experiments. The number of cells analyzed in each experiment is described in the corresponding figure legend. Data analysis was performed using GraphPad Prism 8.

Shapiro Wilk normality tests were performed prior to the statistical test to inform whether a parametric or non-parametric test should be used. The statistical tests used are indicated in the figure legends. The significant threshold was defined as *p* < 0.05 and depicted as follows on all graphs: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** LINE-1 are upregulated in differentiated LUHMES cells following stress. (A) Experimental paradigm of the differentiation scheme of LUHMES neuronal precursor cells into mature, post-mitotic dopaminergic neurons. At day 5 of differentiation, neurons were subjected to arsenite (NaAsO₂). (B) After 5 days of differentiation, LUHMES cells express the neuronal marker TuJ1 and the dopaminergic marker TH as revealed by immunofluorescence. These neurons express LINE-1 at steady-state as shown by immunostaining for ORF1p protein. (C) Differentiated LUHMES neurons subjected to arsenite treatment, show increased levels of ORF1p protein by Western blot analysis. The three lanes in each condition represent data from 3 independent wells. n = 3 wells per condition from 4 independent experiments, mean ± SEM, two-tailed *t* test. (D) Immunostaining for LINE-1 ORF1p of differentiated LUHMES cells treated with arsenite and quantification of global ORF1p intensity, n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments is shown, mean ± SEM, two-tailed *t* test. (E) Immunostaining of γH2AX following arsenite treatment in the presence or the absence of stavudine and quantification of γH2AX foci number in the nucleus. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 2 independent experiments is shown, mean ± SEM, One-way ANOVA with Tukey's multiple comparisons test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001. Scale bar, 5 µm.</figcaption></figure>
# 3. Results

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

To generate a human neuronal model of endogenous LINE-1 activation, we turned to LUHMES cells, which represent a robust model to investigate the molecular and cellular mechanisms leading to neurodegeneration (Zhang et al., 2014; Harris et al., 2017; Harischandra et al., 2020). Differentiation of LUHMES cells into post-mitotic neurons was initiated by the complete shut-down of v-Myc activity according to the experimental paradigm (Lotharius et al., 2002; Lotharius et al., 2005; Scholz et al., 2011) displayed in Fig. 1A (and Suppl. Fig. 1A). These cells acquired a mature dopaminergic neuronal phenotype as revealed by immunostaining for the neuronal marker TuJ1 (neuron-specific class III beta-tubulin) and the dopaminergic marker TH (tyrosine hydroxylase) at day 5 of differentiation (Fig. 1B and quantified in Suppl. Fig. 1B). All cells in culture were positive for TH and TuJ1 at day 5 of differentiation as shown in Fig. 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 (Blaudin de Thé et al., 2018) and throughout the mouse brain in vivo (Bonnifet et al., 2023), full-length LINE-1 elements are expressed at steady-state in cultured human dopaminergic neurons as revealed by immunostaining for the LINE-1 ORF1p protein (Fig. 1B). Human ORF1p, as mouse ORF1p (Blaudin de Thé et al., 2018); shows a predominant cytoplasmic expression pattern but is also observed in the nuclear compartment. Oxidative stress is a common feature of aging (Junqueira et al., 2004; Romano et al., 2010; Liguori et al., 2018), cancer (Hayes et al., 2020) and NDs (Emerit et al., 2004; Chen et al., 2012; Kim et al., 2015; Fischer and Maier, 2015) and has been associated with LINE-1 activation (Giorgi et al., 2011; Wongpaiboonwattana et al., 2013; Whongsiri et al., 2019; Teneng et al., 2007). We determined the minimal, non-toxic concentration and treatment time of the know oxidative stress-inducing molecule arsenite (NaAsO₂) which led to a robust increase of LINE-1 expression. The 50 µM dose for 1 h showed no cytotoxicity, as indicated by low LDH release levels similar to sham-treated control cells. Significantly higher toxicity was observed at 500 µM, especially after prolonged exposure (Suppl. Fig. 2C). Based on time-course (Suppl. Fig. 2A), dose-response (Suppl. Fig. 2B) and cytotoxicity experiments (Suppl. Fig. 2C), we thus chose the non-toxic dose of 50 µM arsenite for 1 h to induce LINE-1 expression in dopaminergic neurons. This treatment did not affect the neuronal and dopaminergic phenotypes as shown by the quantification of TuJ1 and TH expression (Suppl. Fig. 3A). Arsenite increased the expression of LINE-1 in human neurons as revealed by Western blot and immunofluorescence analysis of LINE-1 ORF1p protein by about 1.5-fold (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 of about 1.5-fold (Suppl. Fig. 3B). The mean increase of ORF1p expression upon arsenite as revealed by immunostainings was consistently and reproducibly in the range of 1.5-fold in seven independent experiments (Suppl. Fig. 3C). These data indicate an overall modest, but rapid and robust increase in LINE-1 expression upon low-dose arsenite. LINE-1 kinetics were similar to what had been observed in mouse primary midbrain neurons challenged with H₂O₂, in mouse dopaminergic neurons in vivo injected with 6-OHDA (Blaudin de Thé et al., 2018) and in BE (2C) neuroblastoma cells after exposure to H₂O₂ (Giorgi et al., 2011), indicating that LINE-1 RNA and protein increase rapidly in neurons upon different stressors.

In order to test LINE-1 encoded protein activity, we analyzed two known consequences of ORF2p activity. Increased LINE-1 ORF2p activity has been previously reported to induce DNA damage (Gasior et al., 2006; Belgnaoui et al., 2006; Blaudin de Thé et al., 2018; Simon et al., 2019; Thomas et al., 2017) which can be at least partially prevented by

reverse transcriptase inhibitors active against LINE-1 (Blaudin de Thé et al., 2018; Simon et al., 2019; Thomas et al., 2017). Upon arsenite treatment, differentiated human dopaminergic neurons displayed increased DNA damage as quantified by γH2AX (phosphorylated histone H2AX)-positive foci, which decreased in the presence of the reverse transcriptase inhibitor stavudine (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 (Simon et al., 2019; Thomas et al., 2017). Indeed, we observed an increase of ssDNA foci volume and intensity in the cytosol of stressed neurons (Suppl. Fig. 4). Full-length LINE-1 and its encoded proteins are thus expressed at steady-state in human dopaminergic neurons, are inducible by arsenite-induced 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.

## 3.2. ORF1p translocates into the nucleus and localizes to the nuclear envelope upon arsenite-induced stress

We next investigated the precise cellular localization and expression levels of ORF1p upon arsenite-induced stress using confocal imaging (Fig. 2A). ORF1p was predominantly cytoplasmic and showed both diffuse and dot-like patterns in steady-state as well as in stress conditions (Fig. 2A). Surprisingly and against the current assumptions (Mita et al., 2018), ORF1p was also readily detectable in the nucleus and at the NE (Fig. 2A). Both cytoplasmic and nuclear ORF1p increased under stress conditions (Fig. 2B). ORF1p intensities were further quantified in the cytoplasmic and nuclear compartment 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 like the nucleus and the cytosol. The increase in staining intensities in both compartments 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. The localization of ORF1p at the NE (pointed by arrowheads, Fig. 2A) under stress conditions was striking and caught our attention. The script was therefore 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 increased ORF1p intensities, dot number and volume in both the inner and outer NE as well as in the nucleoplasm under stress conditions (quantified in Fig. 2F and Suppl. Fig. 5A, B). In summary, arsenite induced a general increase of ORF1p and the translocation of ORF1p into the nucleus including an accumulation at the inner NE.

## 3.3. ORF1p targets nuclear envelope components upon arsenite-induced stress

To confirm ORF1p localization at 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 was 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). Possible binding of ORF1p to Lamin B1 was further examined by proximity ligation assay (PLA) which confirmed direct interaction between these two proteins at the nuclear periphery upon stress (Fig. 3C) in line with the colocalization signals observed by co-immunostainings (Fig. 3A). Interaction of ORF1p and Lamin B1 by PLA was also detected in the nucleoplasm (Fig. 3C). Quantification of PLA signal confirmed an increase of the number of dots (= direct
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**Fig. 2.** ORF1p increases, changes its conformational pattern and translocates to the nucleus following arsenite-induced stress. (A) Immunostaining for LINE-1 ORF1p of differentiated LUHMES cells treated with arsenite. (B) Quantification of ORF1p intensity both in the cytoplasm and the nucleus in the presence and the absence of stress. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 8 independent experiments is shown, mean ± SEM, two-tailed *t* test (C) Scheme of the script (for details see Materials and Methods) for ORF1p quantification in the cytoplasmic, nuclear (nucleoplasm = nucleus – inner NE) and inner and outer NE compartments. (D) Quantification of the number and volume of ORF1p positive dots shown in ROI (region of interest) (A) in the cytoplasm and the nucleus using the same script, n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 8 independent experiments, mean ± SEM, two-tailed *t* test. (F) Quantification of ORF1p signal at the NE by an in-house script as schematized in (E) (for details see Materiel and Methods) shows increased ORF1p intensity and dot number at the inner NE and outer NE, n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 5 independent experiments is shown, mean ± SEM, two-tailed *t* test (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001. Scale bar, 5 µm.

interaction events) at the inner NE but also in the nucleoplasmic compartment with arsenite (Fig. 3D). This suggests a possible scenario in which the stress-induced nuclear translocation of ORF1p and direct ORF1p binding to Lamin B1 could induce 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) (Freeman et al., 2019; Idica et al., 2017), which could mediate its nuclear translocation using nuclear pore complexes (NPCs). To confirm that nuclear translocation of ORF1p in neurons upon stress is also mediated by this nuclear import pathway, we performed co-immunostaining of ORF1p with KPNB1 and NUP153 (a nucleoporin of the nuclear basket), a core NPC protein reported to interact with KPNB1 (Walther et al., 2001; Kapinos et al., 2017). The results shown in Fig. 3E indicate the colocalization of ORF1p with KPNB1 and NUP153 at the NE indicating that nuclear translocation of ORF1p in human neurons upon stress might indeed be mediated by these proteins. In addition, several ORF1p-positive cytoplasmic foci colocalized with KPNB1 (Fig. 3E) which might jeopardize normal KPNB1 function. Co-immunoprecipitation experiments confirmed the interaction between ORF1p and KPNB1 under stress conditions (Fig. 3F). Additionally, direct interaction of ORF1p with KPNB1 and NUP153 was further confirmed by PLA (Fig. 3G). PLA signal of ORF1p and KPNB1 interaction was not affected by stress (Fig. 3H), whereas the direct interaction between ORF1p and NUP153 was increased in stress condition as indicated by increased number of PLA foci at the inner NE and the nucleoplasm (Fig. 3I). Altogether, these results reveal a direct interaction of ORF1p with NE components and prompted us to examine NE morphology and nucleocytoplasmic transport (NCT) functioning in this neuronal model.

### 3.4. Nuclear ORF1p accumulation correlates with nuclear envelope alterations

Lamin B1 staining of the inner nuclear lamina of human differentiated neurons revealed various NE dysmorphologies including nuclear invaginations and blebbing which appeared upon stress (exemplified in Fig. 4A). It is noteworthy that nearly 70 % of stressed cells presented such deformations (Fig. 4B). Co-immunostaining of ORF1p and Lamin B1 revealed the accumulation of ORF1p at the level of NE invaginations and blebs, which was clearly observable in STED acquired 3D images (Fig. 4A, upper right panel). Nuclear dysmorphology was accompanied by increased Lamin B1 levels at the inner NE but even more so in the nucleoplasm of stressed cells (Suppl. Fig. 5C), again suggesting a delocalization of Lamin B1 possibly as a result of direct interaction with ORF1p. The degree of NE distortion (expressed as a circularity index) was also analyzed using an automated script (detailed in the Materiel and Methods section) which measures nuclear circularity using Hoechst staining as a mask (Fig. 4D). This analysis showed a significant decrease of nuclear circularity in stressed cells (Fig. 4C). 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 (p < 0.0001; Spearman r = -0.8230; 95 % CI -0.8578–-0.7807) and at the inner NE (p < 0.0001; Spearman r = -0.9169; 95 % CI -0.9339–-0.8958, Fig. 4E). This correlation suggests that nuclear ORF1p translocation and

accumulation in the nucleoplasm and at the inner NE might be involved, at least partly, in the occurrence of nuclear dysmorphologies observed in neurons upon stress.

Human dopaminergic neurons are particularly vulnerable to neurodegeneration observed in PD (Hirsch et al., 1988), express ORF1p more abundantly than surrounding neurons and their degeneration can be driven by LINE-1 expression in mice (Blaudin de Thé et al., 2018). In addition, NE alterations have been previously described in dentate gyrus brain tissues of sporadic and LRRK2-linked PD samples (Liu et al., 2012). In order to establish whether ORF1p is expressed in neurons in the human brain, and if so, whether nuclear ORF1p levels correlate with NE deformations, we turned to human post-mortem brain tissues of PD patients and non-PD affected controls some of whom had signs of early AD pathology (Braak 1–2; Table 1). We chose the cingulate gyrus, a region secondarily affected in PD, reasoning that dopaminergic neurons of the *substantia nigra pars compacta*, were most probably already lost at these advanced stages of the disease. We performed immunostainings using ORF1p, the pan-neuronal marker NeuN and either Hoechst to identify the nucleus or Lamin A/C to identify the NE and used an unbiased machine-learning assisted cell detection workflow which (a) identifies neurons based on the presence of NeuN, (b) delimits the nucleus based on Hoechst staining, (c) quantifies ORF1p within this delimitation and (d) measures the circularity of the nuclei. Immunostainings of ORF1p and Lamin A/C or Hoechst showed a mostly cytoplasmic but also nuclear localization of ORF1p in neurons in samples from both, PD patients and non-PD controls (Fig. 4F, G). Importantly, analysis of a correlation of nuclear ORF1p intensity with nuclear circularity in each identified neuron identified a significantly negative correlation between the two parameters in PD samples (p = 0.0021; Spearman r = -0.1949; 95 % CI -0.32–0.07) but not in controls (p = 0.4725; Spearman r = -0.06458; 95 % CI -0.24–0.12, Fig. 4H). As in human dopaminergic neurons in culture, this suggests a relationship in human post-mortem PD-affected neurons in which the higher the concentration of ORF1p, the more pronounced the deformation of the nucleus.

In an attempt to establish causality between an increase in nuclear ORF1p and nuclear dysmorphologies, we used siRNA to knock-down LINE-1 expression. However, cytoplasmic ORF1p and nuclear ORF1p were not efficiently decreased using this approach. We therefore turned to a different approach by blocking nuclear import of ORF1p using nuclear import inhibitors. As mentioned above, ORF1p nuclear import is mediated by a KPNB1/KPNA2-dependent process (Freeman et al., 2019; Idica et al., 2017). KPNA2 is an importin alpha family member, which recognizes a nuclear localization signal (NLS) of cargo proteins and serves as an adapter for KPNB1, which provides the import activity of the complex into the nucleus (Goldfarb et al., 2004; Conti et al., 1998). KPNA2 has been shown to be involved in nuclear import of ORF1p in addition to KPNB1 (Freeman et al., 2019; Idica et al., 2017). Using ivermectin, a KPNB1 inhibitor (Bird et al., 2013; Soderholm et al., 2011), and importazole, an inhibitor of KPNA2 (Wagstaff et al., 2012; Yang et al., 2020), we obtained a significant decrease in nuclear ORF1p upon stress (Fig. 5A), which we did not observe with either drug alone (Suppl. Fig. 5D, E). ORF1p nuclear import in human neurons is thus dependent on both, KPNB1 and KPNA2. Importantly, the decrease in nuclear circularity upon stress was significantly rescued by blocking the
<figure><img src="image_12.png" /><figcaption>**Fig. 3.** ORF1p association with nuclear envelope components and nuclear transport proteins increases upon arsenite-induced stress. (A) Co-immunostaining of ORF1p and Lamin B1 reveals their colocalization (yellow). (B) Validation of ORF1p interaction with Lamin B1 by co-immunoprecipitation and Western blotting. The immunoprecipitating antibody used was directed against ORF1p and the Western blot was done using a LaminB1 antibody. (C) Direct interaction of ORF1p with Lamin B1 was revealed by PLA and quantified in (D), n > 50 neurons were quantified per condition, (3 wells per condition). (E) Co-immunostaining of ORF1p and KPNB1 or NUP153. (F) Validation of ORF1p interaction with KPNB1 by co-immunoprecipitation and Western blotting. The immunoprecipitating antibody used was directed against ORF1p and the Western blot was done using a KPNB1 antibody. (G) Validation of a direct interaction between ORF1p and KPNB1 or NUP153 by PLA. The quantification in separate cellular compartments is shown in (H, I), n > 50 neurons were quantified per condition, (3 wells per condition), mean ± SEM, two-tailed *t* test (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001). Scale bar, 5 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

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<figure><img src="image_14.png" /><figcaption>**Fig. 4.** Stress induced association of ORF1p to the nuclear envelope leads to nuclear envelope dysmorphology. (A) NE dysmorphology in the arsenite stress model revealed by co-immunostaining of ORF1p and LaminB1 quantified in (B). The percentage of deformed cells was quantified in > 10 images per condition, a representative experiment (3 wells per condition) of 3 independent experiments, mean ± SEM, two-tailed *t* test. (C) Quantification of nuclear dysmorphology by analyzing nuclear circularity using a dedicated script (for details see Materiel and Methods) based on Lamin B1 as a mask (D), shows decreased circularity under stress conditions. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 5 independent experiments is shown, mean ± SEM, two-tailed *t* test. (E) Correlation of ORF1p intensity per cell in the nucleus or at the inner NE with nuclear circularity. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments is shown; Spearman correlation (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001). (F) Co-immunostaining of ORF1p and Lamin A/C in neurons of the cingulate gyrus of post-mortem PD and non-PD tissues. (G) Immunostaining of NeuN, Lamin A/C and ORF1p in the cingulate gyrus of post-mortem PD and non-PD tissues. (H) Correlation of nuclear ORF1p intensity and circularity per NeuN-positive cell in the cingulate gyrus of post-mortem tissues from PD patients (n = 5) and non-PD affected individuals (n = 4); Spearman correlation (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001).</figcaption></figure>
<table><caption>**Table 1**
PD and non-PD post-mortem tissues used in this study.</caption><thead><tr><th>Diagnosis</th><th>PMI (hr)</th><th>Gender</th><th>Age at death</th><th>Pathological staging</th></tr></thead><tbody><tr><td>Parkinson's Disease</td><td>35</td><td>M</td><td>72</td><td>Diffuse Lewy Body Disease</td></tr><tr><td>Parkinson's Disease</td><td>36</td><td>M</td><td>82</td><td>Diffuse Lewy Body Disease</td></tr><tr><td>Parkinson's Disease</td><td>nd</td><td>F</td><td>62</td><td>Diffuse Lewy Body Disease</td></tr><tr><td>Parkinson's Disease</td><td>48</td><td>F</td><td>94</td><td>Diffuse Lewy Body Disease</td></tr><tr><td>Parkinson's Disease</td><td>20</td><td>M</td><td>83</td><td>Diffuse Lewy Body Disease</td></tr><tr><td></td><td>34.8 (+/-11.2)</td><td>2F / 3 M</td><td>78.6 (+/-12.1)</td><td></td></tr><tr><td>Non-PD</td><td>23</td><td>M</td><td>78</td><td>Alzheimer lesion Braak II</td></tr><tr><td>Non-PD</td><td>15</td><td>M</td><td>84</td><td>—</td></tr><tr><td>Non-PD</td><td>10</td><td>M</td><td>73</td><td>Alzheimer lesion Braak II</td></tr><tr><td>Non-PD</td><td>21</td><td>F</td><td>92</td><td>Alzheimer lesion Braak I</td></tr><tr><td>Non-PD</td><td>10</td><td>M</td><td>85</td><td>Alzheimer lesion Braak II</td></tr><tr><td></td><td>15.8 (+/-6.1)</td><td>1F / 4 M</td><td>82.4 (+/-7.2)</td><td></td></tr></tbody></table>

PMI, post-mortem interval.

import of ORF1p into the nucleus (Fig. 5A). Therefore, the correlation of an increase of nuclear ORF1p with a decrease in nuclear circularity as well as the restoration of nuclear deformations by blockade of ORF1p nuclear import in stress conditions indicates that nuclear ORF1p causally participates in the loss of NE integrity. It is noteworthy that ORF2p reverse transcriptase 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. 6A–C). This indicates that the effect observed might not involve ORF2p activity.

Since the nuclear lamina plays an important role in anchoring heterochromatin at the inner NE (= nuclear lamina) (Misteli, 2007) and since the alteration of nuclear membrane proteins disrupts the peripheral localization of heterochromatin and can lead to its detachment (Chang et al., 2022); we examined whether the observed alteration of the nuclear lamina was accompanied by heterochromatin disorganization by using immunostaining of the heterochromatin markers HP1 (heterochromatin 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. 5B) and H4K20me3 levels decreased in stress conditions (Fig. 5C) suggesting possible decondensation or detachment of heterochromatin from the inner NE and favoring conditions allowing further activation of LINE-1 expression(Ren et al., 2021).

### 3.5. Nuclear envelope targeting by ORF1p is accompanied by nucleocytoplasmic 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 (Suppl. Fig. 7A) 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 (Zhang et al., 2015). RanGAP1 accumulated in the cytoplasm, shown by increased intensity, number and dot volume in the cytoplasm (Suppl. Fig. 7B) which is indicative of an alteration of NCT. Disruption of NCT can lead to the abnormal localization of the RNA binding protein TDP-43 (TAR DNA-binding protein 43), a predominantly nuclear protein. Mutations in TDP-43 gene (TARDBP) are causal in familial forms of FTD (Frontotemporal dementia) and ALS and a change from its predominantly nuclear to cytoplasmic localization has been observed in several NDs (Gao et al., 2018; Lee et al., 2019). Current consensus suggests that TDP-43 misdistribution leads to a pathological gain-of-function in the cytoplasm and loss-of-function in the nucleus (Neumann et al., 2006; Aizawa et al., 2019) 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 (Suppl. Fig. 7C). NUP153 is a nucleoporin which is involved in NCT and its cytoplasmic mislocalization and/or intranuclear accumulation are associated with NCT defects in various neurodegenerative contexts (Zhang et al., 2015; Chou et al., 2018; Lin et al., 2021). The quantification of NUP153 also showed an abnormal intranuclear and cytoplasmic accumulation of NUP153 in stress conditions (Suppl. Fig. 7D).

### 3.6. ORF1p overexpression leads to nuclear envelope dysmorphology and nucleocytoplasmic transport defects in the absence of stress

In order to evaluate whether NE dysmorphology induced by an increase in nuclear ORF1p could also be observed in the absence of arsenite-induced stress and was directly related to ORF1p, we performed gain of function experiments by nucleofection of a plasmid expressing HA-tagged ORF1p but not ORF2p. Dopaminergic neurons were nucleofected at day 3 of differentiation with a plasmid encoding either HA-ORF1 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 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 and establish a causal link between NE abnormalities and nuclear ORF1p content.

### 3.7. Rescue of nuclear envelope dysmorphology under stress conditions by remodelin

NE morphology is disrupted during aging (Scaffidi and Misteli, 2006) and in premature aging syndromes caused by laminopathies (Worman, 2012; Stiekema et al., 2020) including progeria syndromes (De Sandre-Giovannoli et al., 2003). In Hutchinson-Gilford progeria syndrome (HGPS), nuclear integrity was shown to be restored by the small molecule remodelin, an inhibitor of the acetyl-transferase protein NAT10 (Larrieu et al., 2018). 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 arsenite-induced stress in human neurons could be restored by remodelin. NE deformations (i.e
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<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**Fig. 5.** Restauration of nuclear circularity upon blockade of ORF1p nuclear import and analysis of chromatin repressive marks upon stress. (A) Quantification of ORF1p both in the nucleus and the cytoplasm and the nuclear circularity in the presence or the absence of import inhibitor drugs ivermectin (IV) and importazole (IMP) in stressed and control cells. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 2 independent experiments is shown, mean ± SEM, One-way ANOVA with Tukey's multiple comparisons test. (B) Immunostaining of HP1 in stressed cells and the ratio of HP1 intensity in the nucleoplasm over the nuclear periphery (inner NE). n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments is shown, mean ± SEM, two-tailed t test. (H) Immunostaining of H4K20me3 repressive histone mark in stressed and control cells and its quantification in the nucleus. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments is shown, mean ± SEM, two-tailed t test (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001). Scale bar, 5 µm.</figcaption></figure>

invaginations) were indeed 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 Fig. 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 (Larrieu et al., 2018; Larrieu et al., 2014), we examined H4K20me3, known to repress LINE-1 elements (Ren et al., 2021; Rangasamy, 2013). As shown in Fig. 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 blockade of nuclear ORF1p translocation.

4. Discussion

In recent years, TEs have emerged as possible new pathogenic players in NDs (Blaudin de Thé et al., 2018; Li et al., 2012; Sun et al., 2018; Rekaik et al., 2015; Peze-Heidsieck et al., 2022; Casale et al., 2021; Tam et al., 2019; Ravel-Godreuil et al., 2021). 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 described as an RNA chaperone with cis-preference for its own RNA and a necessary component of the LINE-1 ribonucleoprotein particle (RNP) for retrotransposition (Martin, 2006). Previous work suggests that
<figure><img src="image_20.png" /><figcaption>B</figcaption></figure>

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

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

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

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

<figure><img src="image_25.png" /></figure>

**Fig. 6.** LINE-1 over-expression following transfection leads to nuclear envelope deformation and nucleocytoplasmic transport defects in the absence of stress. (A) Co-Immunostaining of HA and Lamin B1 in cells transfected with pcDNA3-ORF1-HA or with pmaxGFP as a control. (B) Quantification of the nuclear circularity index, Lamin B1 intensity in the nucleoplasm (left panel; mean ± SEM, two-tailed *t* test) and correlation of HA-ORF1p intensity per cell in the nucleus with nuclear circularity (right panel; Spearman correlation). n > 90 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments is shown; (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001). (C) Immunostaining of HA and RanGAP1 in cells transfected with pcDNA3-ORF1-HA or with pmaxGFP as a control. (D) Quantification of cytoplasmic RanGAP1 intensity in transfected cells with pcDNA3-ORF1-HA or pmaxGFP as a control. n > 90 neurons were quantified per condition (3 wells per condition), mean ± SEM, two-tailed *t* test. (E) Immunostaining of HA and TDP-43 in cells transfected with pcDNA3-ORF1-HA or with pmaxGFP as a control. (F) Quantification of cytoplasmic and nuclear TDP-43 signal in cells transfected with pcDNA3-ORF1-HA or pmaxGFP as a control. n > 100 neurons were quantified per condition (3 wells per condition), mean ± SEM, two-tailed *t* test (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001). Scale bar, 5 µm.

ORF2p is not only a source of genomic instability (Belgnaoui et al., 2006; Blaudin de Thé et al., 2018; Simon et al., 2019), but can also trigger the innate immune response through reverse transcription of RNA generating cytoplasmic ssDNA (Simon et al., 2019; De Cecco et al., 2019;

Thomas et al., 2017). 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
<figure><img src="image_26.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>D</figcaption></figure>

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

**Fig. 7.** Restauration of nuclear alterations by the small molecule remodelin. (A) Immunostaining of ORF1p, Lamin B1 and H4K20me3 of neurons with or without remodelin pre-treatment 24 h before addition of arsenite or sham. (B) Quantification of nuclear circularity and Lamin B1 intensity in the nucleoplasm. (C) Quantification of ORF1p intensity in the nucleus, in the inner NE and in the cytoplasm. (D) Quantification of H4K20me3 intensity in the nucleus. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments is shown, mean ± SEM, One-way ANOVA with Tukey's multiple comparisons test (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p < 0.0001). Scale bar, 5 µm.

(Vylegzhanina et al., 2023) and/or its lesser abundance compared to ORF1p (1 ORF2p for ≈ 240 ORF1p molecules per LINE-1 RNP (Dai et al., 2014; Basame et al., 2006)). 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 important as accumulating data suggests that full-length LINE-1 are expressed, predominantly in epithelial cancers (Rangasamy et al., 2015) but also at steady-state in mouse neurons (Blaudin de Thé et al., 2018; Bonnifet et al., 2023), human neurons (Macia et al., 2017) and post-mortem brain tissues (Sur et al., 2017).

In this study, we used a human neuronal model of increased LINE-1 activity as monitored by an increase in endogenous ORF1p and LINE-1 RNA expression and ORF2p-dependent genomic instability 1 h after treatment. This rapid increase in LINE-1 expression upon cell stress is similar to that observed in other neuronal cells and experimental

models. Indeed, a rapid increase in LINE-1 levels was also observed in our previous study. In mouse primary midbrain neurons, LINE-1 RNA increased significantly as early as 10 min and DNA damage appeared as early as 30 min. In mouse dopaminergic neurons *in vivo*, LINE-1 RNA was significantly increased at 1 h after stereotaxic injection of the toxin 6-OHDA (Blaudin de Thé et al., 2018). In BE(2)C neuroblastoma cells (Giorgi et al., 2011), addition of H₂O₂ for 1 h induced an increase in retrotransposition and expression of endogenous LINE-1 transcripts accompanied by an increase in γH2AX foci. These data suggest that rapid LINE-1 activation in neurons triggered by different stressor might be an early stress-response similar to the one elicited by immediate early genes.

We observed not only an increase of ORF1p in the cytoplasm, but also a striking translocation of ORF1p to the nucleus upon exposure to sodium arsenite. Current consensus postulates that LINE-1 encoded proteins enter the nucleus only during cell cycle (Mita et al., 2018)
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 of human cells (Mita et al., 2018; Idica et al., 2017; Macia et al., 2017; Kubo et al., 2006; Shi et al., 2007; Xie et al., 2013; Taylor et al., 2013) in a neurodegenerative context (Pereira et al., 2018) and in post-mortem human brain tissues (Sur et al., 2017). 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 (Blaudin de Thé et al., 2018), and arsenite-induced stress dramatically increases the nuclear translocation of ORF1p. The most probable mode of import for ORF1p is the importin pathway. Indeed, ORF1p, as we show here in neurons and others have shown in HeLa cells (Freeman et al., 2019) binds directly to the importin alpha and beta family members, KPNA2 and KPNB1, which are transport proteins carrying cargo proteins through the NPC. This importin-assisted mode of nuclear entry is widely used by several viral proteins (Twyffels et al., 2014; Kimura et al., 2013), certain retrotransposons (i.e Ty1 (Manhas et al., 2018), LINE-1 ORF1p itself (Idica et al., 2017) but also a pathological form of Tau which aggregates in neurodegeneration (Candia et al., 2022). 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, i. e. the interaction with a KPNA family member most probably via a non-canonical NLS. Previously, Freeman et al. (Freeman et al., 2019) demonstrated using mutation and deletion analyses that charged amino acids in the C-terminus of ORF1p influence its subcellular localization and its interaction with KPNA family of nuclear pore proteins. Mutating different amino acids yielded similar effects on ORF1p localization, indicating the importance of overall charged residues for ORF1p nuclear localization. These findings supported the presence of a non-canonical NLS and redundancy in the ORF1p interaction with the nuclear pore machinery. The idea of a non-canonical NLS is supported by our data. It is noteworthy that ORF1p, in a stress-dependent manner, accumulates at the NE and co-localizes with and binds directly to the NPC protein NUP153, which might also contribute to nuclear import of ORF1p upon stress.

The small transcriptional increase in LINE-1 RNA following arsenite treatment suggests that the overall increase in ORF1p could be partially transcriptional and partially post-transcriptional. Once ORF1p translocates to the nucleus, it accumulates at the inner NE by directly binding to Lamin B1 in a stress-specific manner. The nuclear translocation of ORF1p is paralleled by NE dysmorphology including invaginations and blebs which significantly correlated with ORF1p intensity, both in the nucleus and the inner NE. This suggests that it is the stress-dependent nuclear translocation of ORF1p that drives NE dysmorphology. This conclusion is also supported by the fact that nuclear dysmorphologies under stress are prevented when ORF1p nuclear import is blocked by nuclear import inhibitors or, through unknown mechanisms, by remodelin. A direct functional role of ORF1p in NE dysmorphology, independent of arsenite-induced stress, is further supported by 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 is not dependent on the presence of the entire RNP or ORF2p. Nuclear localization and the correlation of ORF1p nuclear intensity with the loss of nuclear circularity in neurons of PD patients suggest that ORF1p might fulfil a similar pathological function in neurodegenerative diseases. We speculate that NE dysmorphology is mediated by the direct binding of ORF1p to Lamin B1 and its delocalization from the NE to the nucleoplasm. Indeed, LaminB1 increase and its displacement from the nuclear periphery to the nucleoplasm, as observed in the context of Huntington's disease (HD), are sufficient to induce NE dysmorphology (Alcalá-Vida et al., 2021).

NE dysmorphology is associated with NCT deficiencies (Paonessa et al., 2019) and both have been described to be disturbed in the context of NDs (Liu et al., 2012; Aizawa et al., 2019; Chou et al., 2018; Kim and

Taylor, 2017; Kinoshita et al., 2009; Sheffield et al., 2006; Gasset-Rosa et al., 2017; Grima et al., 2017). Indeed, nuclear translocation of ORF1p in neurons was accompanied by NCT deficiencies as revealed by the mislocalization and abnormal or compartmentalized accumulation of certain proteins involved in NCT function, some of which interact with ORF1p directly (KPNB1 and NUP153). Possible KPNB1 sequestration by binding to ORF1p might alter nuclear import but could also inhibit its important disaggregate function (Guo et al., 2019). 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 (Savas et al., 2012; Toyama et al., 2013) and rely on cell division for their renewal. Post-mitotic neurons do not divide and nucleoporins therefore are not renewed (D'Angelo et al., 2009; Hetzer, 2010). 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 (D'Angelo et al., 2009). Abnormal intranuclear and cytoplasmic accumulation of certain nucleoporins, as we observed for NUP153 in stressed neurons, has also been described in the context of other NDs (i.e, HD) (Grima et al., 2017). Possible cytoplasmic sequestration of NUP98 by tau has also been suggested to lead to NCT perturbation in AD (Eftekharzadeh et al., 2018). Similarly, delocalization and abnormal cytoplasmic accumulation of RanGAP1 we observed in stressed neurons, have also been described in several ND-affected tissues (Zhang et al., 2015). Our results suggest that ORF1p might act as a sponge to alter the activity of certain proteins involved in NCT. Together, our results suggest a possible scenario in which ORF1p might sequester and/or mislocalize NE components such as Lamin B1 or NUP153.

NCT deficiencies have been consistently linked to the abnormal subcellular distribution of TDP-43, a nuclear protein involved in RNA metabolism (Buratti and Baralle, 2010; Colombrita et al., 2012), mutated in ALS/FTD (Barmada et al., 2010; Benajiba et al., 2009; Kabashi et al., 2008; Sreedharan et al., 2008) and mislocalized to the cytoplasm in numerous NDs (Zhang et al., 2015; Neumann et al., 2006; Aizawa et al., 2019) not linked to TDP-43 mutations. Indeed, in our model, NCT deficiencies resulted in the cytoplasmic accumulation of TDP-43. TDP-43 exits the nucleus through passive (Duan et al., 2022) or active nuclear export (Winton et al., 2008) 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 (Ederle and Dormann, 2017). A direct functional role of ORF1p in NCT disruption, independent of arsenite-induced stress, was further corroborated by ORF1p gain of function experiments showing that neurons over-expressing ORF1p (and not ORF2p) present NCT defects. We propose that the mechanism behind NCT defects involves ORF1p directly binding to nuclear import (KPNB1), the nuclear pore complex (NUP153), potentially resulting in their delocalization and/or sequestration.

NE alteration and NCT defects are emerging pathogenic features in several NDs (Kim and Taylor, 2017; Eftekharzadeh et al., 2018; Frost, 2016) including HD (Gasset-Rosa et al., 2017), ALS/FTD (Chou et al., 2018), PD (Liu et al., 2012), AD (Eftekharzadeh et al., 2018; Frost, 2016) and more generally in the context of tauopathies (Prissette et al., 2022). For example, aggregated Tau directly interacts with the nuclear pore complex (Eftekharzadeh et al., 2018) and oligomeric Tau with Lamin proteins (Jiang and Wolozin, 2021) resulting in loss of nuclear integrity including NCT failure (Paonessa et al., 2019), 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 (Martins et al., 2020) including in the brain (Mertens et al., 2015) with associated changes in chromatin organization and NCT dysfunction (Frost, 2016; Mertens et al., 2015; Oberdoerffer and Sinclair, 2007; Jovićić et al., 2015). Progerin, a mutant form of Lamin A, is the cause of HGPS. Similar to mutant Tau, it accumulates at the NE and disrupts NE morphology, NCT and chromatin organization (Larrieu et al., 2018). In
cellular and mouse models of accelerated aging, these alterations can be successfully corrected by treatment with the small molecule remodelin (Larrieu et al., 2018; Larrieu et al., 2014). Remodelin acts by inhibiting the acetyltransferase NAT10 (Larrieu et al., 2018). In our model of 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 (Chang et al., 2022) and potentially cell type specific gene expression (Perovanovic et al., 2016). 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 (Ren et al., 2021). Indeed, arsenite-induced stress triggered 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 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 (Traxler et al., 2023). Indeed, mutated Tau can lead to chromatin relaxation, re-expression of certain fetal genes in the brain in a fly model (Frost et al., 2014) and activation of TEs (Guo et al., 2018; Frost et al., 2014). 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 (Richards and Elgin, 2002; Black et al., 2012)) 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 stress-induced 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 (Denais et al., 2016; Nava et al., 2020; Raab et al., 2016).

Recent studies have shown that aggregated proteins such as Tau (Candia et al., 2022; Hochmair et al., 2022), mutant Huntingtin (Gasset-Rosa et al., 2017) and C9orf72 poly-GA proteins (Zhang et al., 2015) 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 (Gasset-Rosa et al., 2019). 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 (Sil, 2022). 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.

Our data suggests that ORF1p might play a pathogenic role by targeting the NE. LINE-1 activation is a recurring observation in several neurological diseases (Shpyleva et al., 2018; Bundo et al., 2014; Doyle et al., 2017; Liu et al., 2016; Muotri et al., 2010; Zhao et al., 2019; Coufal et al., 2011), in aging (Simon et al., 2019) and more generally upon stress (Blaudin de Thé et al., 2018) and if this activation includes full-length LINE-1 elements with coding potential, this will result in increased levels of 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 could trigger the nuclear translocation of ORF1p causally contributing to NE deformation and NCT dysfunction. One consequence we identified is the cytoplasmic accumulation of TDP-43 which could favor its aggregation (Van Eersel et al., 2011), one of the hallmarks of several NDs (Zhang et al., 2015; Neumann et al., 2006; Aizawa et al., 2019).

Activation of LINE-1 elements in neurons, through ORF2p-induced genomic instability (Blaudin de Thé et al., 2018), ORF2p-induced inflammation (Simon et al., 2019; De Cecco et al., 2019; Thomas et al., 2017) or LINE-1 RNA mediated heterochromatin erosion (Della Valle et al., 2020) and, as we show here, through the ORF1p-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.

### CRediT authorship contribution statement

**Rania Znaidi:** Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. **Olivia Massiani-Beaudoin:** Investigation. **Philippe Mailly:** Methodology, Software. **Héloïse Monnet:** Methodology, Software. **Tom Bonnifet:** Investigation. **Rajiv L. Joshi:** Conceptualization, Supervision, Writing – original draft, Writing – review & editing, Project administration. **Julia Fuchs:** Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

RZ acknowledges the Fondation Recherche Alzheimer (FRA) for a Ph.D. fellowship and was enrolled with the Ecole Doctorale ED3C at Sorbonne University. We are very grateful to Raphaël Rodriguez and Ludovic Colombeau for having synthetized and provided the small molecule remodelin. We sincerely thank Dr. J. Moran and Dr. Y. Ariumi for kindly providing the pcDNA3-ORF1-HA plasmid. 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, the National French Agency for Research (ANR-20-CE16-0022 NEUR-AGE) and the Fondation Alzheimer. 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. Human post-mortem brain material and associated data were obtained from the "Brainbank Neuro-CEB Neuropathology Network" which is supported by ARSLA, CSC, France DFT, Fondation ARSEP, Fondation Vaincre Alzheimer, France Parkinson.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.brainres.2025.149579.

### Data availability

Data will be made available on request.
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