# Severe dynein dysfunction in cholinergic neurons exacerbates ALS-like phenotypes in a new mouse model

Fabio A Simoes, Eleni Christoforidou, Raphaelle Cassel, Luc Dupuis, Majid Hafezparast

## ► To cite this version:

Fabio A Simoes, Eleni Christoforidou, Raphaelle Cassel, Luc Dupuis, Majid Hafezparast. Severe dynein dysfunction in cholinergic neurons exacerbates ALS-like phenotypes in a new mouse model. Biochimica et Biophysica Acta - Molecular Basis of Disease, 2025, 1871 (1), pp.167540. 10.1016/j.bbadis.2024.167540. inserm-05509171

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# Severe dynein dysfunction in cholinergic neurons exacerbates ALS-like phenotypes in a new mouse model

Fabio A. Simoes<sup>a,1</sup>, Eleni Christoforidou<sup>a,1</sup>, Raphaelle Cassel<sup>b</sup>, Luc Dupuis<sup>b</sup>, Majid Hafezparast<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Dynein

Amyotrophic lateral sclerosis

ALS

TDP-43

Neuromuscular junctions

Motor neurons

MND

## ABSTRACT

Cytoplasmic dynein 1, a motor protein essential for retrograde axonal transport, is increasingly implicated in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this study, we developed a novel mouse model that combines the *Legs at odd angles* (*Loa*, F580Y) point mutation in the dynein heavy chain with a cholinergic neuron-specific knockout of the dynein heavy chain. This model, for the first time, allows us to investigate the impact of *Loa* allele exclusivity in these neurons into adulthood. Our findings reveal that this selective increase in dynein dysfunction exacerbated the phenotypes observed in heterozygous *Loa* mice including pre-wean survival, reduced body weight and grip strength. Additionally, it induced ALS-like pathology in neuromuscular junctions (NMJs) not seen in heterozygous *Loa* mice. Notably, we also found a previously unobserved significant increase in neurons displaying TDP-43 puncta in both *Loa* mutants, suggesting early TDP-43 mislocalisation – a hallmark of ALS. The novel model also exhibited a concurrent rise in p62 puncta that did not co-localise with TDP-43, indicating broader impairments in autophagic clearance mechanisms. Overall, this new model underscores the fact that dynein impairment alone can induce ALS-like pathology and provides a valuable platform to further explore the role of dynein in ALS.

## 1. Introduction

Cytoplasmic dynein 1 (hereafter referred to as dynein), is a large (~1.5 MDa) ATP-dependent motor protein complex assembled around a conserved homodimer of heavy chains encoded by the *DYNC1H1* gene [1]. The N-terminal tail of each heavy chain interacts with an intermediate chain and a light intermediate chain, with the former further binding three light chains [2–4]. Towards the C-terminus of each heavy chain lies the motor domain consisting of six ATPase domains associated with diverse cellular activities (AAA+) and a microtubule binding stalk region, allowing dynein to move along microtubules towards their minus end. However, efficient dynein processivity along microtubules also requires the binding of adaptor proteins, of which dynactin is the most critical [4,5]. In neurons, dynein is responsible for the retrograde axonal transport of various cargoes including membranous organelles like mitochondria, signalling complexes, proteins, and mRNA [6].

Both retrograde and anterograde axonal transport deficits have been

observed as an early pathology in amyotrophic lateral sclerosis (ALS) [7]. ALS is a late onset, rapidly progressive and invariably fatal motor neuron disease (MND) characterised by the degeneration of both upper and lower motor neurons [8]. Approximately 10 % of ALS cases exhibit mendelian inheritance (familial ALS; fALS), with the remaining 90 % of cases occurring sporadically (sporadic ALS; sALS), and of these, only about 15 % are explained by mutations in known ALS genes [9].

Axonal transport deficits are seen in transgenic mice overexpressing disease-causing mutant forms of superoxide dismutase 1 (SOD1) [10–17]. Similar observations have also been made in mice over-expressing wildtype or disease-associated mutant forms of the gene encoding TAR DNA binding protein 43 (TDP-43) [16,18,19]. In patients, proximal axonal swellings and axonal spheroids in motor neurons are highly indicative of axonal transport deficits [20–24]. Additionally, mutations in the *KIF5A* gene, a member of the anterograde motor kinesin family, have been linked to the development of ALS [25]. Specifically, mutations are clustered on the C-terminal cargo-binding

<footnote><sup>a</sup> Department of Neuroscience, School of Life Sciences, University of Sussex, Brighton, United Kingdom</footnote>

<footnote><sup>b</sup> University of Strasbourg, INSERM, UMR-S1329, Strasbourg, France</footnote>

<footnote>* Corresponding author.
E-mail address: M.Hafezparast@sussex.ac.uk (M. Hafezparast).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
domain of the KIF5A protein. On the other hand, the hallmark deposition of aggregated proteins in ALS, the most common of which is TDP-43, indicates a failure of clearance mechanisms such as autophagy which is intimately reliant on retrograde transport [26–29].

Given that age is a major risk factor for ALS and that dynein function has been shown to attenuate with age, it is possible that dynein is able to act as a disease modifier in sporadic ALS [30–33]. Significantly, ALS-associated mutations have been identified in the dynactin 1 (also called p150<sup>Glued</sup>) subunit (DCTN1) of dynactin [5,34,35]. Furthermore, mutations in *DYNC1H1* are known to cause an autosomal dominant congenital motor neuron disease (spinal muscular atrophy, lower extremity predominant 1; SMALED1) [36].

An important tool in investigating the role of dynein in the degeneration of motor neurons has been the *Legs at odd angles* (*Loa*) mouse model, which harbours a F580Y substitution in *DYNC1H1*. *Loa* heterozygous mice exhibit an age-dependent progressive muscle atrophy and reduced locomotor ability [37]. The *Loa* mutation causes a loss of spinal motor neurons in homozygous embryos and trigeminal motor neurons in adult heterozygotes [37,38], which also involves proprioceptive sensory neuron loss [39–41]. At the molecular level, the *Loa* mutation leads to impaired dynein complex integrity and defective retrograde transport [37,42–44]. However, *Loa* homozygous mice do not survive beyond 24 h after birth [37], precluding the possibility of determining if ALS-like pathology emerges with age in these mice.

Recently, we generated a floxed allele of *Dync1h1* [45], that allows for the deletion of the ATP-binding module of dynein heavy chain, thereby knocking out dynein motor activity. Here, we took advantage of this new allele to generate mice which are hemizygous for the *Loa* mutation in cholinergic motor neurons using Chat-CRE expressing mice.

This approach has allowed us, for the first time, to study cholinergic neurons including motor neurons that exclusively express the *Loa* mutant dynein into adulthood (*Dync1h1<sup>-/Loa</sup>* mice) and thus enabling us to examine the dose-dependent correlation of loss of dynein function with ALS pathology. As such, our investigation in this study primarily focused on comparative analysis of the phenotypes between *Dync1h1<sup>-/Loa</sup>* versus *Dync1h1<sup>+/-Loa</sup>* mice, and how their phenotypes compare with the wildtype (*Dync1h1<sup>+/+</sup>* mice. We found that restricted *Loa* hemizygosity to cholinergic neurons was sufficient to cause a more severe phenotype including reduced pre-wean survival, greater weight loss, and weakened grip strength. Additionally, analysis of NMJs and TDP-43 distribution for the first time in either *Loa* mutant showed that only the more severe *Dync1h1<sup>-/Loa</sup>* mouse model exhibited denervation and fragmentation of NMJ endplates and an increase in neurons with TDP-43 puncta. This is particularly significant given that these resemble pathologies in motor neuron diseases like ALS.

## 2. Materials and methods

### 2.1. Animals

Procedures involving animals were approved by the UK Home Office under the UK Animals (Scientific Procedures) Act 1986 and the University of Sussex Animal Welfare and Ethics Review Board. Mice were on a C57Bl/6 J background and were housed under a 12-h light-dark cycle (lights on at 7 am) with free access to food and water and with up to five littermates. To generate mice with hemizygous *Loa* mice (*Dync1h1<sup>-/Loa</sup>*) a 3-step breeding program was carried out (Fig. 1A). *Loa* heterozygous mice [described in [37]] were bred with Chat-*IRES-Cre* knock-in mice

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Survival and body weight of male mice with varying levels of functional dynein. (A) Percentage survival of pups of undetermined sex up to 21 days old (weaning age). Data is from all the mice born and genotyped since the creation of these mouse lines until the experiments were completed. Log-rank Mantel-Cox test, *P* < 0.0001. Pairwise comparisons (Marascuilo Procedure): *Dync1h1<sup>+/+</sup>* vs *Dync1h1<sup>+/-Loa</sup>*; *P* < 0.01, *Dync1h1<sup>+/+</sup>* vs *Dync1h1<sup>-/Loa</sup>*; *P* < 0.0001, *Dync1h1<sup>+/-Loa</sup>* vs *Dync1h1<sup>-/Loa</sup>*; *P* < 0.01. (B) Body weight of male mice with varying levels of functional dynein. Measurements were taken once a month up to 52 weeks of age. Data is shown as mean ± SEM and was analysed by fitting a mixed model using Restricted Maximum Likelihood (REML) with Geisser-Greenhouse correction and Tukey's post-hoc. *N* = 10–20 mice (N-numbers per age and genotype are shown in Table S1). (C) Relationship between age and mean male body weight increase (*Dync1h1<sup>+/-Loa</sup>* – *Dync1h1<sup>-/Loa</sup>*). Second-order (quadratic) polynomial regression fit (solid curve) and 95 % confidence bands (dotted curves). Adjusted R² = 0.8404. Outliers are shown in red.</figcaption></figure>
(JAX#006410), which carry the *Cre* transgene after the *Chat* stop codon in all cells, to transition them to a homozygous *Chat-IRES*-Cre background (*Dync1h1<sup>+</sup>/Loa; Chat<sup>Cre/Cre</sup>*). These mutant offspring were then bred to mice carrying one floxed *Dync1h1* allele (*Dync1h1<sup>+</sup>/Flx*) [described in [45]]. Briefly, the latter mice contain LoxP sites flanking exons 24 and 25 of the *Dync1h1* gene in all cells. Therefore, since expression of the Cre recombinase enzyme is conditional to the expression of the *Chat* gene, this breeding scheme generated mice with one dynein allele knocked out only in *ChAT<sup>+</sup>* cells. This final cross generated either wildtypes (*Dync1h1<sup>+</sup>/*+*), heterozygous mutants (*Dync1h1<sup>+</sup>/Loa*), hemizygous wildtypes (*Dync1h1<sup>-/+</sup>*) or hemizygous mutants (*Dync1h1<sup>-/Loa</sup>*) on a *Chat-Cre* background.

## 2.2. Genotyping

All genotyping was performed from DNA extracted from ear biopsies aside from when performed post-mortem to determine pre-wean mortality in which case a tail biopsy was used. DNA was used in a 1:10 dilution following extraction by overnight incubation at 55 °C in 200 µL lysis buffer (100 mM Tris-HCl at pH 8.0, 5 mM EDTA, 0.2 % SDS and 200 mM NaCl) and 2 µL Proteinase K (20 mg mL⁻¹)]. The presence of the *Loa* mutation was confirmed by PCR using the HS Taq/Pfu PCR Mix (Apto-Gen, 414,500.5.1A25; forward primer: 5'-AAG CAC CTG AAC CCC AAG AC-3', reverse primer: 5'-GTA GGA CTC CCA CAC CAA CG-3') followed by a restriction digest using *RsaI* restriction enzyme, as the *Loa* mutation introduces a second *RsaI* restriction site in the amplicon. For confirmation of the presence of the *Cre* transgene a multiplex PCR was used in which a forward primer (5'-GCA AAG AGA CCT CAT CTG TGG A-3') and a reverse primer (5'-CAA AAG CGC TCT GAA GTT CCT-3') targeted exon 16 of the *Chat* gene and flanked the transgene, while an internal forward primer (5'-CAG GGT TAG TAG GGG CTG AC-3') targeted the transgene itself and worked in combination with the reverse primer. PCR was also used to confirm the presence of the LoxP sites (forward primer: 5'-GGA TTT TGA TGG CCA ACG CAC TGA G-3', reverse primer: 5'-CTC ATA CAA ACA TCT GCC AGG GCC TG-3').

## 2.3. Confirmation of recombination of the floxed *Dync1h1* allele

DNA was extracted from ~10 mg of thoracic (L3-L6) spinal cord tissue using the Monarch® Genomic DNA purification kit (New England Biolabs, #T3010) as per the manufacturer's instructions. PCR was performed using a forward primer (5'-GCT GCC TGA ACT CCC AGT TTT CCG-3') that anneals to intron 23 of *Dync1h1* and a reverse primer (5'-CTG TGT CAG TTG CAG ACA GTT TCT ACG-3') that anneals to intron 25. Without recombination and excision of exons 24 and 25 the product from the conditional knockout is 1435 bp, while following the excision of these exons the product from the knockout is 372 bp. The product of wildtype DNA is 1164 bp.

## 2.4. Grip strength and rotarod testing

Mice underwent grip strength testing and rotarod testing every 4 weeks starting at 4 weeks of age until 52 weeks of age. Due to attrition from natural causes unrelated to the study, not all mice completed tests at all age points. To maintain adequate sample sizes, newly born mice were enrolled in the study as they reached the starting age. Both tests were performed on the same day with a rest period of at least 15 min in between, and mice were weighed prior to testing. Mice were placed in the experimental room for 15 min prior to the start of testing for acclimatisation. Additionally, to mitigate potential stress, mice were handled using a tunnel when being placed on and removed from the rotarod.

Grip strength was tested using Kondziela's inverted screen test [46]. Mice were acclimatised to the grid and the procedure 2 days prior to the beginning of testing. For testing, mice were placed in the centre of a wire mesh grid and the grid was inverted slowly until the mice were suspended upside down. Mice were left suspended for up to 60 s and the

time taken to fall was recorded. For each mouse, the time was an average of three trials performed with 10-min rest intervals between trials.

For rotarod testing, mice underwent two pre-training sessions to mitigate anxiety caused by the procedure. In these sessions, mice were placed on the stationary rod and then underwent two training trials with the rod accelerating. Experimental tests were performed with the rotarod accelerating up to 40 rpm over the course of 5 min, with time taken to fall recorded. Each test was an average of three trials separated by 15-min rest intervals. A mouse holding on to the rotarod for two complete revolutions was counted as a fall.

## 2.5. Tissue collection

At either 8 weeks or 52 weeks of age, animals were anaesthetised with an overdose of pentobarbital and transcardially perfused using a peristaltic pump with oxygenated artificial cerebrospinal fluid (125 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 2 mM MgCl₂·6H₂O, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄·H₂O, 25 mM C₆H₁₂O₆). The vertebral column was dissected and cut cross-sectionally between vertebrae T9 and T10 and between vertebrae L6 and S1. The spinal cord was dissected out of the cervical and thoracic portion of the vertebrae and snap-frozen in liquid nitrogen. The lumbar portion of the vertebral column (T10-L6) was drop-fixed in 4 % formaldehyde for 24 h, followed by removal of the spinal cord from the vertebrae which was then stored in 30 % sucrose with 0.2 % sodium azide at 4 °C until cryosectioning. The hind limbs were collected for dissection of lumbrical muscles and a tail biopsy was taken for genotype confirmation.

## 2.6. Immunostaining of neuromuscular junctions in whole mounted lumbrical muscles

Lumbrical muscles were dissected from 8- and 52-week-old mice as previously described [47] and fixed for 2 min with 4 % formaldehyde. Four random muscles per animal were immunostained as indicated in [47], with some modifications. Briefly, innervating axons were labelled by targeting neurofilaments with a mouse anti-2H3 antibody (DSHB, AB_2314897; 1:50 dilution), while pre-synaptic nerve terminals were labelled by staining for synaptic vesicles using a mouse anti-SV2 antibody (DSHB, AB_2315387; 1:100 dilution). Both were visualised together with an anti-mouse AlexaFluor 647 secondary antibody (Invitrogen, A32728; 1:250 dilution). Post-synaptic acetylcholine receptors were labelled with Tetramethylrhodamine-Alpha-bungarotoxin (α-BTX; Cambridge Biosciences, BT00012; 1:500 dilution).

## 2.7. Morphometric analysis of neuromuscular junctions

Individual motor endplates were cropped, and the z-stacks were converted to maximum projection images, with the AChR and SV2 colour channels separated. The morphology of each endplate was automatically measured using custom ImageJ and MATLAB code written specifically for this data set [available at [48]]. The morphology parameters included: (1) area of the endplate, (2) area of AChR staining, (3) compactness of the endplate, calculated as the percentage of the AChR convex hull area that is occupied by AChR staining, (4) endplate fragmentation, which is the number of individual AChR islands within a single endplate, and (5) endplate coverage, calculated as the percentage of the AChR staining that co-localises with SV2 staining. An illustration of each parameter is shown in Fig. S1A-J. For the quantification of endplate coverage, a subset of endplates was excluded due to inadequacies in presynaptic staining, which manifested as either too faint or too intense (overexposed). These exclusions were necessary because the binary images derived from the original microscopy images did not adequately represent the original staining in these cases and could thus not be used for accurate quantification of the endplate coverage. The exclusion was conducted blinded to mouse genotype. A minor percentage (8 % at 8 weeks old and 4 % at 52 weeks old) of endplates were
affected by this criterion.

## 2.8. Cryosectioning

Fixed spinal cords were mounted in optimal cutting temperature (OCT) compound (Agar Scientific, AGR1180) and sliced cross-sectionally in 30 µm-thick sections using a cryostat at -17 °C. Each section was kept in antifreeze buffer (30 % ethyleneglycol, 30 % glycerol, 192.5 mM NaOH, 244 mM $NaH_2PO_4$) at 4 °C until immunostaining.

## 2.9. Immunohistochemistry of spinal cord sections

One section per lumbar L3-L6 segment was immunostained for TDP-43, p62, and ChAT per animal. Briefly, antigen retrieval was performed with sodium citrate buffer (10 mM Sodium citrate, 0.05 % Tween 20, pH 6.0) prewarmed to 80-95 °C for 30 min. Permeabilization was performed with 0.1 % Triton X-100 for 10 min at 100 rpm. Blocking was performed with PBS containing 0.5 % Triton X-100 and 5 % horse serum (Fisher, 11,520,516) for 90 min at 100 rpm. Each section was then probed overnight in a humidity chamber at 4 °C in PBS containing blocking solution (0.1 % Triton X-100, 1 % horse serum) for TDP-43 (1:100, Proteintech, 10,782-2-AP), ChAT (1:50, Merck/Sigma, AB144P) and the C-terminal domain (20 amino acids: C-NYD IGA ALD TIQ YSK HPP PL) of p62 (1:500, Progen, GP62-C). Following PBS washes, secondary probing was performed for 3 h in the dark in PBS containing 0.1 % Triton X-100 and the relevant secondary antibodies for TDP-43 (1:500, Alexa Fluor 647, Jackson Immuno research, 711-605-152), ChAT (1:500, Alexa Fluor 488, Life Technologies, A11055), and p62 [1:500, Rhodamine (TRITC), Jackson Immuno research, 706-025-148-JIR]. Final washes were performed, with the addition of DAPI stain (Sigma, D9542) at 2 µg mL⁻¹ final concentration during the second wash only, and the slides were sealed with mounting medium (Fisher, 11,539,306).

## 2.10. Analysis of protein distribution in neurons from spinal cord tissue

Z-stack images of immunohistochemically labelled spinal cord sections were converted to maximum projection images. Cholinergic spinal motor neurons were identified by their location in the ventral horn and strong ChAT staining. The soma of $ChAT^+$ cells and their nuclei ($DAPI^+$) were outlined manually using the freehand selection tool in ImageJ, and regions of interest were created to represent the soma area, nucleus area, and cytoplasm area (i.e., soma area minus nucleus area). Then, the fluorescence intensity values of proteins of interest were calculated (following background subtraction which was performed by measuring and subtracting the fluorescence intensity from manually selected background regions of interest) using custom ImageJ and MATLAB code written specifically for this data set (available at [48]). For analysis of punctate staining, the images were thresholded using the Minimum method [49] and all cytoplasmic puncta with area $≥ 0.36 µm^2$ were measured.

## 2.11. Motor neuron counts

Five sections from L3, ten from L4, twenty from L5, and five from L6 per animal were Nissl-stained. Briefly, sections were baked overnight at 37 °C then de-myelinated through a series of alcohol washes as follows: 50 % ethanol, 70 % ethanol, 95 % ethanol (x2), 100 % ethanol (x3), all for 3 min, then xylene (x2) for 5 min. Sections were then rehydrated with 100 % ethanol (x3), 95 % ethanol (x2), 70 % ethanol, all for 3 min, rinsed in tap water for 30 s, then stained for 10 min with 0.1 % cresyl violet acetate solution (Fisher, 10,501,481) prewarmed to 37-50 °C. The stain was removed by rinsing in tap water and quickly dipping in 70 % ethanol, then the tissue was differentiated in 95 % ethanol, 1 % glacial acetic acid for 15 min, before dehydrating with 95 % ethanol for 1 min, 100 % ethanol for 1 min, and 100 % ethanol (x2) for 3 min. Finally, sections were cleared in xylene (x2) for 5 min, and mounted in

VectaMount (2bscientific, H-5000-60).

The counting procedure was based on a previously published protocol [50]. Two investigators performed the counting while blinded to genotype, and their counts were then averaged. As illustrated in Fig. S1K, a horizontal line below the central canal was drawn to separate the dorsal and ventral horns. Within the ventral horn, a line at a 45° angle towards the apex of the grey matter was drawn to create a triangle encompassing the side of the ventral horn containing the sciatic motor pool. Within this boundary, motor neurons were counted if they satisfied the following criteria: (1) multipolar (three or more projections), (2) large size, and (3) darker staining. The motor neurons were counted from both ventral horns of each spinal cord tissue section.

## 2.12. Microscopy

Fluorescent images were acquired blinded to genotype using a Leica SP8 confocal scanning electron microscope and captured using the LAS X version 3.4 software. For morphometric analysis of neuromuscular junctions, maximum intensity projections of z-stacks providing full coverage of motor endplate volume were acquired. For experiments using histologically stained spinal cord tissue, a single-frame image of both ventral horns was taken to allow for motor neuron counts. For experiments using immunostained spinal cord tissue, only one of the ventral horns per section was imaged at random.

## 2.13. Statistical analysis

Statistical analysis was performed using GraphPad Prism version 10.2.3 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com). Outliers were identified by the ROUT test and removed prior to statistical analysis. Survival analyses were conducted using Kaplan-Meier curves, and differences in survival between genotypes were assessed using the Log-rank (Mantel-Cox) test. Pairwise comparisons of pre-weaning survival rates among the four different genotypes were performed using the Marascuilo Procedure implemented through custom-written R code (version 4.2.2, [51]). Body weight, grip strength, and rotarod data were analysed by fitting a mixed model using Restricted Maximum Likelihood (REML) with Geisser-Greenhouse correction and Tukey's post-hoc. Neuromuscular junction data and immunohistochemistry data were analysed using nested one-way analysis of variance (ANOVA) with Tukey's post-hoc, with individual endplate/cell measurements nested within each mouse, and mice nested within their respective genotypes. For analysis of the proportion of cells exhibiting TDP-43 or p62 puncta, ordinary one-way ANOVAs were used. The threshold for significance was set to $P ≤ 0.05$. The relationship between age and the mean difference in body weight between $Dync1h1⁺/Loa$ and $Dync1h1⁻/Loa$ mice was modelled using a second order (quadratic) polynomial regression model in R. The age variable was included in the model both in its original form and squared, to account for potential nonlinear effects. The goodness of fit for the model was assessed by the coefficient of determination ($R^2$), and the statistical significance of the model parameters was determined using t-tests. Assumptions of the regression model, including linearity, independence, homoscedasticity, and normality, were visually and formally checked using residual plots, Durbin-Watson test for autocorrelation, and Q-Q plots, respectively.

# 3. Results

## 3.1. Generating hemizygous *Loa* mice

To generate mice which are hemizygous *Loa* in cholinergic neurons including motor neurons, we crossed $Dync1h1Flx$ mice [45] with Chat-CRE knock-in mice [52] that express the CRE recombinase in cholinergic neurons using the endogenous Chat promoter (Fig. S2). We previously used this CRE mouse line and showed its specific activity of
recombination in motor neurons [53,54]. When we generated homozygous *Dync1h1*<sup>Flx/Flx</sup> *Chat*<sup>+/-Cre</sup> mice, we observed death of pups within the first week of birth (median survival 4.5 days; Fig. S3), suggesting that proper dynein function was required for postnatal development of cholinergic neurons, including motor neurons. Additionally, we have previously shown that recombination of the floxed *Dync1h1* allele results in an approximately 50% reduction in protein production [45].

To confirm successful recombination of the floxed *Dync1h1* allele, PCR was performed on DNA extracted from the thoracic spinal cord, as the recombination is limited to cholinergic cells. As shown in Fig. S4, the recombined knockout allele (372 bp) was successfully detected in *Dync1h1<sup>-/Loa</sup>* mice.

## 3.2. Hemizygous *Loa* mice have a more severe reduction in survival, body weight, and grip strength

As shown in Fig. 1A, analysis of pre-weaned survival shows a significantly lower survival rate among pups of pre-wean age (21 days) in *Dync1h1<sup>-/Loa</sup>* (26% mortality), compared to *Dync1h1<sup>+/-</sup>* mice (1.7% mortality, *P* < 0.0001). Importantly, there is also a significantly reduced survival rate between *Dync1h1<sup>-/Loa</sup>* and *Dync1h1<sup>+/-Loa</sup>* mice (*P* < 0.01).

These findings further highlight the importance of fully functional dynein in cholinergic neurons in early postnatal development.

Following these initial survival assays, we will first present the results from male mice, followed by a comparison with the results from the female mice in the final section. Thus, our analysis of longitudinal assessment to determine the strength and motor phenotypes of male mice over 52 weeks revealed that *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice broadly exhibited a reduction in body weight relative to *Dync1h1<sup>+/-</sup>*

mice (Fig. 1B). At 4 weeks of age, only the *Dync1h1<sup>-/Loa</sup>* mice had a statistically significant reduction in body weight (*P* = 0.0023) in comparison to *Dync1h1<sup>+/-</sup>* mice. From 8 to 52 weeks of age, both *Loa* genotypes reached statistical significance, compared to *Dync1h1<sup>+/-</sup>* mice. Importantly, from 8 weeks onwards *Dync1h1<sup>-/Loa</sup>* mice appeared to diverge from the *Dync1h1<sup>+/-Loa</sup>* mice with ageing, finally reaching a statistically significant reduction in body weight at 40 (*P* = 0.0481), 48 (*P* = 0.0301) and 52 weeks of age (*P* = 0.0251). To further investigate this divergence, a quadratic polynomial regression model was fitted to the body weight data, and this showed that age explained 84.04% of variance in weight differences between *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice (adjusted R² = 0.8404). Importantly, this analysis allowed us to conclude that there is a significant increase in the difference in weight between *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice from about 14.28 weeks of age (Fig. 1C). Overall, this indicates a more severe early deficit in *Dync1h1<sup>-/Loa</sup>* mice in comparison to *Dync1h1<sup>+/-Loa</sup>* which, while admittedly subtle, progressively worsens as mice age.

Interestingly, a similar pattern was observed in the male mice when assessing grip strength using Kondziela's inverted screen test [46]. Briefly, mice were placed on a mesh screen which was then inverted, forcing the mice to hang upside down. The time taken to fall was measured as an indicator of strength. As expected, both *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice performed significantly worse than *Dync1h1<sup>+/-</sup>* mice throughout (Fig. 2A). However, *Dync1h1<sup>+/-Loa</sup>* mice showed a decline from 4 weeks to 16 weeks of age before reaching a plateau, while the decline in *Dync1h1<sup>-/Loa</sup>* mice was faster and the same plateau was reached by 8 weeks of age. Consequently, *Dync1h1<sup>-/Loa</sup>* mice performed significantly worse compared to *Dync1h1<sup>+/-Loa</sup>* mice at 8 and 12 weeks of age (*P* = 0.0157 and *P* = 0.007, respectively). While no difference is seen

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Motor function of male mice with varying levels of functional dynein. (A) Grip strength in males assessed by Kondziela's inverted screen test. If the mouse had not fallen by 60 s, the test was stopped. Lack of error bars for some *Dync1h1<sup>+/-</sup>* data points is due to all mice not falling during the duration of the test. (B) Motor coordination in males assessed by the accelerating rotarod test. If the mouse had not fallen by 5 min, the test was stopped. Data in A and B is shown as mean ± SEM and was analysed by fitting a mixed model using Restricted Maximum Likelihood (REML) with Geisser-Greenhouse correction and Tukey's post-hoc. *N* = 10–19 mice for the inverted screen test and *N* = 9–19 mice for the accelerating rotarod test (*N*-numbers per age and genotype are shown in Table S1). ns = not significant. Due to non-significant fixed effects of age for panel B, no significance symbols are shown at each age point. Instead, main genotype effects are shown next to panel B.</figcaption></figure>
between the two mutants between 16 and 48 weeks of age, by 52 weeks of age *Dync1h1<sup>-/Loa</sup>* mice had significantly lower latency to fall than *Dync1h1<sup>+/-Loa</sup>* mice (*P* = 0.0101). Importantly, these trends remained even after the data was normalised to body weight (Fig. S5), thereby suggesting that weight changes with ageing do not confound these genotype differences.

To further assess motor function and coordination, rotarod testing was performed. No significant interaction between genotype and age was found, with only genotype having significant main effect (*P* < 0.0001; Fig. 2B). Both male *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice performed significantly worse than *Dync1h1<sup>+/-</sup>* mice (*P* < 0.0001) but performed similarly to each other (*P* = 0.5459). Overall, this suggests that deficits in motor function and coordination are not progressive in the mutant mice.

Overall, in comparison to *Dync1h1<sup>+/-Loa</sup>* mice, *Dync1h1<sup>-/Loa</sup>* mice exhibited higher pre-weaning mortality, and in male mice, a more rapid decline in grip strength (most notably between 4 and 8 weeks, with a further widening in performance at 40, 48 and 52 weeks of age), and a subtle but progressively more severe weight loss. These findings, for the first time, reveal the consequences of *Loa* allele exclusivity in cholinergic neurons in adulthood.

### 3.3. Mice with reduced dynein function display NMJ morphological abnormalities

Increasing evidence suggests that early pathological changes occur at neuromuscular junctions (NMJs) in ALS [55,56]. Changes in muscle innervation and NMJ morphology may underpin the differences seen in grip strength and motor coordination. To investigate this, we analysed NMJs from lumbrical muscles of the hind limbs as these are the most distal and consequently most likely to show axonal transport related pathology. Additionally, they are predominantly composed of fast twitch glycolytic type IIX muscle fibres [57,58] which have been shown to be selectively vulnerable in ALS [59].

Since a difference in grip strength between male *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice was seen at 8, 12, and 52 weeks of age, with the largest difference at 8 weeks, NMJs from these mice were analysed at both 8 and 52 weeks of age to determine if there is a progressive phenotype. As shown in Fig. 3A, whole mounted lumbrical muscles were stained with tetramethylrhodamine conjugated α-bungarotoxin (magenta) to label post-synaptic acetylcholine receptors (AChRs), while innervating fibres were labelled with antibodies directed against pre-synaptic vesicles (SV2, cyan) and the neurofilaments of innervating axons (2H3, cyan).

As an indicator of overall NMJ size, the endplate area was calculated, which encompasses the AChR postsynaptic staining and the internal perforations which form during the maturation of NMJs [47,60,61]. At 8 weeks of age, no significant differences were seen in either *Loa* mutants (Fig. 3B). At 52 weeks of age, there was a trend towards a reduction in endplate area in *Dync1h1<sup>-/Loa</sup>* mice, but this did not reach statistical significance (Fig. 3C). A similar trend was also seen in the area of the AChR-stained clusters (excluding the unstained perforated area) at 52 weeks of age but not at 8 weeks of age (Fig. 3D-E).

Next, a compactness value was calculated from the AChR area and endplate area which gives a measure of the amount of AChR receptors in a given endplate. The compactness value can be used as an indication of the potential of an NMJ to adapt to neuronal activity, since it is thought that an increase in compactness occurs to ensure a more efficient communication between the pre- and postsynaptic components [62]. At 8 weeks of age (Fig. 3F), *Dync1h1<sup>-/Loa</sup>* mice not only showed a reduction in AChR compactness in comparison to both *Dync1h1<sup>+/-Loa</sup>* (*P* = 0.0007) and *Dync1h1<sup>+/-</sup>* (*P* = 0.0008) mice, but they also exhibited fragmentation (*P* = 0.0074 when compared to *Dync1h1<sup>+/-Loa</sup>* and *P* = 0.0064 when compared to *Dync1h1<sup>+/-</sup>*; Fig. 3G). While no reduction in compactness was seen in *Loa* mutants at 52 weeks of age (Fig. 3H), there remained significant fragmentation of the endplates in *Dync1h1<sup>-/Loa</sup>*

mice at this age compared to *Dync1h1<sup>+/-</sup>* mice (*P* = 0.0367; Fig. 3I). The proportion of endplates which were fragmented (had >1 cohesive AChR-positive element to the endplate) was significantly increased at 8 weeks of age in *Dync1h1<sup>-/Loa</sup>* mice (*P* = 0.194 and *P* = 0.0375, in comparison to *Dync1h1<sup>+/-</sup>* and *Dync1h1<sup>+/-Loa</sup>*; respectively; Fig. 3J), but while the same trend was present at 52 weeks, it did not reach statistical significance (Fig. 3K).

Finally, endplate coverage was calculated as the percentage of postsynaptic AChR area occupied by SV2 staining, as a measure of the level of innervation. *Dync1h1<sup>-/Loa</sup>* mice showed a significant deficit in endplate coverage at both 8 weeks (*P* = 0.0029; Fig. 3L) and 52 weeks of age (*P* = 0.0256; Fig. 3M).

Overall, these findings show that in the male mice at 8 weeks of age, there are morphological abnormalities in NMJ compactness, fragmentation, and most importantly in NMJ innervation of *Dync1h1<sup>-/Loa</sup>* mice which are absent in *Dync1h1<sup>+/-Loa</sup>* mice. Significantly, in *Dync1h1<sup>-/Loa</sup>* mice at 52 weeks of age, not only is increased fragmentation and reduced innervation of the endplates still present, but there are also trends towards smaller endplates with lower densities of AChRs. These alterations not only underscore the critical role of dynein in development and maintaining neuromuscular integrity, but also provide potential indicators for the onset and progression of neuromuscular disorders such as ALS.

### 3.4. Reduced dynein function causes TDP-43 and p62 abnormalities

TDP-43 ubiquitin-positive inclusions are found in over 97 % of all ALS cases, indicating a failure of clearance mechanisms such as autophagy [63]. Importantly, these ubiquitin inclusions are also positive for the autophagy receptor p62 [64]. Given that dynein plays an important role in autophagy [63,65–73], L3-L6 spinal cord sections from 52-week-old male mice were immunostained for TDP-43 and p62 to determine if reduced dynein function recapitulates this ALS pathology in ChAT⁺ neurons (Fig. 4A).

As shown in Fig. 4B, neither *Loa* mutant showed an increase in the number of TDP-43 puncta per cell, but both *Dync1h1<sup>+/-Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice showed an increase in the percentage of neurons with TDP-43 puncta (*P* = 0.0169 and *P* = 0.0355, respectively; Fig. 4C). On the other hand, *Dync1h1<sup>-/Loa</sup>* mice had significantly more p62 puncta per cell than both *Dync1h1<sup>+/-Loa</sup>* (*P* = 0.0090) and *Dync1h1<sup>+/-</sup>* (*P* = 0.0076) mice (Fig. 4D). However, there were no differences in the proportion of neurons with p62 puncta between genotypes (Fig. 4E), and the p62 puncta did not co-localise with TDP-43 puncta (Fig. 4F).

This study shows a previously unreported increase in cells exhibiting TDP-43 puncta in *Loa* mutants, which could be an early sign of TDP-43 mislocalisation and aggregation. Interestingly, this is accompanied by an increase in p62 puncta per cell in *Dync1h1<sup>-/Loa</sup>* mice, which did not appear to co-localise with TDP-43. Given the role of p62 as an autophagic receptor, this may indicate a broader failure or imbalance in autophagic clearance [74].

### 3.5. Reduced dynein function does not lead to motor neuron loss

Nissl staining of serial sections of L3-L6 spinal cord tissue from male 52-week-old mice was used to determine the number of ventral horn motor neurons in the sciatic motor pool. The total number of motor neurons did not significantly differ between the four genotypes, although there is a trend in reduced motor neuron numbers in *Dync1h1<sup>-/Loa</sup>* mice (Fig. 5). These data suggest that the differences in NMJ morphology as well as the motor phenotypes observed in mice with reduced dynein function are less likely to be due to a loss of spinal motor neurons.

### 3.6. Sex differences

Given the well-documented sex differences in ALS incidence and
<figure><img src="image_5.png" /></figure>

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

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

<figure><img src="image_8.png" /></figure>
**Fig. 3.** Morphological abnormalities in hind limb NMJs of male mice with varying levels of functional dynein. (A) Representative images of motor endplates. Postsynaptic acetylcholine receptors (AChRs) are labelled by α-BTX in magenta, presynaptic axon terminals are labelled by SV2 in cyan, and neurofilaments are labelled by 2H3 in cyan. Scale bar = 10 µm. (B) Endplate area at 8 weeks of age. (C) AChR area at 8 weeks of age. (D) Endplate compactness at 8 weeks of age. (E) Endplate fragmentation at 8 weeks of age. (F) Endplate area at 52 weeks of age. (G) AChR area at 52 weeks of age. (H) Endplate compactness at 52 weeks of age. (I) Endplate fragmentation at 52 weeks of age. (J) Percentage of fragmented endplates per mouse at 8 weeks of age. (K) Percentage of fragmented endplates per mouse at 52 weeks of age. (L) Endplate coverage at 8 weeks of age. (M) Endplate coverage at 52 weeks of age. Data in B-M is shown as mean ± SD and is from 3 to 4 mice per genotype and 3-5 muscles per mouse. The total number of endplates analysed is shown in Tables S2–3. Nested one-way ANOVAs with Tukey's post-hoc (B—K), Welch's ANOVA with Dunnett's T3 post-hoc (L), or ordinary one-way ANOVA with Tukey's post-hoc (M). \*P < 0.05, \*\*P < 0.001. Frequency distributions of the data in this figure are provided in supplementary file 1.

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

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

**Fig. 4.** TDP-43 and p62 immunohistochemistry in ChAT⁺ cells of L3-L6 spinal cord segments at 52 weeks of age in males. (A) Representative images showing TDP-43 and p62 staining, with prominent ChAT⁺ puncta (yellow arrows) representing clusters of choline acetyltransferase. The presence of these large ChAT⁺ puncta is indicative of the distribution and concentration of this enzyme within the neurons, serving as a hallmark of motor neuron identification. Examples of some of the TDP-43 and p62 puncta quantified are indicated by cyan and magenta arrowheads, respectively. Yellow outlines represent the nuclei, as identified by DAPI staining. Scale bar = 10 µm. (B) Number of cytoplasmic TDP-43 puncta normalised to cytoplasm area. (C) Number of cytoplasmic p62 puncta normalised to cytoplasm area. (D) Percentage of cells with cytoplasmic TDP-43 puncta. (E) Percentage of cells with cytoplasmic p62 puncta. Data is shown as mean ± SD from three mice per genotype. (F) Representative example of a ChAT⁺ *Dync1h1⁻/Loa* cell showing lack of colocalisation between TDP-43 (cyan) puncta (yellow arrowheads) and p62 (magenta) puncta (white arrows). The total number of cells analysed is shown in Tables S4-S5. Nested one-way ANOVAs (B, C) or ordinary one-way ANOVAs (D, E) with Tukey's post-hoc. \*P < 0.05, \*\*P < 0.01. Frequency distributions of the data in this figure are provided in supplementary file 1.

prevalence [75,76], we also performed similar analysis on female mice. Like their male equivalents, female *Dync1h1⁺/Loa* and *Dync1h1⁻/Loa* mice exhibited a reduction in body weight relative to *Dync1h1⁺/+* mice (*P* < 0.0001; Fig. 1 in Supplementary file 2). However, unlike the males, whose body weights between the *Dync1h1⁻/Loa* and *Dync1h1⁺/Loa* genotypes showed increasing divergence that became statistically significant at later ages, female *Dync1h1⁻/Loa* and *Dync1h1⁺/Loa* mice showed a significant difference in body weight between 8 and 20 weeks (Fig. 1 in Supplementary file 2), highlighting some sex differences.

In addition, grip strength assessment of the females showed that both *Loa* strains performed significantly worse than wildtypes (*P* < 0.0001; Fig. 2A in Supplementary file 2), consistent with the observations in males. Unlike males, where *Dync1h1⁻/Loa* showed a decline in performance compared to *Dync1h1⁺/Loa* between 4 and 16 weeks, that

remained unaffected even after body weight normalisation, a similar pattern was not observed in females prior to normalisation. When normalised to body weight, *Dync1h1⁺/Loa* females performed worse at 8 weeks compared to *Dync1h1⁻/Loa* females (*P* = 0.032; Fig. 3 in Supplementary file 2) but then matched the *Dync1h1⁻/Loa* females at subsequent ages. The accelerating rotarod test did not reveal any sex differences, since both female *Loa* strains performed significantly worse than wildtypes (*P* < 0.0001), mirroring the male results (Fig. 2B in Supplementary file 2).

The NMJs of females were assessed only at 52 weeks of age. While a non-significant trend towards a reduction in endplate and AChR areas was seen in male *Loa* mutant strains, this reached statistical significance in female mice (Fig. 4A-C in Supplementary file 2). In females, there were no significant differences in endplate compactness between
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Percentage of motor neurons per male mouse relative to the mean *Dync1h1<sup>+/+</sup>*. Motor neurons in the sciatic motor pool were counted every 60 µm along the lumbar spinal cord from a total of 40 tissue sections per mouse. *N* = 3 biological replicates (52 weeks old) per genotype. Data is shown as mean ± SD. Ordinary one-way ANOVA with Tukey's post-hoc.</figcaption></figure>

genotypes (Fig. 4D in Supplementary file 2) but there was a dose-
dependent trend in increased fragmentation of the endplates with
reduced functional dynein, although this did not reach statistical sig-
nificance as it did with the male data (Fig. 4E in Supplementary file 2). In
addition, we observed the same dose-dependent trend towards an in-
crease in the proportion of endplates which were fragmented in females
as that we observed in males (Fig. 4F in Supplementary file 2). Finally, a
deficit in endplate coverage was not observed in females as was
observed in males (Fig. 4G in Supplementary file 2). The frequency
distributions of the female NMJ data are shown in Fig. 5 in Supple-
mentary file 2.

Resource limitations prevented motor neuron counts and analysis of
TDP-43 and p62 puncta in the spinal cord tissue in female mice. None-
theless, our findings emphasise the importance of considering sex as a
variable when examining ALS-related phenotypes in *Dync1h1* mutant
mice, as subtle sex differences were observed in body weight, motor
performance, and NMJ morphology.

**4. Discussion**

In this study, for the first time we were able to show the impact of *Loa* allele exclusivity into adulthood in cholinergic neurons including motor neurons. To achieve this, we used a floxed allele of *Dync1h1* combined with a Chat-CRE expressing mouse model to knock out the wildtype *Dync1h1* allele in cholinergic neurons of *Dync1h1<sup>+/Loa</sup>* mice. Importantly, we used these mice to determine if this results in greater pathology than that seen in *Dync1h1<sup>+/Loa</sup>* mice, and whether *Dync1h1<sup>-/Loa</sup>* mice model pathology associated with ALS with greater fidelity.

In our previous observations, we found that mice homozygous for the
*Loa* mutation in *Dync1h1* fail to survive beyond the first day of birth,
likely due to an inability to feed [37]. An observation supported by a
non-quantitative, direct visual inspection, where the absence of milk in
their stomachs is noticeable through their semi-transparent skin,
consistent with what has been reported previously [37]. In the present
study, we extended these findings by showing that both *Dync1h1<sup>+/Loa</sup>*
and *Dync1h1<sup>-/Loa</sup>* mice exhibit reduced survival during the pre-weaning

period (up to 21 days of age) compared to *Dync1h1<sup>+/+</sup>* controls. Notably,
survival was significantly lower in *Dync1h1<sup>-/Loa</sup>* mice than in *Dync1h1<sup>+/-</sup>*
*mice*, indicating that a further reduction in dynein function, even
when confined to cholinergic neurons, exacerbates mortality. The
observed decrease in early-life survival is consistent with the essential
role of dynein in numerous cellular processes, including neural devel-
opment and the transport of organelles and macromolecules, all of
which are vital for organismal development and survival [77–80]. The
importance of dynein during development has been previously high-
lighted in *Loa* homozygotes which were found to have defective
neocortical lamination and neuronal migration and arborisation due to a
decreased rate of radial migration of bipolar neurons [37,81].

In the longitudinal assessments, both *Dync1h1<sup>+/Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* male mice demonstrated reductions in body weight and grip strength in all limbs. Significantly, the difference in body weight between *Dync1h1<sup>+/Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice became more pronounced as the mice aged. Notably, male *Dync1h1<sup>-/Loa</sup>* mice exhibited an earlier and steeper decline in grip strength, with a significant drop occurring between 4 and 8 weeks of age, reaching a level of impairment that was only matched at 16 weeks of age in the *Dync1h1<sup>+/Loa</sup>* mice. While reductions in both body weight and hind limb grip strength have been previously reported in *Dync1h1<sup>+/Loa</sup>* mice [83], we show that further loss of dynein function restricted to cholinergic neurons, including motor neurons, exacerbates this phenotype.

Interestingly, while the female mice of both *Loa* mutant strains
showed a similar reduction in body weight and grip strength perfor-
mance, the body weight difference between the two *Loa* strains did not
appear to change with age and was instead more prominent at earlier
ages before converging. Additionally, the faster decline in grip strength
seen in male *Dync1h1<sup>-/Loa</sup>* mice compared to *Dync1h1<sup>+/Loa</sup>* mice was not
found in female mice. Instead, the two genotypes broadly performed
similarly, apart from at 8 weeks of age when the *Dync1h1<sup>+/Loa</sup>* female
mice performed worse than *Dync1h1<sup>-/Loa</sup>* female mice when data was
normalised to body weight. Overall, this underscores the importance of
considering sex as a critical factor when examining ALS-related phe-
notypes, as even subtle differences can provide insights into the diverse
mechanisms underlying disease progression in males and females.

The decline in body weight may suggest underlying muscle wasting, particularly given that mutations in *DYNC1H1* have been linked to SMALED1 in humans, a condition characterised by significant muscle weakness and atrophy [36,84]. Although we did not directly assess muscle atrophy in this study, the parallels in weight reduction could indicate similar underlying processes, warranting further investigation in future studies. Interestingly, despite these differences in weight and grip strength, we did not observe a significant difference in motor function and coordination between *Dync1h1<sup>+/Loa</sup>* and *Dync1h1<sup>-/Loa</sup>* mice when measured using an accelerating rotarod. Both mutants exhibited significant deficits compared to *Dync1h1<sup>+/+</sup>* controls, consistent with previous reports in *Dync1h1<sup>+/Loa</sup>* mice [83].

NMJ abnormalities have been previously reported in the *Dync1h1<sup>+/-</sup>* *Cra1* mouse model of dynein dysfunction and a mutant *Dync1h1* knock-in model of Charcot-Marie-Tooth disease [85,86]. Similarly, NMJ abnormalities are a common feature in models with impaired dynein co-factor or adaptor function [87–90]. However, until now, NMJ integrity and morphology has not been studied in mice harbouring the *Loa* mutation. Our findings show that only *Dync1h1<sup>-/Loa</sup>* mice exhibit NMJ abnormalities. Specifically, we found increased motor endplate fragmentation at both 8 and 52 weeks of age in male mice in comparison to *Dync1h1<sup>+/+</sup>* controls which was absent in *Dync1h1<sup>+/Loa</sup>* mice. Importantly, we also found that only *Dync1h1<sup>-/Loa</sup>* mice had significant denervation of motor endplates at both ages.

Additionally, while a reduction in endplate compactness was seen in
male mice at 8 weeks of age, there were trends towards smaller end-
plates and a reduction in AChR area also at 52 weeks of age in male
*Dync1h1*<sup>-/Loa</sup> mice. Given that there is evidence of increased NMJ
fragmentation in rodents beyond 52 weeks of age, this suggests a
developmental defect resembling aged NMJs [91–104]. NMJ fragmentation has also previously been noted in an ALS murine model in which the DCTN1 component of the dynein co-factor dynactin was conditionally knocked out in postnatal neurons [89]. Similarly, fragmentation was also observed in a transgenic model overexpressing mutant DCTN1 [87]. While previous research has demonstrated that dynein plays a role in post-synaptic AChR clustering [105], our findings reveal that a more pronounced pre-synaptic loss of dynein function in *Dync1hl*⁻¹/Loa mice is sufficient to cause NMJ fragmentation and denervation. Importantly, this ALS-like pathology does not occur in *Dync1hl*⁺¹/Loa mice, where dynein dysfunction is widespread but less severe in cholinergic neurons. This finding underscores the significance of motor neuron-specific dynein dysfunction in driving NMJ fragmentation, aligning with the understanding that ALS primarily targets motor neurons.

Changes in NMJ structure can serve as early pathology indicators, predicting muscle function decline. Such changes have been observed in ALS, spinal muscular atrophy, Charcot-Marie-Tooth disease, and muscular dystrophies prior to denervation onset [93,106–114]. As regards ALS, human studies have found trends towards reduced endplate and nerve terminal areas in people with ALS [115,116] and in transgenic mouse models of ALS expressing mutant human TDP-43, SOD1, and C9orf72 [91,117–122]. Overall, our findings of significantly altered postsynaptic integrity and denervation of the lumbrical muscle NMJs in *Dync1hl*⁻¹/Loa mice, but not *Dync1hl*⁺¹/Loa mice, suggest that the former may serve as a better model for studying the role of dynein in NMJ integrity in these diseases.

Interestingly, we observed subtle differences in some NMJ measures between males and females. Sex differences in NMJs have also been observed in mouse models of autosomal recessive spinal muscular atrophy (SMA) and could be associated with genetic modifiers related to sex, as has been postulated for SMA [123,124]. However, while some measures reached statistical significance in one sex and not the other, the overall pattern of the data was similar. For example, both *Loa* mutant strains showed reductions in endplate and AChR areas in females, whereas males only exhibited a non-significant trend. Additionally, females demonstrated a dose-dependent trend towards increased endplate fragmentation with reduced functional dynein, which mirrored the statistically significant changes seen in the males.

In addition to the above pathology, the alterations in TDP-43 and p62 found in our study highlight potential ways in which dynein may contribute to ALS pathology. We observed that more neurons exhibited TDP-43 puncta in both *Dync1hl*⁺¹/Loa and *Dync1hl*⁻¹/Loa mice. However, only *Dync1hl*⁻¹/Loa mice had a concomitant increase in p62 puncta, which did not appear to co-localise with the TDP-43 puncta. It is possible that the increase in the frequency of motor neurons exhibiting TDP-43 puncta may represent an early stage of TDP-43 mislocalisation prior to aggregation. TDP-43 mislocalisation and its aggregation along with p62 are common in ALS, thus, our observations in mice with reduced dynein function suggest a possible link between dynein and these processes. Non-fibrillar diffuse puncta have been suggested as being the earliest TDP-43 pathology in ALS [125]. Indeed, we have also recently shown that expression of mutant dynein in combination with wildtype human TDP-43 lead to p62 upregulation and TDP-43 aggregation [126]. With evidence indicating a direct interaction between TDP-43 and the dynactin complex [127], the TDP-43 changes observed might be a secondary effect of dynein dysfunction. Alternatively, TDP-43 accumulation might result from autophagy impairment, as motor neurons from *Dync1hl*⁻¹/Loa mice also exhibited a higher number of p62 puncta, an autophagy receptor [74].

Further investigation should aim to determine if the TDP-43 puncta within *Dync1hl*⁺¹/Loa and *Dync1hl*⁻¹/Loa mice are localised to stress granules as this is suggested as an initiation site of TDP-43 pathology [128]. Moreover, it remains unclear whether the TDP-43 abnormalities are restricted to the cell body or also present in the axon, dendrites, and axon terminals, which were unobservable in our spinal cord sections. Understanding this may prove important, as recent studies hint at TDP-

43 mislocalisation in the axon potentially disturbing the synthesis of essential presynaptic proteins and leading to NMJ degeneration [129]. Thus, the NMJ alterations we noted in our mice might be related to the changes in TDP-43 distribution within their spinal motor neurons, especially if this is widespread within the neurons. Investigating whether the TDP-43 and p62 aggregations seen at 52 weeks also occur at 8 weeks and worsen with age would also be prudent, considering we observed NMJ structural changes in the younger mice.

Finally, we did not observe a statistically significant decrease in spinal motor neurons in either of the *Loa* mutants. Our current data align with the findings indicating no significant spinal motor neuron loss in *Dync1hl*⁺¹/Loa mice [39,40] or in *Dync1hl*⁺¹/Cra1 mice [41,85]. Previously, denervation of NMJs has been observed in geriatric mice (29 months old) without motor neuron loss [130]. These results suggest that the changes in NMJ morphology and motor phenotypes in our dynein-deficient mice are not driven by a decrease in motor neuron quantity but may result from other cellular processes influenced by dynein dysfunction.

## 5. Conclusion

Overall, the novel *Dync1hl*⁻¹/Loa mouse model allowed us for the first time to examine the consequences of *Loa* allele exclusivity in cholinergic neurons into adulthood and found these mice to exhibit a more severe phenotype than the previously available *Dync1hl*⁺¹/Loa model. Many of the observed pathologies are relevant to ALS, reinforcing the critical role of dynein. This model not only enhances our understanding but may also serve as a more effective tool for studying the role of dynein in this disease.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbadis.2024.167540.

## CRediT authorship contribution statement

**Fabio A. Simoes:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Eleni Christoforidou:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Raphaelle Cassel:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Luc Dupuis:** Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Conceptualization. **Majid Hafezparast:** Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization.

## Declaration of competing interest

The author EC holds shares in Thermo Fisher Scientific and various index funds which may include companies whose products were used in the research reported in this article. Additionally, EC has been engaged in a compensated collaboration with Thermo Fisher Scientific for promotional activities unrelated to this research. These potential financial interests do not influence the design, execution, or interpretation of the research findings presented in this article. All other authors declare no competing interests.

## Acknowledgements

We would like to thank Dr. Bernadett Kalmar at Queen Square Institute of Neurology, University College London, for guidance on the preparation of the lumbrical muscles. We are also grateful to Marion Brownridge for her generous funding of part of this study. This work was supported by the Motor Neurone Disease Association project grant awarded to LD and MH (Dupuis/Apr16/852-791).
Data availability

The data that support the findings of this study are available from the corresponding author, MH, upon reasonable request. The computer code used for data analysis in this study is available at https://zenodo.org/record/7938481.

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