Phenotypical, genotypical and pathological characterization of the moonwalker mouse, a model of ataxia

Gabriella Sekerková; Sumeyra Kilic; Yen-Hsin Cheng; Natalie Fredrick; Anne Osmani; Haram Kim; Puneet Opal; Marco Martina

doi:10.1016/j.nbd.2024.106492

. Author manuscript; available in PMC: 2024 Jun 1.

Published in final edited form as: Neurobiol Dis. 2024 Apr 2;195:106492. doi: 10.1016/j.nbd.2024.106492

Phenotypical, genotypical and pathological characterization of the moonwalker mouse, a model of ataxia

Gabriella Sekerková ^a,^*, Sumeyra Kilic ^a, Yen-Hsin Cheng ^a, Natalie Fredrick ^b, Anne Osmani ^a, Haram Kim ^a, Puneet Opal ^b, Marco Martina ^a,^*

PMCID: PMC11089908 NIHMSID: NIHMS1989410 PMID: 38575093

Abstract

We performed a comprehensive study of the morphological, functional, and genetic features of moonwalker (MWK) mice, a mouse model of spinocerebellar ataxia caused by a gain of function of the TRPC3 channel. These mice show numerous behavioral symptoms including tremor, altered gait, circling behavior, impaired motor coordination, impaired motor learning and decreased limb strength. Cerebellar pathology is characterized by early and almost complete loss of unipolar brush cells as well as slowly progressive, moderate loss of Purkinje cell (PCs). Structural damage also includes loss of synaptic contacts from parallel fibers, swollen ER structures, and degenerating axons. Interestingly, no obvious correlation was observed between PC loss and severity of the symptoms, as the phenotype stabilizes around 2 months of age, while the cerebellar pathology is progressive. This is probably due to the fact that PC function is severely impaired much earlier than the appearance of PC loss. Indeed, PC firing is already impaired in 3 weeks old mice. An interesting feature of the MWK pathology that still remains to be explained consists in a strong lobule selectivity of the PC loss, which is puzzling considering that TRPC is expressed in every PC. Intriguingly, genetic analysis of MWK cerebella shows, among other alterations, changes in the expression of both apoptosis inducing and resistance factors possibly suggesting that damaged PCs initiate specific cellular pathways that protect them from overt cell loss.

Keywords: Apoptosis, Calcium, Cerebellum, Endoplasmic reticulum, Neurodegeneration, Purkinje cells, Spinocerebellar ataxia, TRPC3, Unipolar brush cell

1. Introduction

Spinocerebellar ataxias (SCAs) are autosomal dominant genetic diseases characterized by cerebellar and brainstem degeneration (Ashizawa et al., 2018; Correia et al., 2023; Mitoma et al., 2019). They are caused by mutations in distinct genes with >40 different types of SCAs identified to date. Most SCAs are caused by protein misfolding and cause toxicity by loss or gain of function mechanisms (Robinson et al., 2020; Rodriguez and Todd, 2019). The most common SCAs are repeat disorders in proteins of unknown functions, and it has been difficult to mechanistically understand downstream pathways of pathogenesis (Coarelli et al., 2023; Diallo et al., 2021). Several rare SCAs, however, are caused by mutations in proteins with known functions that are proving to be useful in this regard (Ashizawa et al., 2018; Coarelli et al., 2023; Cunha et al., 2023). One disease recently identified is SCA41 (Fogel et al., 2015), which is caused by a single-point gain of function mutation (T635A) in transient receptor potential channel 3 (TRPC3), a calcium permeable ion channel (Cole and Becker, 2023; He et al., 2017), which in the cerebellum is selectively expressed in two cell types: unipolar brush cells (UBCs) and Purkinje cells (PCs) (Hartmann et al., 2008; Huang et al., 2007; Sekerková et al., 2013). Remarkably, prior to its discovery in humans, a TRPC3 mutation had been identified in a mouse ENU ataxia mutagenesis screen and dubbed the Moonwalker (MWK) mouse (Becker, 2014; Becker et al., 2009).

MWK mice are part of a much larger group of ataxias characterized by dysfunctions in calcium homeostasis (Kasumu and Bezprozvanny, 2012; Prestori et al., 2019). Despite large heterogeneity in the identity of the causal gene mutations, some phenotypic and cellular features of MWK mice are remarkably similar across most SCAs (Kasumu and Bezprozvanny, 2012; Shimobayashi and Kapfhammer, 2018; Wu and Kapfhammer, 2021; Wu and Kapfhammer, 2022), including the observation that PC dysfunction precedes overt pathological findings (Chang et al., 2022; Cook et al., 2021; Jayabal et al., 2015; Kapfhammer and Shimobayashi, 2023), possibly because the high metabolic rate of these cells (Howarth et al., 2010) makes them particularly vulnerable.

MWK mice have undergone some behavioral and morphological characterization when they were first identified (Becker, 2014; Becker et al., 2009). It was shown that the TRPC3 MWK mutation causes ataxia in 3 weeks old mice and a slow and gradual PC loss after 4 months of age. Thus, the time course of PC loss and the development of ataxia do not overlap as the ataxic phenotype precedes the PC loss by months. In contrast the UBC loss in MWK mice is massive (>95%) at 1 month of age (Sekerková et al., 2013). Our electrophysiological analysis further showed that PCs are dysfunctional in 4-week-old MWK mice (Sekerková et al., 2013). Importantly it has been shown that the gain of function TRPC3^mwk mutation causes altered calcium signaling pathways (Dulneva et al., 2015). These previous papers however did not investigate the natural course of the disease, the cellular pathology, and the possible disease mechanisms. With the recent identification of human disease with a similar TRPC3 gain of function (Fogel et al., 2015), the model warrants a more comprehensive analysis from a behavioral, pathological, and genetic perspective that may provide a clearer understanding of the progression of the diseases and thus lead to the identification of potential therapeutic targets (Manto et al., 2020; Prestori et al., 2019).

Here we provide a detailed characterization of the cellular, morphological, and phenotypic features, and of the natural history of the MWK ataxia, and investigate changes in gene expression caused by TRPC3^mwk mutation. We show that the TRPC3^mwk mutation affects the cerebellar circuitry much earlier than originally thought. We also show that the TRPC3^mwk mutation interferes with PC and UBC development and causes irreversible changes in PCs by postnatal day (P) 40. We also show that unlike many SCAs (e.g. SCA1 and SCA2) the MWK ataxia is non-progressive, resembling SCA29. Interestingly, SCA29 is caused by missense Iptr3 (inositol phosphate 3 receptor 1; IP3R1) mutation that interferes with calcium homeostasis and is characterized by a motor development delay that develops into a non-progressive ataxia (Huang et al., 2012; Zambonin et al., 2017) thus sharing with MWK the calcium dysregulation pathway. Accordingly, our gene analysis in MWK cerebellum revealed multiple differentially regulated genes involved in calcium homeostasis regulation and ER stress.

2. Material and methods

2.1. Animals

The study was carried out in accordance with protocols approved by the Northwestern University Animal Care and Use Committee. All animals were bred and kept in the Northwestern University vivarium with 12 h light/dark cycle and tightly regulated temperature and humidity. Each cage environment was enriched with a running wheel and crinkled paper strips. Mice had access to food and water ad libitum. MWK and WT were housed together, with max density of 5 mice/cage. All studies were performed during the light phase.

We crossed heterozygous MWK male (ENU-mutagenized mice originally kept on C3H/HeH; Becker et al., 2009) with CD1 wild-type (WT) females through >7 generations. The CD1 background provided us with larger litter size compared to the original background strain (12–15 pups vs 5–8 pups; (Hsieh et al., 2017; Ramsey et al., 2023). Between P20 to P40 the MWK mice appear malnourished and weak, and they show slowed weight gain when compared to WT siblings. We supplement their usual diet with Diet Gel 67 (ClearH2O, Westbrook, USA) on the floor of the cage for easy access. In our experience however this temporary phase corrects naturally by P40 when the animals rapidly gain weight even if they are not provided with supplementary diet. The heterozygous MWK mice were marked by ear punch on the day of the weaning (around P25–28). At this age they are unequivocally distinguishable from the WT littermates by smaller body weight and ataxic behavior. Additionally, the presence of the TRPC3 mutation was confirmed by extracting genomic DNA and sequencing the mutated TRPC3 region. The MWK mice carry an A-to-G transition at nucleotide position 1903 in the TRPC3 gene (Fig. 1A).

Fig. 1. — A) Heterozygous MWK mice were identified by A1903G transition (arrow) in the TRPC3 locus using DNA sequence analysis. B) MWK mice are ~50% lighter than the WT at 1 month of age. At later age points the weight difference between the WT and MWK decreases to ~20%. These differences are similar in female and male mice. C) In grip strength test the MWK mice fell off from a wire top within seconds after inversion. Young WT mice clung to the inverted wire top for the whole duration of the test (3 min). 3-month-old WT mice performed slightly worse than the younger WT, possibly because of their increasing body weight (see panel B). D) Rearing count assessed in a cylinder during 3 min testing showed that MWK mice rearing ability is severely impaired at all age points tested. E) Accelerating rotarod test of 2- and 3- month-old MWK and WT mice. MWK mice underperformed at every time point when compared to WT mice. However, 2-month-old MWK mice show a slight motor learning ability on the 3rd day of the testing. **F—I)** Footprint analysis of 2- and 3- month-old MWK and WT mice. F) The left schematic illustrates the measured parameters: a, stride length; b, step width; c, print length; d, contralateral paw placement distance measured at the ipsilateral stride length. The right panel shows representative paw prints of a WT and a MWK mouse. Hind- are fore-limbs are shown in blue and orange, respectively. G) MWK mice have shorter stride length, wider step width and longer print length in comparison to WT mice. H) Stride Length/Step Width (SL/SW) ratio is significantly reduced in MWK mice in comparison to WT. I) Contralateral paw placement (“d” in panel F schematic) expressed as absolute deviation from the expected “50%” placement along the ipsilateral step (exact middle of the ipsilateral stride length; distance “a” in schematic). MWK mice have a wider range of contralateral paw placements compared to WT. *Asterisks in B-E and G-H represent p < 0.05 obtained by One-Way-ANOVA and Tukey’s* post hoc *analysis. The exact p values are provided in* Supplementary Table 1.

2.2. Behavioral analysis

2.2.1. Phenotype scoring system

Mice were first observed in their home cage and then individually recorded for 3 min in a new cage and for another 3 min in a clear plexiglass cylinder (13 [diam.] x 17 [H] cm). We used the in-cylinder recordings to score the behavioral profile of each mouse. As the confined space of the cylinder might affect the behavior of the mouse we used in-cage recordings to confirm our observations. We adapted an assessment system previously used in other ataxic mice (Guyenet et al., 2010; Yerger et al., 2022). We scored the following parameters: 1) tremor during moving and resting phase, 2) circling behavior, 3) time spent grooming, 4) time spent resting (when animals stayed at the same spot for >3 s, not counting grooming time), 5) Straub tail, and 6) hindlimb clasp (holding animals by tail for about 15 s). The tremor and circling behaviors were scored on separate scales, each spanning 0 to 3; 0 = none, 1 = low tremor; clear but not continuous circling, 2 = moderate tremor; continuous circling with short interruptions, and 3 = severe tremor; constant circling with no interruption.

2.2.2. Grip strength

Mice were placed on a wire cage top which was then inverted and held ~50 cm above the cage for 3 min. We recorded the time when the mice fell from the cage top.

2.2.3. Rearing ability

Mice were placed in a clear plexiglass cylinder (13 [diam.] x 17 [H] cm) for 3 min. We counted the number of rears defined as extending upward from hindlimbs and placing both fore paws on the wall of the cylinder.

2.2.4. Accelerating rotarod

Mice were placed on a rotating rod (Ugo Basile, Gemonio, Italy) that accelerates linearly from 4 to 40 rotations per minute over a maximum duration of 5 min. We measured the time it took for each mouse to fall off the rotating rod. The first day training session was followed by 3–4 consecutive testing days. We performed 4 trials/day with 10–15 min rest between each trial.

2.2.5. Footprint analysis

We constructed a 10 cm wide, and 60 cm long channel surrounded by tall walls. At the end of the channel, we placed a dark box with an opening to entice the animals to walk toward it. The channel floor was lined with white paper. The forepaws and hind paws of the mice were painted with nontoxic food-dye. The mice were then placed at the beginning of the channel and let walk toward the dark box. We performed this analysis 3 times for each animal with 5 min resting intervals.

2.3. Immuno- and TUNEL Staining

2.3.1. Tissue preparation

Mice were deeply anesthetized with sodium pentobarbital (60 mg/kg body weight) and then perfused through the ascending aorta with saline followed by 4% freshly depolymerized formaldehyde (PFA) in 0.12 M phosphate buffer (PB), pH 7.4. The brains were dissected out and cry-oprotected in 30% sucrose in phosphate buffered saline (PBS) for cry-osectioning. The cerebella were sectioned in the sagittal or coronal planes at 30 μm on a freezing-stage microtome.

2.3.2. Bright field microscopy

Sections were immunostained using an avidin/biotin amplification (ABC) protocol. Briefly, the endogenous peroxidase activity was blocked in 0.3% H₂O₂ and 10% methanol in Tris-buffered saline (TBS; pH 7.4). Unspecific binding was suppressed in a blocking solution containing 3% normal goat serum (NGS) and 1% bovine serum albumin (BSA) in TBS with 0.2% Triton X-100. Sections were then incubated overnight, or up to 2 days at 4 °C with primary antibodies. Bound antibodies were detected using biotinylated anti-mouse or anti-rabbit IgG (GE Healthcare), ABC Elite kit (Vector) and 3,3″-diaminobenzidine (DAB).

2.3.3. Fluorescence microscopy

Sections were incubated in 3% NGS and 1% BSA in TBS with 0.2% Triton X-100 for 1 h followed by overnight, or up to 2 days, incubation with primary antibodies at 4 °C. Bound primary antibodies were visualized by secondary antibodies coupled to Alexa 488 or Alexa 594 (Invitrogen). Sections were coverslipped with Mowiol based mounting medium.

2.3.4. Primary antibodies

The following primary antibodies were used: mouse anti-calretinin (CR; 1:2000; Millipore), rabbit anti-metabotropic glutamate receptor 1á (mGluR1á 1:750, Frontier Institute, Japan), mouse anti-calbindin (CaB, 1:5000; Sigma), rabbit anti-cleaved caspase 3 (cCasp3; 1:200, Cell Signaling), and guinea pig anti-tyrosine hydroxylase (TH; 1:750; SYSY).

2.3.5. TUNEL labeling

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay was performed using In Situ Cell Death detection kit (POD, Roche diagnostics), according to the manufacturer’s protocol. In addition to the WT control, slices were used without the TUNEL reaction mix, as a negative control.

2.4. Silver impregnation

Mice were perfused with 4% PFA in 0.12 M PB. After the dissection, the brains were kept in the same fixative overnight and then cry-oprotected in 30% sucrose in PBS. We prepared 40 μm-thick sagittal sections on a freezing-stage microtome. The sections were kept in 4% PFA in 0.12 M PB at 4 °C, for >7 days before processing. The sections were processed with FD Neurosilver^™ Kit II (FD Neurotechnologies Inc., Ellicott City, MD, USA) according to the manufacturer’s instructions.

2.5. Transmission electron microscopy

P40 and P85 mice (n = 3 MWK; n = 2 WT at each age point) were perfused as described above with 2% PFA and 1.25% glutaraldehyde in 0.12 M PB. The brains were dissected 1 h after perfusion and postfixed overnight in the same fixative. One hour after the perfusion the cerebellum was removed and sliced on a vibratome at 150 μm in sagittal plane. The slices were collected in PBS, postfixed with 2% OsO₄ in PBS for 1 h, rinsed with distillated water, contrasted with 1% aqueous uranyl acetate for one hour in dark, dehydrated in passages of 35–50–70-85-90-100% alcohol, and infiltrated with Epon/propylen oxide mixture overnight. The sections were flat embedded in Epon and cut on an Ultra-microtome (Leica Ultracut UTC). Ultrathin sections, 50–80 nm thick, were contrasted with lead citrate and uranyl acetate. The sections were analyzed and photographed using either a Zeiss EM-10 or a FEI Tecnai Spirit G2 electron microscopes operated at 80 kV.

2.6. Electrophysiological recordings

2.6.1. Slice preparation

P19 (n = 2 MWK) and 3-month-old mice (n = 2 WT; n = 3 MWK) were used for electrophysiological recordings. They were deeply anesthetized using isoflurane and brains were dissected. 300-μm thick sagittal brain slices containing the cerebellum (in the range of lateral −1.0 mm to +1.0 mm from the midline) were cut using a vibratome (Leica VT-1200) in ice-cold artificial cerebrospinal fluid (ACSF). The recording ACSF was (in mM): 125 NaCl, 25 NaHCO₃, 4 KCl, 1.25 NaH₂PO₄, 25 glucose, 2 CaCl₂, 1 MgCl₂, saturated with 95% O₂ and 5% CO₂ to pH 7.4. Slices were recovered at 34 °C for 15 min and then transferred to room temperature (24–26 °C) to be incubated in the same solution for >30 min.

2.6.2. Cell-attached recordings

PCs in lobules VIII to X were visually identified using an upright microscope with a 60× water-immersion objective (Olympus, Tokyo, Japan). 4–6 MΩ glass pipettes filled with ACSF were used to obtain a stable giga-seal (>1 GΩ) at 30–32 °C. To measure the frequency of spontaneous activity, no current was injected, and the spontaneous current fluctuations were recorded for 1 min. Signals were filtered at 2 kHz and sampled at 10 kHz. All measurements were performed using an Axopatch 200B amplifier and pClamp9 software (Axon Instruments, Union City, CA, USA) in the “I₀ mode”, and spikes were detected using Clampfit10 (Axon Instruments).

2.6.3. Whole-cell recordings

These recordings were performed on a separate set of cells. The recording pipettes were filled with potassium gluconate-based solution (in mM): 140 K-gluconate, 8 NaCl, 2 MgATP, 0.2 Na₃GTP, 0.1 EGTA, 10 HEPES (pH 7.3 with KOH). In these experimental conditions, PC membrane resistance was 40–70 MΩ. In current clamp mode, cells were held at ~ −70 mV by negative current injection and then stimulated using 1-s positive current steps (100 pA increments) until the spikes were evoked or to a maximum current injection of 1.6 nA.

2.7. RNA-Seq analysis

2.7.1. RNA isolation

The whole cerebellum was dissected from P21 MWK (n = 3) and WT (n = 3) littermate mice. RNA was isolated from the tissue using the RNeasy Plus Universal Mini Kit (cat# 73404, Qiagen). Briefly, the tissues were homogenized in QIAzol Lysis Reagent then treated with gDNA Eliminator Solution and chloroform for subsequent phase separation. After centrifugation, the aqueous phase was removed and loaded onto a spin column for washing and elution of RNA.

2.7.2. RNA-Seq analysis

mRNA-Seq was conducted in the Northwestern University NUSeq Core Facility. The quality of the RNA was checked using Agilent Bioanalyzer 2100. The Illumina TruSeq Stranded mRNA Library Preparation Kit was used to prepare sequencing libraries from 800 ng of total RNA (the RNA quantity was determined with Qubit fluorometer). This procedure includes mRNA purification and fragmentation, cDNA synthesis, 3′ end adenylation, Illumina adapter ligation, library PCR amplification and validation. Lllumina NextSeq 500 Sequencer was used to sequence the libraries with the production of single-end, 75 bp reads. The quality of DNA reads was evaluated using FastQC and reads of inadequate quality or those aligning to rRNA sequences were filtered. The cleaned reads were aligned to the Mus musculus genome (mm10; obtained from UCSC (University of California Santa Cruz; http://genome.ucsc.edu) using STAR (Spliced Transcripts Alignment to a Reference; Dobin et al., 2013). Read counts for each gene were calculated using HTSeq-count (Anders et al., 2015). Normalization and differential expression were determined using DESeq2 (Love et al., 2014). The statistical cutoff for determining differentially expressed genes was a false discovery rate (FDR)-adjusted p-value ≤0.05. Gene Ontology (GO) pathway analysis was performed on both gene lists using MetaScape (http://metascape.org; Zhou et al., 2019) to identify pathways that are enriched with genes that are upregulated and downregulated. Enrichment of GO pathways were analyzed using DAVID (Database for Annotation, Visualization and Integrated Discovery, National Institute of Allergy and Infectious Disease, NIH, (Huang da et al., 2009a; Huang da et al., 2009b).

The top 200 differently expressed genes were manually curated using PubMed to identify genes associated with cerebellar motor dysfunction. Next, we identified multiple review papers that compiled ataxia related genes (Ashizawa et al., 2018; Coarelli et al., 2023; Hoxha et al., 2018; Lalonde and Strazielle, 2007; Prestori et al., 2019; Robinson et al., 2020; Shimobayashi and Kapfhammer, 2018; Wu and Kapfhammer, 2022) as well papers that analyzed gene changes in ataxic mice (Arsović et al., 2020; Borgenheimer et al., 2022; Cunha et al., 2023; Eidhof et al., 2019; Halbach et al., 2017; Lin et al., 2000; Mezey et al., 2022; Wu and Kapfhammer, 2021). The later also included a paper analyzing gene changes in laser captured MWK PCs at P18 (Dulneva et al., 2015). Once we identified ataxia related genes in our dataset, we checked their cell type specificity. Primarily we used the in-situ hybridization histochemistry (ISHH) database of Allen Brain Atlas, but we also crosschecked published data in cerebellum.

2.8. Quantification of morphological parameters

2.8.1. Cerebellum/cortex ratio

We used ImageJ (Schneider et al., 2012) to measure the cortical and cerebellar areas from brain micrographs of WT and MWK mice.

2.8.2. Cerebellar cortex area

The area of the cerebellar cortex was measured in 4 sagittal sections from each animal sampled at levels corresponding to the following stereotaxic coordinates for WT cerebella (Paxinos and Franklin, 2013): 0.0–0.3 mm, 0.6–0.8 mm, 1.2–1.4 mm, and 1.8–2 mm lateral to the midline. The (smaller) MWK cerebella were matched with WT counterpart based on morphological features. Stereoinvestigator (Micro-BrigthField) was used for measurement.

2.8.3. Molecular layer thickness

We used the sagittal sections 0.6–0.8 mm lateral to the midline and measured the area of the molecular layer and the length of the PC layer (PCL) using Stereoinvestigator. The thickness was calculated as molecular layer area/PCL length.

2.8.4. Granule cell layer area

We used sagittal midline sections (0.0–0.3 mm) to measure the granule cell layer area in individual lobules. The data were normalized per 100 μm PCL.

2.8.5. PC density

We used the same sagittal sections used for the measurement of the cerebellar cortex area. In each section we drew a line along the PCL in each lobule, and the Neurolucida program automatically calculated the length of these lines. As a next step we partitioned the lines into 300-μm-long segments labeled by a thick mark at each end. Thick marks of neighboring segments overlapped and were considered as one. The center of a 100 μm diameter circle was placed at each thick mark. All PC cell bodies situated within the circle were counted, and PCs that touched the left side of the circle were counted, but not those touching the right side. The density was then calculated as PC number/PCL unit length.

2.8.6. PC numbers

We calculated the number of PCs based on PCL length and stereological estimation of PC density.

2.8.7. Axonal torpedoes

In the same sections as above, we counted the number of axonal torpedoes in each cerebellar lobule directly in the microscope with 20× lens.

2.8.8. Synaptic contacts and swollen ER in PC dendritic spines

We prepared TEM samples from lobule VII of 3 months-old MWK (n = 3) and WT (n = 2) mice and acquired images at 4800× magnification from the molecular layer at proximal, mid, and distal level of the PCs dendrites. For each level we took 6 fields containing 20 to 40 dendritic spines. For each field, we calculated the ratio of the spines receiving synaptic contacts. The same images were used to quantify the swollen ER in the dendritic spines.

2.8.9. Statistical analysis

The data were tabulated and analyzed in Excel. Statistical analysis was performed in Prism 9 (GraphPad).

2.9. Photography

Whole-mount images were taken using a Nikon DN100 digital camera mounted on an Olympus SZH10 stereomicroscope. Sections were visualized using Nikon Eclipse E800 or Zeiss Axioplan2 microscopes. Bright field and immunofluorescence images were acquired with a Spot RT CCD video camera (Diagnostics Instruments). Laser scanning confocal analysis was performed with a Nikon PCM 2000 Confocal Microscope. Electron microcopy (TEM) imaging was performed using a Zeiss EM-10. Electron micrographic negatives were scanned and imported into Adobe Photoshop CS (Adobe Systems Inc., San Jose, CA, USA). All images were processed with Adobe Photoshop CS or 2022. The images were cropped and corrected for brightness and contrast.

3. Results

3.1. Behavioral phenotype of the MWK mice

MWK mice show an ataxic phenotype with onset around P17. Until P17 the MWK mice are indistinguishable from WT littermates. However, after the onset of ataxia their weight gain is slowed and by P21 they appear emaciated and weak compared with WT littermates. At 1 month of age both female and male MWK mice show ~50% reduction in body weight compared with WT littermates (Fig. 1B), but they rapidly gain weight around P35–40 and by 2 months of age they are only ~20% lighter than the WT littermates of the same sex (Fig. 1B). This 20% weight difference between the MWK and WT mice is maintained throughout the rest of their lifetime (Fig. 1B). Notably, we did not observe premature death of MWK mice. MWK in our breeding colony live beyond 1 year of age.

Next, we evaluated the motor behavior of MWK mice at different ages (Table 1). We observed significant differences between WT and MWK mice but not between female and male mice of the same strain. All MWK mice showed resting tremor (a repeated rhythmic twitch of the front body and head bobbing), a wobbly ataxic gait, and pronounced hindlimb splay (or ducked feet) during locomotion. The tremor was especially prominent in the significantly underweight 1-month-old MWK mice and was often accompanied by decreased locomotion (Table 1). By 3 months of age the tremor was less prominent, and the MWK mice showed increased locomotion. Indeed, ~40% of the MWK mice were hyperactive during testing (Table 1). The tremor/ataxia was never as severe as to hinder the voluntary movement of the animals. We did not observe the MWK mice dragging their hindlimbs, falling, or rolling over the side during movement which would be signs of severe ataxia.

Table 1. Phenotypic assessment of MWK and WT mice.

The behavioral parameters (tremor, grooming, resting, and the presence of kyphosis and Straub’s tail) were scored from a 3-min video recorded in a cylinder (13 [diam.] x 17 [H] cm). Circling behavior was also assessed from the cylinder recordings but, additionally, it was confirmed using in-cage recordings. The percentage (%) represent fraction of the animals with each behavior. The numerical data (severity for circling, seconds for resting and grooming) was collected only from animals that showed these behaviors. Resting tremor was not assessed in some MWK mice (* n = 3; ** n = 2) as they never stopped moving.

Age	Strain	N	Tremor (severity)		Circling		Resting (> 5 s)		Grooming		Straub tail
Age	Strain	N	Resting	Moving	%	Severity	%	Second	%	Second	Straub tail
1 month	WT	8	0	0	0%	0	100%	21.55	100%	32.74	0%
1 month	MWK	17	2.12	1.91	53%	0.89	94%	52.19	59%	3.06	65%
2 months	WT	6	0	0	0%	0	100%	24.92	83%	17.45	0%
2 months	MWK	14	1.82*	1.43	50%	0.93	79%	24.85	50%	7.72	57%
3 months	WT	6	0	0	0%	0	100%	23.13	100%	15.49	0%
3 months	MWK	12	1.35**	1.33	83%	1.10	58%	35.70	67%	6.49	50%

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The grip test showed that MWK mice are unable to cling to the inverted cage lid at any age (Fig. 1C) and we did not observe hindlimb clasping when the animals were held by the tail for ~15 s. At 1 month of age, MWK mice are unable to rear (Fig. 1D), but they gain some rearing ability with increasing age.

The motor coordination and motor learning ability of the MWK mice was tested using an accelerating rotarod. MWK mice significantly underperformed in comparison to WT siblings at any tested age. 1 month old MWK mice with small body weight and little locomotion perform poorly on rotarod (Supplementary Fig. 1). MWK mice show limited and transient motor learning at 2 months of age, but it completely disappears by 3 months of age (Fig. 1E).

Footprint analysis (Fig. 1F-H) of 2- and 3-month-old MWK mice revealed shorter stride length, wider step width, and longer print length compared with WT mice (Fig. 1G). Because MWK mice are smaller than the age-matched WT, we calculated the ratio between the stride length and step width (SL/SW) to account for the size difference between the 2 strains. The SL/SW ratio is decreased in MWK animals (Fig. 1H) indicating that the changes were not simply due to size differences. We also observed irregular paw placement in MWK mice. While WT mice place their contralateral paw approximately at the middle of the ipsilateral step (Fig. 1F), MWK mice place the paw with a wider range in both directions (Fig. 1I). The gait patterns are similar in 2- and 3-month-old MWK mice.

In addition to the ataxic symptoms, most MWK mice also show stereotypic circling behavior. Unlike the tremor that become less pronounced with age, the circling behavior worsens in older MWK mice (Table 1). Additionally, we observed Straub’s tail (tail erection) in most young MWK mice (Table 1) and decreased self-grooming (Table 1).

3.2. The MWK mutation affects cerebellar size but not lobular morphology

MWK brain size is similar to WT with the exception of the cerebellum. (Supplementary Table 2). MWK cerebella are smaller in comparison with WT (Fig. 2A,B). We calculated the ratio between cerebellar area and cortical area and found that the cerebellar/cortical area ratio was reduced in MWK mice (but not affected by age, body weight, or sex within strain; Fig. 2C, Supplementary Table 2). By measuring the cerebellar areas in whole-mount brain images we found an~21% reduction in the MWK cerebellar area. The cerebellar size reduction was confirmed in cerebellar sections taken at 4 different levels along the medio-lateral axis of the brain and that included both vermal and hemispheral cortex. We observed ~24% reduction in the cerebellar cortex area at every level of the cerebellar sections irrespective of the age of the animals (Fig. 2D). This reduction in cerebellar size is similar across the cerebellar cortex and we did not observe any major changes in lobular morphology at any age (Figs. 3, 5, and 6B).

Fig. 3. — **A-F)** mGluR1α immunolabeling of coronal cerebellar sections shows even immunostaining (brown labeling) of PC dendrites in the molecular layer of WT and MWK mice. WT cerebella contain high densities of mGluR1α-positive type II UBCs in lobule X (A,E), ventral lobule IX (A), flocculus (Fl; C), and ventral paraflocculus (PFl; C). Type II UBCs are rare in MWK cerebellum (B,D,F). **G,H)** Calretinin (CR) immunolabeling of coronal cerebellar sections illustrates the reduction of the calretinin-positive type I UBCs in the MWK lobule X. Arrowheads in E-H point to UBCs that are enlarged in the insets of the same image.

Fig. 5. — CaB immunolabeling of WT (A,E) and MWK cerebella (B—D, F-G) illustrates the gradual PC loss in cerebellar lobules. **A-D)** Lobules VI-VIII in sagittal sections. At P40 the density of the PCs (arrowheads) in MWK cerebella (B) is similar to WT (A). At P120 the PC loss is clearly noticeable in lobule VII (C) and becomes prominent at P180 in lobules VII and VIII (D). *gcl, granule cell layer; ml*, *molecular layer; wm, white matter.* **E-F)** Coronal sections illustrating flocculus (Fl), paraflocculus (PFl), and crus I (CI). PC loss is more prominent in the PFL than in the other lobules (F,G). *CN, cerebellar nuclei.* H) Coronal section from a P100 MWK illustrates overt PC loss in Copula (Cop) and Crus II/Paramedian lobule (CII/PM). PC loss is not obvious in the other lobules shown in the image; lobules, IV/V, VI, X, Simplex (Sim), and Crus I (CI). *CN, cerebellar nuclei.*

Fig. 6. — A) At P40, PC density is similar in WT and MWK mice. At P120 and P180 PC density is significantly reduced in the posterior, but not in the anterior, lobules and in lobule X. The anterior lobules show minor PC density decrease at P180. This pattern in PC density changes is observed in both vermal and hemispheral regions. We grouped lobule simplex with anterior lobules (Ant+Sim) as they both show similar PC density. *Asterisks represent p < 0.05 obtained by One-Way-ANOVA and Tukey’s* post hoc *analysis. The exact p values are shown in* Supplementary Table 1. B) Heatmap of PC densities illustrates PC loss in cerebellar lobules. The colour code represents PC loss using dark blue (0 PC loss) to red (100% PC loss). The exact PC density values in individual lobules are provided in Supplementary Fig. 3. *Roman numerals I-X*, *cerebellar lobules; A, anterior; CI, crus I; CII, crus II; Cop, Copula; PM, Paramedian lobule; S, Simplex; S* + *C, Simplex and Crus.*

Next, we measured the molecular layer area in cerebellar sections immunolabeled with calbindin (CaB; Fig. 2E). In MWK mice, the molecular layer is thinner than in WT at every studied age (Fig. 2F, top panel). The difference slightly increases with age from ~15% (P40) to ~26% (P180), which is attributable to the fact that, contrary to the WT, in MWK mice the molecular layer width does not increase with age (Fig. 2F; Supplementary Fig. 2). We also measured the granule cell layer area and found no significant decrease in the granule cell layer area MWK mice (Fig. 2F, bottom panel; Supplementary Fig. 3).

3.3. Loss of UBCs but not PCs in early postnatal MWK cerebellum

We first examined the cerebellar morphology at P18, a time point when MWK mice exhibit ataxic phenotype. MWK cerebella are smaller, but we did not observe obvious PCs loss (Fig. 3A-D), and PCs appear morphologically intact in CaB and mGluR1α immunostained sections (Fig. 3A-D). UBCs, however, are already severely affected (Fig. 3E-H). Areas normally enriched in UBCs, such as lobule X, ventral lobule IX and flocculus (Fig. 3A,C,E,G), are almost devoid mGluR1α-positive type II UBCs (~98% reduction; Fig. 3B,D,F), while the calretinin (CR)-positive type I UBC are reduced in number (>60% reduction; Fig, 3H).

Next, we utilized cleaved caspase3 (cCasp3) immunostaining and TUNEL staining to investigate possible apoptosis in MWK cerebella (Fig. 4). We did not detect any cCasp3 labeled PCs at P18, and cCasp3 labeled PCs were extremely rare even at the later age points (Fig. 4B).

At P18, we found several cells (presumed UBCs) in the granule cell layer of lobule X and ventral lobule IX that were distinctly labeled with cCasp3 antibody (Fig. 4A,D) or TUNEL staining (Fig. 4F). We counted ~1.4 cCasp3-postive cell/0.01mm² area in the granule cell layer of lobule X in MWK mice (n = 3) and none in WT mice (n = 3; Fig. 4C). TUNEL staining showed about the same number of apoptotic cells (Fig. 4D,F). The number of apoptotic UBCs decreases with age, and by P26 only a few apoptotic cells were seen in lobule X (Fig. 4G) and none at later ages (Fig. 4H).

3.4. Purkinje cell loss is slow and incremental

While UBCs are eliminated from the MWK cerebella before 1 month of age, PCs are spared at this early stage. PC loss is not evident until 3 months of age (Fig. 5) and even after this age it is slow and only partial. At P40, MWK PCs are properly aligned just as in the WT cerebella (Fig. 5A,B) and we only found rare ectopic or misplaced PC in both WT and MWK mice (not shown), indicating proper PC migration and positioning during development in both strains. However, the MWK cerebella contain ~10% less PCs than the WT when counted in comparable sagittal cerebellar sections (Table 2). While PC density in most lobules is similar in MWK and WT mice (Fig. 6; Supplementary Fig. 4), there are exceptions such as the vermal lobule VIII, and hemispheral paramedian lobule and copula, where PC density in MWK cerebella is ~20% decreased (Fig. 6B; Supplementary Fig. 4).

Table 2. PC number in WT and MWK mice.

The PC numbers estimated in 4 sagittal cerebellar sections taken at different levels along the medio-lateral axis. The lateral distance indicated in the table is based on Paxinos and Franklin (2013) and applies only to the WT cerebella. Since the MWK cerebella are smaller than the WT, we matched them with WT counterparts based on morphological landmarks such as cerebellar nuclei. Data shown with SEM. (n = 3 WT for all ages, 4 MWK for P40 and P120, and 5 MWK for P180).

Age	Lateral 0.0–0.3 mm			Lateral 0.6–0.8 mm			Lateral 1.2–1.4 mm			Lateral 1.8–2.0 mm
Age	WT	MWK	Difference	WT	MWK	Difference	WT	MWK	Difference	WT	MWK	Difference
P40	1399.2 ± 40.2	1274.9 ± 14.6	9%	1189.2 ± 43.3	1046.8 ± 27.6	12%	668.4 ± 27.1	626.9± 44.0	6%	712.7 ± 66.0	642.5 ± 33.1	10%
P120	1407.4 ± 62.5	12050 ± 58.8	14%	1132.1± 77.4	894.7 ± 72.8	21%	704.3 ± 18.3	483.0± 15.6	31%	720.2 ± 26.8	530.0 ± 33.2	26%
P180	1489.2 ± 33.2	1104.0 ± 89.2	26%	1300.5 ± 31.0	839.5 ± 110.2	35%	697.8± 52.2	443.8 ± 63.2	36%	717.4 ± 68.7	409.2 ± 84.4	43%

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PC loss becomes more noticeable after 3 months of age (Fig. 5F,H). At P120, MWK mice show ~23% decrease in PC number, while at P180 the loss is ~35% (Table 2). We did not calculate PC density at later stages, although a coarse estimation from visual inspection of 1 year old MWK coronal cerebellar sections (n = 3) suggests that ~50% PC are still present and that the pattern of PC loss is similar to that observed in P180 MWK mice (Fig. 5G; Supplementary Fig. 5).

Remarkably, the PC loss is not uniform across the cerebellum. At every age analyzed, the posterior lobules (lobule X excluded) show greater PC loss than the anterior lobules or lobule X (Figs. 5 and 6; Supplementary Fig. 4 and 5). A detailed lobular analysis revealed that the TRPC3 MWK mutation predominantly affects PC in vermal lobules VII and VIII, hemispheral crus II, paramedian lobule and copula, and paraflocculus (Fig. 5 and 6; Supplementary Fig. 4 and 5). PC in anterior lobules show little PC loss until P180 and even after that it is not as prominent as in the posterior cerebellum (Fig. 3F). PCs in lobule X, ventral lobule IX and flocculus show remarkable resilience with slight PCs loss even at 1 year of age (Fig. 5E-H and 6; Supplementary Fig. 4 and 5).

3.5. Axonal torpedoes and degeneration

CaB-labeled MWK sections revealed an increased occurrence of PC axonal swellings (Fig. 7A). These swellings, also known as torpedoes, are associated with PC degeneration, but they also occur transiently during development in healthy animals (Lang-Ouellette et al., 2021; Ljungberg et al., 2016). In WT, we only observed small torpedoes, and only at P40 (Fig. 7A-C). In contrast, the torpedoes are prominent in MWK, and they increase in size and number with mouse age (Fig. 8). By P180 some torpedoes are as large as a PC soma. Some axons contain multiple torpedoes and are unusually thick (Fig. 7C). As expected, the occurrence of torpedoes in different cerebellar lobules coincides with the degree of PC loss (Fig. 7D).

Fig. 8. — **A,B)** Low magnification images of lobule X, IX and cerebellar nuclei (CN) from WT (A) and MWK (B) mice at P40. **C-G)** Higher magnification images from the boxed areas in panels A and B (C,D,F,G) and from lobule III (E). In WT cerebella (C,F) only non-specific, fine silver precipitates (bluish staining; magenta arrows in F) are detectable, mostly over cerebellar nuclei (CN) neurons (F). This precipitate is also observed in MWK (magenta arrows in G) cerebella and is considered as a staining artefact. In MWK cerebella the granule cell layer (gcl) in lobule X (D) contains multiple degenerating cells (black staining; arrows). A degenerating PC (arrowhead) in MWK cerebellum (E). The remnants of the degenerating PC arbor are still recognizable in the molecular layer (ml). The MWK cerebellar white matter (wm) and cerebellar nuclei (CN) contain several degenerating fibers (bluish-black staining; arrowheads in G). *ml, molecular layer; Pcl, Purkinje cell layer.*

The presence of these torpedoes suggests an axonal pathology that could lead to PC degeneration. Therefore, we next looked at the degeneration in MWK cerebella utilizing silver impregnation. We found degenerating cells and fibers in the MWK but not WT cerebella (Figs. 8 and 9). At P40, degenerating cells are sparse in most lobules, but the granular cell layer of lobule X, ventral region of lobule IX, and flocculus contain numerous degenerating cells (Fig. 8D). As the areas are typically enriched in UBCs, the degenerating cells are likely UBCs. Overall, PC degeneration is modest considering the degree of PC loss in MWK cerebellum (Fig. 6). At P40 we found only rare PCs (~1 PC in 30 μm-thick section) identified either by degenerating soma and dendritic arbor (Fig. 8E) or just by degenerating PC dendrites (the sectioning angles were not always ideal to capture both soma and dendritic arbor). Degenerating PCs are also rare in 2- and 3- month-old MWK cerebella, when only ~2 degenerating PCs/30 μm-thick section are detectable. Degenerating PCs occur randomly in every lobule at every age.

Fig. 9. — **A,B)** In the anterior lobules I-V the degenerating fibers in the white matter (wm) are not detectable until P67 (bluish-black staining; arrows in B). **C,D)** The white matter of the posterior lobules VI-IX contained multiple degenerating fibers (arrows in C) already at P40. The number of the degenerating fibers (arrows in D) visibly increased at P67. **E,F)** At P40, the degenerating fibers are present in the white matter of the posterior cerebellar lobules (arrows in E) as well as in the cerebellar and vestibular nuclei (VeN). The anterior interposed cerebellar nucleus (IntA) contained the least of the degenerating fibers (E). At P67 the degeneration was massive and present in the white matter of all lobules (arrows in F). The degenerating fibers appear to be evenly distributed in all cerebellar nuclei (F). A thin band of *posterior interposed cerebellar nucleus; Med, medial cerebellar nucleus; ml, molecular layer; wm, white matter.*

While degenerating PC bodies and dendrites are rare, degenerating PC axons are frequent in MWK and their occurrence increases with age (Figs. 8G and 9). At P40 most degenerating fibers appear in the white matter of the posterior (Fig. 9C), but not anterior lobules (Fig. 9A). Degenerating fibers are also found in cerebellar and vestibular nuclei with the anterior interposed cerebellar nucleus showing the least amount of degeneration (Fig. 9E). This observation is in line with our result showing differential PC loss in the posterior versus anterior MWK cerebellum (Fig. 6). However, by P67 the PC axonal degeneration is prominent in every lobule and in cerebellar and vestibular nuclei (Fig. 9B,D,F). The same pattern of degenerating fibers was observed in 3-month-old MWK mice (not shown).

3.6. MWK Purkinje cells are electrophysiologically silent

We performed cell-attached recordings from MWK PC at P19, around the onset of the ataxic symptoms. Only 4 of 11 recorded PCs showed spontaneous firing (at 30–32 °C; Fig. 10A-C), while 7 of 11 PCs were silent. Because in the SCA1 mouse model PCs can regain spontaneous firing ability through PC atrophy/remodeling (Dell’Orco et al., 2015), we also recorded spontaneous activity in 3-month-old MWK PCs. At this age, all WT PCs showed spontaneous activity during cell-attached recordings (Hu et al., 2019; Womack and Khodakhah, 2002), and the average firing frequency was 26.9 ± 5.0 Hz at 30–32 °C, (n = 6 PCs; Fig. 10A-C). Under the same conditions ~80% of MWK PCs (12 of 15 PCs; Fig. 10A-C) were silent, while the other 3 PCs exhibited spontaneous activity with higher frequency compared with WT (63.5 ± 4.7 Hz; Fig. 10A-C), suggesting the presence of an enhanced depolarizing conductance in MWK PCs.

Fig. 10. — A) Cell-attached recordings from acute slices reveal 3 types of electrophysiological activity in PCs from MWK mice. B) The relative frequency of occurrence of these firing patterns in MWK slices is age dependent. In contrast, all WT PCs show spontaneous regular firing. C) The average firing frequency in WT cells was 26.9 ± 5.0 Hz (6 cells). At P19, 7 of 11 MWK PCs were silent; the 4 (of 11) that were spontaneously active, fired at a frequency similar to WT cells. At P90 MWK PCs showed either no spontaneous activity (12 of 15 cells) or fired at higher frequency (63.5 ± 4.7 Hz; 3 of 15 cells). D) All silent MWK PCs could fire in response to depolarizing current injections when held at hyperpolarized membrane potential with injection of bias current.

In line with this hypothesis, the average (integrated) membrane potential (measured in whole cell configuration) is depolarized in MWK PCs compared with WT (−36.4 ± 2.9 mV vs −48.1 ± 1.4 mV, 15 and 8 PCs, respectively; p < 0.01). This finding suggests that the lack of spontaneous firing in cell-attached recordings results from decreased sodium channel availability due to partial channel inactivation. If so, MWK PCs should be able to fire action potentials when held at more hyperpolarized potentials via injection of bias current. This was the case, as all tested MWK PCs fired action potentials with typical shape and amplitude in response to positive current injections when held at ~ −70 mV (Fig. 10D). This finding suggests that the functional properties and density of voltage-gated ion channels are by and large unaffected in MWK PCs.

3.7. RNA seq analysis reveals large, ataxia-specific, changes in gene expression

MWK is caused by a single point mutation in a single gene; yet the cerebellum shows massive cellular and morphological alterations caused by homeostatic and/or maladaptive responses. To gain insights into the complexity of cerebellar rearrangement we performed RNA profiling of P21 MWK and WT cerebella (3 mice in each group). We identified 2067 genes that were differentially expressed between the 2 strains. A full list of the significantly FDR (p ≤ 0.05) upregulated (1052) and downregulated (1016) genes is provided as Supplementary Table 5. TRPC3 transcript expression was not affected in MWK mice. Somewhat unexpectedly the genes that showed the largest differential expression are not typically associated with motor functions. Opsin3 (Opn3; Fig. 11A, Table 3) shows the largest fold change of all genes, but little is known about its function in the cerebellum, except that it is expressed in PCs (Lein et al., 2007). The second highest upregulated gene in the MWK cerebella, is TH (Th; Fig. 11A, Table 3), which is only transiently expressed in the PC during development. However, TH expression was previously detected in the PCs of some mutant mice (Fureman et al., 1999; Fureman et al., 2003; Jeong et al., 2001; Sawada and Fukui, 2010; White et al., 2016; White et al., 2014).

Fig. 11. — A) Volcano plot showing the genes up- and downregulated in MWK compared with WT cerebellum. Mutation or lack of the genes depicted on the plot were reported to cause motor dysfunction/ataxia in several human SCA, in SCA mouse models, and ataxic mutant rodents. Many genes are also differentially regulated in SCA models (SCA1 and SCA2) and P18 laser captured MWK PC (e.g. *Opn3, Svc2, Stk17b*, *Dgkh, Fgf7 and Doc2b;* Dulneva et al., 2015). Magenta and green dots represent PCs and granule cells, respectively. **B,C)** Top 15 affected biological processes and cellular component identified using DAVID (Database Annotation Visualization Integrated Discovery) enrichment analysis of genes differentially expressed in MWK cerebellum. Additional terms for both enrichment categories and the exact p values are shown in Supplementary Tables 3 and 4.

Table 3. Downregulated ataxia-related genes in MWK.

Mutation or knockout (KO) of several listed genes are known to cause SCA in humans as well as motor dysfunction and ataxia in mice. The list also includes genes downregulated in P18 MWK mice with exception of Shank2, which is upregulated in P18 MWK. Cell type expression is based on Allen Brain Atlas ISHH images and published papers. CN-cerebellar nuclei, BG-Bergman glia, GC-granule cells, GoC-Golgi cells, PC-Purkinje cells, S/BC-stellate and basket cells. The list shows genes with FDR Adjusted p < 0.003.

Official Symbol	Official Full Name (also known as)	Cell type expression	FDR Adj p Value	SCA, SCA models and mouse mutants	References
Fgf7	fibroblast growth factor 7	PC	3.2E-45	downregulated in P18 MWK	Dulneva et al., 2015
Doc2b	double C2, beta	PC	6.3E-39	downregulated in P18 MWK	Dulneva et al., 2015
Shank2	SH3/ankyrin domain gene 2	PC, GoC, S/BC, CN	3.2E-32	PC-specific KO; downregulated in SCA2 model; upregulated in P18 MWK	Dulneva et al., 2015; Peter et al., 2016; Arsović et al., 2020
Grid2ip	glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein 1	PC	5.8E-27	listed under Gid2
Stk17b	serine/threonine kinase 17b (apoptosis-inducing) (DRAK2)	PC	1.2E-24	downregulated in SCA1, SCA7, SCA14, SCA41 models; and P18 MWK	Dulneva et al., 2015; Wu and Kapfhammer, 2021, 2022
Hipk2	homeodomain interacting protein kinase 2	PC	1.2E-18	KO mouse	Anzilotti et al., 2015
Camk2a	calcium/calmodulin-dependent protein kinase II alpha	PC	3.1E-15	downregulated in SCA2 model	Arsović et al., 2020
Camk1d	calcium/calmodulin-dependent protein kinase ID	no signal	4.3E-15	downregulated in SCA2 model	Arsović et al., 2020
Rora	RAR-related orphan receptor alpha	PC, S/BC, CN	1.5E-14	staggerer mice; RORA−/− mouse; human mutation	Guissart et al., 2018; Lalonde and Strazielle, 2007, 2019; Halbach et al., 2017
Ptpn4	protein tyrosine phosphatase, nonreceptor type 4	PC, GoC, CN	1.6E-14	KO mouse	Kina et al., 2007
Grm4	glutamate receptor, metabotropic 4 (mGluR4)	GC, CN	3.2E-14	KO mouse; downregulated in SCA2 model	Pekhletski et al., 1996; Arsović et al., 2020
Frrs1l	ferric-chelate reductase 1 like (6430704M03Rik)	PC	6.2E-13	KO mouse	Wang et al., 2022a
Dgkh	diacylglycerol kinase, eta	PC	1.5E-12	KO mouse; downregulated in SCA2 model and P18 MWK	Dulneva et al., 2015; Hozumi et al., 2017
Bean1	brain expressed, associated with Nedd4, 1	PC	2.2E-12	SCA31	Ishikawa and Nagai, 2019; Ishikawa, 2023
Atp2b3	ATPase, Ca++ transporting, plasma membrane 3 (PMCA3)	PC, GC, S/BC, CN	2.8E-11	spontaneous shaker mutant rat; human mutation	Calì et al., 2015; Figueroa et al., 2016; Erekat, 2017
Gabra1	gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1	PC, S/BC, CN	4.1E-10	KO mouse	Nietz et al., 2020
Far2	fatty acyl CoA reductase 2	PC, GoC	6.4E-10	downregulated in P18 MWK	Dulneva et al., 2015
Cdc42ep1	CDC42 effector protein (Rho GTPase binding) 1	BG	2.4E-09	KO mouse	Ageta-Ishihara et al., 2015
Cdkl5	cyclin-dependent kinase-like 5	GC, CN	4.7E-09	KO mouse	Sivilia et al., 2016
Kcnc3	potassium voltage gated channel, Shaw-related subfamily, member 3	PC, CN	6.9E-09	SCA13	Waters, 1993; Duarri et al., 2015
Cbln1	cerebellin 1 precursor protein	GC, CN	9.5E-09	KO mouse	Hirai et al., 2005; Matsuda and Yuzaki, 2012
Caln1	calneuron 1	PC, GoC, CN	1.4E-08	downregulated in SCA2 model	Arsović et al., 2020
Cacna2d2	calcium channel, voltage-dependent, alpha 2/delta subunit 2	PC, GoC, S/BC, CN	1.7E-08	Ducky mutation mouse	Barclay et al., 2001; Brodbeck et al., 2002
Ptprr	protein tyrosine phosphatase, receptor	PC	2.7E-08	PTPRR^−/− mouse	Chirivi et al., 2007; Hendriks et al., 2009
Igfbp5	insulin-like growth factor binding protein 5	GC	8.8E-08	downregulated in SCA2 model	Arsović et al., 2020
Icmt	isoprenylcysteine carboxyl methyltransferase (PCCMT)	PC	2.5E-07	downregulated in SCA1 model and Atxn2 KO mouse	Halbach et al., 2017; Lin et al., 2000
Atp8a2	ATPase, aminophospholipid transporterlike, class I, type 8 A, member 2	PC, CN	8.6E-07	mutation in humans	Narishige et al., 2022; Onat et al., 2013
Cbln3	cerebellin 3 precursor protein	GC	9.6E-07	downregulated in SCA2 model	Arsović et al., 2020
Car8	carbonic anhydrase 8	PC, CN	2.0E-06	Waddles mutant mouse	White et al., 2016; Shimobayashi and Kapfhammer, 2018
Sacs	sacsin	PC	2.3E-06	Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)	Engert et al., 2000; Márquez, 2021
Cacna1g	calcium channel, voltage-dependent, T type, alpha 1G subunit (Cav3.1)	PC, GoC	3.3E-06	SCA42	Chemin et al., 2018; Hashiguchi et al., 2019; Berecki et al., 2020
Atp2b2	ATPase, Ca++ transporting, plasma membrane 2 (PMCA2)	PC, GoC, CN	4.1E-06	KO mouse	Empson et al., 2010; Kozel et al., 1998
Itpka	inositol 1,4,5-trisphosphate 3-kinase A	PC	4.3E-06	downregulated in SCA2	Arsović et al., 2020
Scn2a1	sodium channel, voltage-gated, type II, alpha	GC	5.4E-06	mutations	Liao et al., 2010
Slc1a6	solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6 (EAAT4)	PC	1.3E-05	episodic human ataxia, downregulated in SCA1 model, KO mouse,	Lin et al., 2000; Jen et al., 2005; de Vries et al., 2009; Choi et al., 2017; Perkins et al., 2018
Shank1	SH3/ankyrin domain gene 1	PC	1.5E-05	downregulated in SCA2 model	Arsović et al., 2020
Camk4	calcium/calmodulin-dependent protein kinase IV	GC	2.7E-05	mutation in humans, downregulated in SCA2 model	Zech et al., 2018; Arsović et al., 2020
Cdk5r1	cyclin-dependent kinase 5, regulatory subunit 1 (p35)	PC	3.5E-05	PC specific KO mouse	He et al., 2014
Gpd1	hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)	BG	4.1E-05	downregulated in Atxn2 KO mouse	Halbach et al., 2017
Scn1a	sodium channel, voltage-gated, type I, alpha (Nav1.1)	PC, GoC	5.2E-05	KO mouse; Dravet syndrome	Kalume et al., 2007; Hoffman-Zacharska et al., 2015; Gataullina and Dulac, 2017; Hoxha et al., 2018
Ank1	ankyrin 1, erythroid	PC, GoC, CN	2.1E-04	nb/nb mutation KO mouse, PHARC (polyneuropathy,	Peters et al., 1991
Abhd16a	abhydrolase domain containing 16 A	GC, CN	2.2E-04	hearing loss, ataxia, retinitis pigmentosa, and cataract) in human	Singh et al., 2020
Itpr1	inositol 1,4,5-trisphosphate receptor 1 (IP3R1)	PC	2.4E-04	SCA15, SCA16, SCA29, Gillespie syndrome, downregulated in SCA1 and SCA2 models	Halbach et al., 2017; Lin et al., 2000; Romaniello et al., 2022
Scn4b	sodium channel, type IV, beta (Navβ4)	PC, CN	2.5E-04	KO mouse	Hoxha et al., 2018; Ransdell et al., 2017
Homer3	homer scaffolding protein 3	PC, S/BC, CN	4.4E-04	downregulated in SCA2 model	Arsović et al., 2020
Grm3	glutamate receptor, metabotropic 3 (mGluR3)	GoC	6.4E-04	downregulated in SCA2 model	Arsović et al., 2020
Hpcal1	hippocalcin-like 1	PC	6.4E-04	downregulated in SCA2 model	Arsović et al., 2020
Grid2	glutamate receptor, ionotropic, delta 2	PC	1.2E-03	SCA18; Lurcher mouse; hotfoot mouse; KO mouse	Lalouette et al., 1998; Doughty et al., 2000; Lalonde and Strazielle, 2007, 2019; Hills et al., 2013; Ceylan et al., 2021
Fgf14	fibroblast growth factor 14	GC, CN	1.6E-03	SCA27	Bosch et al., 2015; Shakkottai et al., 2009
Agtpbp1	ATP/GTP binding protein 1 (Nna1)	PC	2.1E-03	Pcd mutant mouse	Lalonde and Strazielle, 2007, 2019; Zhou et al., 2018

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Next, we undertook an extensive PubMed search to identify the genes from our list that are linked with ataxia and cerebellar dysfunction. We used Allen Brain Atlas database and published literature to determine cell-type specificity of the genes. The results of this analysis are summarized in Tables 3 and 4 showing genes with <0.003 FDR p value. One of the top downregulated gene is Stk17b (DRAK2). This gene is also downregulated in SCA1, SCA7, SCA14, and SCA41 (Wu and Kapfhammer, 2021; Wu and Kapfhammer, 2022). Mutations or lack of several genes have causal role in SCA13(kcnc3), SCA15 (Itpr1), SCA16 (Itpr1), SCA18 (Grid2), SCA31 (Bean1), SCA27 (Fgf14), SCA29 (Itpr1), and SCA42 (Cacna1g) (Bosch et al., 2015; Ceylan et al., 2021; Hashiguchi et al., 2019; Ishikawa, 2023; Ishikawa and Nagai, 2019; Shakkottai et al., 2009; Shimobayashi and Kapfhammer, 2018; Waters, 1993) as well as in ataxic phenotype in SCA mouse models and mutants (e.g. staggerer (Rora), RORA^−/− (Rora), hoot-foot (Grid2), Lurcher mice (Grid2), Waddless (Car8), and pcd (Agtpbp1); (Halbach et al., 2017; Lalonde and Strazielle, 2007; Lalonde and Strazielle, 2019; Shimobayashi and Kapfhammer, 2018; Zhou et al., 2018; White et al., 2016). Some of these genes are also downregulated in SCA1 and SCA2 models (Arsović et al., 2020; Diallo et al., 2021; Lin et al., 2000). About 70% of these genes are expressed in PCs and about 20% in the granule cells (Tables 3 and 4). The rest of the genes are expressed in other cell types like Bergman glia and Golgi cells, or they can not be assigned to a cell type based on Allen Brain Atlas.

Table 4. Upregulated ataxia-related genes in MWK.

Tyrosine hydroxylase (Th) is ectopically upregulated MWK. Several of the listed genes were upregulated in P18 MWK mouse Cell type expression is based on Allen Brain Atlas ISH images and published papers. CN-cerebellar nuclei, BG-Bergman glia, GC-granule cells, GoC-Golgi cells, PC-Purkinje cells, S/BC-stellate and basket cells. The list shows genes with FDR Adjusted p < 0.003.

Official Symbol	Official Full Name (also known as)	Cell type expression	FDR Adj p Value	SCA models and mouse mutants	References
Opn3	opsin 3	PC	3.6E-72	upregulated in P18 MWK	Dulneva et al., 2015
Th	tyrosine hydroxylase	none	9.9E-45	ectopic PC expression in Nagoya mutant, tottering mice, pogo mice, Car8 KO, and PC specific VGAT mutant	Fureman et al., 2003; Jeong et al., 2001; Sawada and Fukui, 2010; White et al., 2014; White et al., 2016
Sv2c	synaptic vesicle glycoprotein	2c PC, CN	4.5E-28	upregulated in P18 MWK	Dulneva et al., 2015
Ryr3	ryanodine receptor 3	PC	1.8E-16	upregulated in Nagoya mouse; changed in SCA2	Arsović et al., 2020; Sawada et al., 2008
Prdx6	peroxiredoxin 6	PC	3.3E-11	upregulated in P18 MWK	Dulneva et al., 2015
Osbpl1a	oxysterol binding protein-like 1 A	not conclusive	4.8E-06	downregulated in P18 MWK	Dulneva et al., 2015
Atp2a2	ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (SERCA)	PC, GoC, CN	2.0E-05	downregulated in SCA1, SCA2 models and Atxn2 KO mouse	Arsović et al., 2020; Halbach et al., 2017; Lin et al., 2000
Inpp5a	inositol polyphosphate-5-phosphatase A	PC	3.6E-05	KO mouse; downregulated in SCA1, SCA2, SCA7, SCA17 models and Atxn2 KO mouse	Lin et al., 2000; Kasumu et al., 2012; Yang et al., 2015; Halbach et al., 2017; Shimobayashi and Kapfhammer, 2018; Arsović et al., 2020; Liu et al., 2020; Wu and Kapfhammer, 2022;
Camk2n1	calcium/calmodulin-dependent protein kinase II inhibitor 1	no data	1.2E-04	downregulated in SCA2 model	Arsović et al., 2020

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3.8. Gene ontology (GO) enrichment analysis suggests that the mutation affects cerebellar development and synaptic connectivity

Gene ontology (GO) enrichment analysis predicts changes in neuronal development, cell adhesion and apoptosis (Fig. 11C; Supplementary Figs. 6 and 7). Expression of Casp3, a key player in apoptosis and Hrk (harakiri) a BCL-2 (B-cell lymphoma 2) interacting protein is highly upregulated in MWK cerebella (Fig. 11A; Supplementary Fig. 5), but the expression of Casp9, one of the key initiators of caspase cleavage (Brentnall et al., 2013; Sahoo et al., 2023), is downregulated (Supplementary Fig. 6). Based on enrichment analysis, expression of genes involved in apoptosis, including the genes involved in negative regulation of the apoptotic process. (Fig. 11B) is significantly changed in the MWK cerebellum.

GO enrichment analysis provides further results that are in line with those expected in general cerebellar dysfunction. Using the cellular component terms (GOTREM_CC_DIRECT; DAVID) on the set of the differentially expressed genes, we found significant changes in glutamatergic synapses as well as in synaptic structures (Fig. 11C). Additionally, GO enrichment analysis for biological processes (GOTREM_BP_DIRECT; DAVID; Fig. 11B), identified changes in synaptic plasticity regulation. Metascape analysis utilizing GO terms glutamatergic synapse (mmu04724) and synaptic signaling (GO:0099537) revealed 190 and 150 differentially regulated genes, respectively (Supplementary Fig. 8).

3.9. TEM analysis reveals pathological changes in Purkinje cell dendritic spines

Our RNA seq analysis suggests changes in synaptic contacts onto PCs. Therefore, we examined cerebellar ultrathin sections from P40 (lobule VII and X) and P85 (lobule VII) WT and MWK mice using TEM (Fig. 12). At P40 about half of the PC dendritic spines in each field were in contact with parallel fiber terminals in WT mice (Fig. 12A), but this was not the case in MWK mice, where contact was reduced (compare panel A with panel B in Fig. 12). In general, the dendritic spines appeared less mature in MWK mice compared to WT, as many dendritic processes looked like filopodia (Fig. 12B). Some dendritic areas also showed higher than usual densities of dendritic processes, as illustrated in Fig. 12B. The molecular layer of lobules VII and X showed similar pathology in the MWK mouse. These findings suggest delays in MWK maturation and thus some of these observations, even at P40, might be due to an immature cerebellum. Therefore, next we looked at P85 cerebellum, more specifically analyzed lobule VII of MWK and WT mice. We have chosen 6 fields with enriched dendritic spines (20–40 spines per field) from each apical, mid, and proximal regions of the molecular layer. Using this selection, we sampled multiple PCs synaptic fields yet avoided areas with missing PCs. Just as in P40 we found reduced synaptic contact between PC dendritic spines and parallel fibers (compare panel C with panel D in Fig. 12) and the reduction was the same for all 3 regions investigated. Quantification of synaptic contacts revealed a significant reduction in every field analyzed (Fig. 12E).

We also noted swollen endoplasmic reticulum (ER) structures in the dendrites and spines of MWK PCs at both age points (Fig. 12B, D). This pathology was not universally present in every dendrite and dendritic spine of the MWK PCs, however, upon closer inspection, most MWK PCs (Fig. 12D) contain some pathological swollen ER. We quantified the swollen ER structures in WT and MWK mice and found a significant increase in swollen ER in every MWK field analyzed (Fig. 12 F). A few PCs showed overt ER pathology as shown in Fig. 12B. Additional experiments will be needed to determine the fraction of PCs with advanced ER pathology and their impact on ataxic phenotype.

4. Discussion

Detailed analysis of the MWK phenotype in CD1 mice shows a non-progressive ataxia that is first detectable around P17 and characterized by a broad spectrum of signs (Fig. 13). The gain of function TRPC3^mwk mutation affects the development and network integration of PCs and UBCs and causes PC dysfunction and ultimately PC loss (although incomplete and lobule-selective). Ultrastructural analysis shows ER dysfunction is a major factor in the PC pathology.

Fig. 13. — *, the minor improvement in ataxic phenotype refers to decreased tremor and increased rearing ability. ML, *molecular layer.*

4.1. MWK ataxic phenotype is non-progressive

The MWK ataxia is characterized by impaired motor learning, a distinct rhythmic tremor, altered gait, and imbalance during rearing, especially in younger animals. During the animals’ life span, the tremor lessens, and the mice gain some rearing ability, but gait and motor coordination/learning do not improve with age.

We also observed circling behavior in the MWK mice. Unlike the ataxic behavior, this behavior worsens with the age of the animals. Circling behavior is often associated with vestibular system dysfunction (Löscher, 2010) but stereotypic repetitive motor behavior is also reported in several psychiatric, neurological, and neurodevelopmental disorders, including Autism Spectrum Disorder (ASD; Brady et al., 2022; Lewis et al., 2007). Notably TRPC3 is expressed in UBCs, a major component of vestibular circuitry and in the inner ear (Quick et al., 2012; Sciarretta et al., 2010); therefore, the circling behavior may be of vestibular origin. An alternative possibility is that this behavior is caused by the mutation affecting extracerebellar neurons expressing TRPC3. For example, stereotypic behaviors such as circling are typically found in autism spectrum disorder models (Kim et al., 2016) and associated with dysfunctions of several brain areas including the basal ganglia, where TRPC3 expression has been described (Um et al., 2021; Zhou et al., 2008), although we did not find any obvious morphological changes in this area, nor in the few other brain regions (e.g. in hippocampus (Amaral and Pozzo-Miller, 2007), caudate putamen, and inferior olive (Allen Brain Atlas)) where extracerebellar expression of TRPC3 has been reported. Notably, the TRPC3c isoform, which shows enhanced calcium signaling, is predominantly expressed in the cerebellum (Cederholm et al., 2019; Kim et al., 2012). This might explain why TRPC3^mwk mutation is so detrimental to cerebellar neurons.

The body weight of the MWK mice is also affected, especially around weaning age (P20-P28) when the animals appear malnourished. The transient early weight deficiency in MWK mice might be caused by ataxia/tremor that hinders their access to food. However, TRPC3 is also expressed in non-neuronal tissues, especially in the cardiovascular system, muscles, and in taste and salivary ductal cells (Cherkashin et al., 2023; Choi et al., 2023; Creisméas et al., 2021; Tiapko and Groschner, 2018) and therefore it is possible that some dysfunction on these tissues may contribute to the observed weight deficiency.

4.2. PC dysfunction rather than loss causes the MWK ataxia

The timeline of PC loss does not match the ataxic behavior that develops early (Fig. 13), while PC loss is not significant until 3 months of age. It is thus tempting to attribute the ataxic symptoms to absence/ death of UBCs, which are already absent at P18, especially since motor dysfunction linked to UBC loss has been described (Kreko-Pierce et al., 2020). Yet, although PCs appear morphologically intact at P18, they lack spontaneous firing. Thus, PC dysfunction rather than PC loss appears to cause MWK ataxia (Becker, 2014; Sekerková et al., 2013). This may explain why the MWK ataxia does not worsen with age despite increasing PC loss. Interestingly, PC firing does not appear to recover in parallel with the transient behavioral improvement of MWK mice ~2 months of age, suggesting that it is simply due to improved muscle mass (gain weight). Altered PC firing has been suggested to cause motor deficiency in other SCAs, episodic ataxia 2 (EA2) and autosomal-recessive spastic ataxia of the Charlevoix-Saguenay (Chang et al., 2022; Cook et al., 2021; Jayabal et al., 2015; Kapfhammer and Shimobayashi, 2023; Shimobayashi and Kapfhammer, 2021). Severe ataxia and tremor without PCs degeneration was also reported in Waddles mouse (Car8^wdl; White et al., 2016).

In MWK, the early PC dysfunction is accompanied by morphological changes. The molecular layer thickness is reduced suggesting shorter PC dendritic tree. This is in agreement with the observation that the TRPC3^mwk mutation affects PC dendritic tree development in organotypic cultures (Becker et al., 2009; Dulneva et al., 2015). Surprisingly, our TEM analysis revealed that PCs lack proper synaptic contact with granule cell terminals and have filopodia-like protrusion instead of typical dendritic spines. The mechanisms underlying this altered connectivity remain to be investigated.

The structures mediating PC output are also severely affected in MWK. PC axonal swellings (torpedoes) and thickened PC axons are detectable weeks before any cell loss. PC torpedoes are part of the pathology of several neurodegenerative diseases (Lang-Ouellette et al., 2021; Lomoio et al., 2011) and even traumatic brain injury (Özen et al., 2022). The occurrence of axonal swellings is also correlated with essential tremor in humans (Babij et al., 2013; Louis et al., 2019; Louis et al., 2023). Intriguingly, in the developing cerebella, PC axonal swellings are transient and associated with enhanced action potential propagation and cerebellar performance (Lang-Ouellette et al., 2021; Ljungberg et al., 2016). Therefore, the formation of torpedoes in MWK mice might represent an adaptive response to defective PC transmission, especially early on in the disease. PC axonal degeneration occurs in the posterior lobules as early as P40 and extends to the whole cerebellum within a few weeks. Thus, the morphological alterations in MWK cerebella also involve the cerebellar and vestibular nuclei.

Considering that the MWK PCs seems to receive a decreased number of excitatory synapses and that their axons undergo degeneration, we suggest that they are isolated from the cerebellar circuitry weeks before PC loss.

4.3. Mechanisms of cell loss in the MWK cerebellum

While UBCs undergo apoptosis, apoptotic and/or degenerating PCs are rare at any studied age. However, because PC loss is slow and incremental over many months, it might be impossible to detect more than a few apoptotic/degenerating PCs at any given time. Hrk, one of the most significantly upregulated genes in our study, is known to inhibit the anti-apoptotic Bcl-2 (Singh et al., 2020; Wilke et al., 2023) which regulates the programmed PC death (Vogel, 2002). The expression of the executioner Casp3 is also highly upregulated in MWK cerebella when UBCs are already absent. However, the activation of the apoptotic pathway requires the cleavage of Casp3 (Walters et al., 2009). Intriguingly, expression of Casp9, one of the key initiators of caspase cleavage (Brentnall et al., 2013; Sahoo et al., 2023), is downregulated. Our gene analysis indicates negative regulation of the apoptotic processes in the MWK cerebellum (Fig. 11B). This suggests that PC are able to survive apoptotic stimuli even after Casp3 activation. While the executioner caspase activation has been considered a point of no-return in apoptotic cell death, recent publications show that cells can overcome caspase activation and survive (Nano et al., 2023; Roux et al., 2015; Sun and Montell, 2017). Although PC death in the MWK may be caused by apoptosis as a failure to counteract apoptotic signals (as shown in Fig. 4B) it is therefore possible that the majority of PC die by non-apoptotic processes such as autophagy (Erekat, 2022; Nikoletopoulou et al., 2015).

Another interesting aspect of the PC loss is its lobule specificity. Considering that in MWK cerebella PCs are dysfunctional and poorly integrated into the circuitry one may expect that their location does not influence their survival. Yet the posterior cerebellum, more precisely vermal lobules VII and VIII, hemispheral paramedian and copula, and paraflocculus show the highest PC loss. In contrast, PC loss is milder in all anterior lobules, lobule X and flocculus. The SCA14 mouse model, which is also characterized by TRPC3 gain of function, shows similar lobule VII-specific PC loss (Trzesniewski et al., 2019). Lobule selective PC loss is not unusual in other SCA and ataxic mutants (Duchala et al., 2004; Liu et al., 2013; Martin et al., 2019; Sarna and Hawkes, 2011; White et al., 2021; Zhou et al., 2018), yet the mechanisms of lobular selectivity in PC loss is not understood, especially since the pattern of PC loss differs in various neurodegenerative diseases. Similar to our findings, lobule X and flocculus are resilient to PC loss in several other models (Chung et al., 2016; Duchala et al., 2004; Martin et al., 2019; Sarna and Hawkes, 2011; White et al., 2021; Zhou et al., 2018). Understanding what differentiates these PCs from those in other lobules may help develop treatments to counter cell loss in other neurodegenerative diseases.

4.4. TRPC3 gain-of-function and calcium homeostasis

Both gain and loss of function in TRPC3-mediated signaling affect cerebellar function, although the TRPC3 gain of function is more deleterious as TRPC3^−/− mice are mildly ataxic, but do not show obvious histological defects (Hartmann et al., 2008). In MWK mice the gain of function TRPC3 mutation increases intracellular calcium which leads to cell loss and ataxia (Dulneva et al., 2015; Hanson et al., 2015). In PCs, mGluR1 activation mediates STIM-1-dependent calcium signals (Hartmann et al., 2014). These signals, in turn, gate TRPC3 channels that mediate the prolonged synaptic response and activation of voltage-gated calcium channels at the parallel fiber-PC synapse (Hartmann et al., 2011). TRPC3-mediated calcium entry modulates IP3R activity at the ER-plasma membrane junction as well as ER-mitochondria calcium transfer (Aslam and Alvi, 2022; Bartok et al., 2019; Curcic et al., 2022; Farfariello et al., 2022). IP3R1 (Itpr1) is especially enriched in PCs (Yamada et al., 1994). Accordingly, IP3R1 dysfunctions cause motor impairment in several diseases (Bezprozvanny, 2011) and are involved in the pathogenesis of SCA15, SCA16 and SCA29 (Prestori et al., 2019; Shimobayashi and Kapfhammer, 2018). Our TEM data show swollen ER in many PC spines. In keeping with the idea that dysfunctional ER has a critical role in the disease, our gene expression analysis show that several genes that play a role in ER calcium signaling (Hartmann et al., 2014; Hozumi et al., 2017; Kaufmann et al., 2009; Prestori et al., 2019; Wu and Kapfhammer, 2021) are downregulated in MWK. These include Stk17b (also known as DRAK2), Dgkh (DGKε), KCNMA1 (BKCa), Itpr1 (IP3R1), and Stim1 (Fig. 11A). Thus, the MWK mouse is a model of calcium dysregulation and shares similarities with other SCAs (Shimobayashi and Kapfhammer, 2018). For example, Stk17b, a downstream effector of protein kinase C (PKC), is downregulated in MWK mice (Fig. 11A; Dulneva et al., 2015) as well as in SCA1, SCA7, SCA14, and SCA41 (Wu and Kapfhammer, 2021). Because PKC modulates TRPC3 function, this finding appears in line with the hypothesis that TRPC3-calcium signaling network dysfunction is a mechanism shared by these ataxias (Liu et al., 2009; Ma et al., 2000; Prestori et al., 2019). SCA2 and SCA3 have been suggested to involve sensitization of the IP3R (Kasumu and Bezprozvanny, 2012), which is also part of the TRPC3-calcium functional network (Clapham et al., 2001). An alteration of this network is also likely at play in SCA5, which is associated with decreased activity of the Slc1a6 (EAAT4) glutamate transporter (Ikeda et al., 2006). This leads to extracellular glutamate accumulation and activation of mGluRs, which are coupled to TRPC3 both in PCs and UBCs (Hartmann et al., 2008; Sekerková et al., 2013).

The toxic interplay between calcium and oxidative stress is considered a cause of cell death in many neurodegenerative diseases (Chaudhari et al., 2014; Sukumaran et al., 2021) and can trigger apoptotic changes or autophagy (Dhaouadi et al., 2023; Lossi et al., 2018; Marchi et al., 2018). Considering that MWK PCs are already dysfunctional at P18, it is remarkable that they survive for months, and that PC loss never exceeds 50%. In contrast UBCs are almost completely wiped out within the first postnatal month. Intriguingly, one major difference between PCs and UBCs is their ability to handle calcium influx. PCs possess an extensive ER network and contain multiple calcium binding proteins (Bastianelli, 2003). In contrast UBCs are poor in ER, and type II UBCs also lack calcium binding proteins (Mugnaini et al., 2011). These differences might explain why the MWK mutation affects UBCs more than PCs. Differential calcium handling might also explain why in MWK the posterior PCs are more vulnerable despite TRPC3 expression being stronger in the anterior cerebellum compared with the posterior cerebellum (Wu et al., 2019). It may be suggested that the anterior PCs are better equipped to deal with TRPC3 mediated calcium influx.

Because intracellular calcium regulation is key in so many ataxias, it is also the target for numerous therapeutic approaches. For example, dantrolene, a calcium stabilizer, alleviates motor deficits and cell loss in SCA2 and SCA3 model mice (Bezprozvanny and Klockgether, 2009; Chen et al., 2008; Liu et al., 2009). Improvement in motor coordination and PC function was also observed in SCA2 after chronic suppression of IP3R-mediated calcium signaling (Kasumu and Bezprozvanny, 2012) and chronic viral-mediated expression of the siRNA targeting STIM1 (Egorova et al., 2023). While a similar approach might be attempted in MWK, the possible window of opportunity for such intervention is likely very narrow, because of the developmental effects of the MWK mutation.

4.5. TRPC3^mwk mutation affects early development of cerebellar neurons

Our gene expression analysis revealed that over 400 genes involved in neurogenesis and development are differentially regulated in the MWK mouse. We studied P18 to 1 year-old MWK mice, but it appears that PCs and UBCs are affected much earlier than P18, suggesting possible developmental effects. In keeping with this idea, MWK cerebella lack about 10–12% of PCs at early age. As we did not detect PC degeneration/apoptosis at P18, the decrease in PC numbers must happen either at early postnatal development or during PC neurogenesis. Physiological apoptotic PC death occurs around the first postnatal weeks (Jankowski et al., 2009; Jankowski et al., 2011; Lossi et al., 2018; Martí-Clúa, 2016), approximately the same time when TRPC3 expression is upregulated in PCs (Huang et al., 2007). It is possible that the calcium dysregulation during the early phases of PC maturation increases PC’s susceptibility to apoptotic changes. On the other hand, proper calcium signaling is essential in embryonic development (Toth et al., 2016) and therefore it is also conceivable that the MWK mutation affects PC neurogenesis, which happens around embryonic day E13–15 in mice (Leto et al., 2016; Sathyanesan et al., 2019). At E13 TRPC3 is present in cortical precursor cells (Boisseau et al., 2009), and a recent paper shows that TRPC3-mediated calcium influx is part of sonic hedgehog (Shh) signaling-dependent pathway (Shim et al., 2023) known to promote cell proliferation, specification of neuronal phenotype and to play a role in cerebellar development (De Luca et al., 2016; Vaillant and Monard, 2009; Wang et al., 2022b).

Finally, we envisage two possible scenarios concerning the absence of UBCs: either (1) it is caused by an early, sudden, and massive cell loss of which we only captured the end tail; or (2) the TRPC3^mwk mutation interferes with UBC neurogenesis and differentiation.

5. Conclusions

The MWK mouse is an intriguing model showing how a point mutation in a single gene leads to an overly complex phenotype. Our data further show that the main symptoms in MWK mice are caused by PC dysfunction rather than cell loss, as supported by the fact that the ataxia symptoms are non-progressive despite the incremental nature of the pathology. Finally, our data suggest that PCs activate specific mechanisms to defend themselves from cell death (possibly in a lobule-dependent fashion). Understanding these mechanisms might help developing innovative strategies to treat neurodegenerative diseases.

Supplementary Material

NIHMS1989410-supplement-1.docx^{(139.1MB, docx)}

NIHMS1989410-supplement-2.docx^{(36.8KB, docx)}

NIHMS1989410-supplement-3.xlsx^{(899.1KB, xlsx)}

Acknowledgments

The authors thank Leigh Kinsler, Nicole Sheman, Annika Hirdesai and Mahmoud Farhan for help with data analysis and animal husbandry. This work was supported by NIH grants NS090346 (MM/PO), NS114738 (SG), NS082351 and NS127204 (PO), and by the Northwestern University NUSeq Core Facility. Part of the EM imaging was performed using an instrument in the Northwestern University Center for Advanced Microscopy (which is supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center).

Footnotes

CRediT authorship contribution statement

Gabriella Sekerková: Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization, Writing – review & editing. Sumeyra Kilic: Formal analysis, Investigation. Yen-Hsin Cheng: Formal analysis, Investigation. Natalie Fredrick: Data curation, Formal analysis, Investigation, Visualization. Anne Osmani: Investigation. Haram Kim: Formal analysis, Investigation. Puneet Opal: Funding acquisition, Methodology, Writing – original draft. Marco Martina: Writing – review & editing, Conceptualization, Funding acquisition, Supervision, Writing – original draft.

Declaration of competing interest

The authors declare no competing financial interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.nbd.2024.106492.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1989410-supplement-1.docx^{(139.1MB, docx)}

NIHMS1989410-supplement-2.docx^{(36.8KB, docx)}

NIHMS1989410-supplement-3.xlsx^{(899.1KB, xlsx)}

Data Availability Statement

Data will be made available on request.

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PERMALINK

Phenotypical, genotypical and pathological characterization of the moonwalker mouse, a model of ataxia

Gabriella Sekerková

Sumeyra Kilic

Yen-Hsin Cheng

Natalie Fredrick

Anne Osmani

Haram Kim

Puneet Opal

Marco Martina

Abstract

1. Introduction

2. Material and methods

2.1. Animals

Fig. 1. Motor deficits in MWK mice.

2.2. Behavioral analysis

2.2.1. Phenotype scoring system

2.2.2. Grip strength

2.2.3. Rearing ability

2.2.4. Accelerating rotarod

2.2.5. Footprint analysis

2.3. Immuno- and TUNEL Staining

2.3.1. Tissue preparation

2.3.2. Bright field microscopy

2.3.3. Fluorescence microscopy

2.3.4. Primary antibodies

2.3.5. TUNEL labeling

2.4. Silver impregnation

2.5. Transmission electron microscopy

2.6. Electrophysiological recordings

2.6.1. Slice preparation

2.6.2. Cell-attached recordings

2.6.3. Whole-cell recordings

2.7. RNA-Seq analysis

2.7.1. RNA isolation

2.7.2. RNA-Seq analysis

2.8. Quantification of morphological parameters

2.8.1. Cerebellum/cortex ratio

2.8.2. Cerebellar cortex area

2.8.3. Molecular layer thickness

2.8.4. Granule cell layer area

2.8.5. PC density

2.8.6. PC numbers

2.8.7. Axonal torpedoes

2.8.8. Synaptic contacts and swollen ER in PC dendritic spines

2.8.9. Statistical analysis

2.9. Photography

3. Results

3.1. Behavioral phenotype of the MWK mice

Table 1. Phenotypic assessment of MWK and WT mice.

3.2. The MWK mutation affects cerebellar size but not lobular morphology

Fig. 2. Cerebellar size is reduced in MWK mice.

Fig. 3. The cerebellum of a P18 MWK mouse lacks UBCs.

Fig. 5. PC loss in the MWK cerebellum is lobule-specific and progressive.

Fig. 6. Quantitative assessment of PC loss in MWK mice.

3.3. Loss of UBCs but not PCs in early postnatal MWK cerebellum

Fig. 4. In MWK cerebellum, UBCs, but not PCs, undergo apoptosis in early postnatal development.

3.4. Purkinje cell loss is slow and incremental

Table 2. PC number in WT and MWK mice.

3.5. Axonal torpedoes and degeneration

Fig. 7. PC axonal swellings (torpedoes) in MWK cerebellum.

Fig. 8. Silver impregnation reveals degenerating neurons in MWK but not in the WT cerebella.

Fig. 9. PC axonal degeneration increases with MWK mice age.

3.6. MWK Purkinje cells are electrophysiologically silent

Fig. 10. TRPC3 MWK mutation impairs PC firing.

3.7. RNA seq analysis reveals large, ataxia-specific, changes in gene expression

Fig. 11. Altered gene expression in MWK mouse cerebellum.

Table 3. Downregulated ataxia-related genes in MWK.

Table 4. Upregulated ataxia-related genes in MWK.

3.8. Gene ontology (GO) enrichment analysis suggests that the mutation affects cerebellar development and synaptic connectivity

3.9. TEM analysis reveals pathological changes in Purkinje cell dendritic spines

Fig. 12. PC dendritic spine pathology in MWK cerebella.

4. Discussion

Fig. 13. Timeline of the behavioral (blue, top) and pathological (green, bottom) changes in MWK mouse.

4.1. MWK ataxic phenotype is non-progressive

4.2. PC dysfunction rather than loss causes the MWK ataxia

4.3. Mechanisms of cell loss in the MWK cerebellum

4.4. TRPC3 gain-of-function and calcium homeostasis

4.5. TRPC3mwk mutation affects early development of cerebellar neurons

5. Conclusions

4.5. TRPC3^mwk mutation affects early development of cerebellar neurons