Sacs R272C missense homozygous mice develop an ataxia phenotype
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 200 SACS mutations have been identified. Most mutations lead to a complete loss of a sacsin, a large 520 kD protein, although some missense mutations are associated with low levels of sacsin expression. We previously showed that Sacs knock-out mice demonstrate early-onset ataxic phenotype with neurofilament bundling in many neuronal populations. To determine if the preservation of some mutated sacsin protein resulted in the same cellular and behavioral alterations, we generated mice expressing an R272C missense mutation, a homozygote mutation found in some affected patients. Though SacsR272C mice express 21% of wild type brain sacsin and sacsin is found in many neurons, they display similar abnormalities to Sacs knock-out mice, including the development of an ataxic phenotype, reduced Purkinje cell firing rates, and somatodendritic neurofilament bundles in Purkinje cells and other neurons. Together our results support that Sacs missense mutation largely lead to loss of sacsin function.
KeywordsARSACS, Purkinje cell, cerebellum Sacsin SACS Ataxia Mouse model
Axon initial segment
Autosomal recessive spastic ataxia of Charlevoix-Saguenay
Coefficient of variation
Polymerase chain reaction
Quantitative reverse transcription polymerase chain reaction
Sacs knock-out mice
Mice homozygous for sacsin R272C mutation
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) was first described in the French Canadian population in 1978 . Since then, ARSACS cases have been reported worldwide  (www.lovd.nl). The original French Canadian ARSACS clinical phenotype consists of a childhood onset progressive spastic ataxia accompanied by sensory-motor polyneuropathy and retinal thickening [3, 4]. French Canadian ARSACS patients become wheelchair-bound on average by the age of 41 and life expectancy is reduced to 61 years . Pathological findings of post-mortem examination of two male ARSACS patient brains show atrophy of the anterior vermis associated with Purkinje cell death, while the cerebellar hemispheres are much less affected [6, 7, 8] ARSACS is the second most common form of recessive ataxia in the Netherlands and Northern UK .
The SACS gene mutated in ARSACS is located on human chromosome 13q12 and encodes sacsin, a 4579 amino acids protein. The enormous size of the SACS gene and its translated protein has considerably hindered functional studies. Sacsin is a multi-domain protein containing an N-terminal ubiquitin-like domain shown to bind to the proteasome . Towards the C-terminus, sacsin contains a DnaJ domain [10, 11] immediately followed by a higher eukaryotes and prokaryotes nucleotide-binding C-terminal (HEPN) domain . The DnaJ domain was demonstrated to bind Hsp70 and to be functional in complementation assay technique using Hsp70 chaperone [10, 11]. The HEPN domain was recognized by bioinformatics analysis to exist in a single copy in the human genome that is exclusive to sacsin . The HEPN domain also mediates dimerization of sacsin . In 2013, Romano et al. described using bioinformatics, three large internal homologous repeating regions, which they named SIRPT1, 2 and 3 . Each SIRPT is divided into sub-repeats namely sr1, sr2, sr3 and srX. The second repeat lacks srX, making SIRPT2 smaller than the others. Each sr1 contains a well-recognizable HATPase_c (Histidine kinase-like ATPases) domain homologous to the nucleotide-binding domain (NBD) of the Hsp90 chaperone. The region combining the sr1 and sr2 corresponds to Anderson and colleagues’ SRR supradomain , which possesses ATPase activity. A missense pathogenic mutation, D168Y, within the sr1 completely abrogates the ability of this domain to hydrolyse ATP. The sr1 sequence in all three SIRPT domains are sites for a number of pathogenic homozygote missense mutations, or single mutations combined on the other allele with macrodeletion, frameshift and stop mutations: D168Y, T201K, R272C, R272H, R276C, L308F, P1583R, H1587R, R1645Q and R2703C [9, 14, 15, 16, 17, 18, 19]. Using crystal structure analysis of the SIRPT1-sr1 encoding construct, the missense mutations R272C, R272H and T201K were demonstrated to affect the structure of protein folding and/or stability of the peptide, whereas the D168Y mutation likely affects chaperone activity by interfering with ATP binding . These findings suggest that distinct mutations will variably affect sacsin protein function.
The organization of the sr1 and sr2 domains matches the structure of an Hsp90-like protein [11, 14, 21], where the sr1 represents the ATP binding domain, whereas the sr2 acts as the Hsp90-like putative middle domain containing an arginine residue accepting phosphate after ATP hydrolysis. As previously observed, there is indeed a phosphor-acceptor arginine in each sr2 domain of sacsin . Significantly, a mutation on one such conserved arginine, namely R474C, was associated with one of the highest clinical severity score after mutations occurring in the DnaJ and HEPN domains . Since, the SIRPT repeats make up more than 80% of the total sacsin protein, exploring their roles will help better understand the function of this large protein.
With this in mind, we generated a mouse model of ARSACS harbouring the missense mutation c.816C > T (p. R272C), the SACSR272C mutation was described in homozygote state in two cases in Canada [2, 17]. Preservation of sacsin was confirmed by Western blot in lymphoblasts of one ARSACS case . The null mice display an early abnormal gait with progressive motor, cerebellar, and peripheral nerve dysfunctions reminiscent of ARSACS pathology. The clinical phenotype is accompanied by an early onset progressive loss of cerebellar Purkinje cells particularly in the anterior cerebellar lobules, which is later followed by spinal motor neuron loss, peripheral neuropathy and muscle atrophy. Loss of sacsin results in distinctive neurofilament (NEF) accumulations most notably in Purkinje cells, deep cerebellar neurons, layer 5 pyramidal cells, thalamic and pontine neurons. Here, we show that R272C homozygous animals develop a clinical and pathological phenotype comparable to the one observed in Sacs−/− animals despite preservation of some sacsin expression.
Ataxia, motor deficit and muscle weakness of Sacs R272C mice
Purkinje cell loss in Sacs R272C mice
Sacs R272C mice exhibit reduction Purkinje cell firing frequency
Since differences in the onset of motor abnormalities have been observed in male and female mice in the past, we wondered whether the changes in firing we observe reflect differences in male and female mice. To examine this, we compared our findings in male and female mice and found that decreases in Purkinje cell firing frequency are observed in both sexes (male WT frequency = 49.0 Hz ± 2.5 Hz, n = 9; male SacsR272C frequency = 29.6 Hz ± 2.6 Hz, n = 8; significantly different, P < 0.0001; female WT frequency = 72.3 Hz ± 4.2 Hz, n = 22; female SacsR272C frequency = 56.0 Hz ± 4.2 Hz, n = 22; significantly different, P = 0.009, data not shown). Thus, we observe a reduction in Purkinje cell firing frequency without any change in firing precision in our SacsR272C mouse model, with low expression levels of mutated sacsin, that are broadly similar to changes previously reported in Sacs−/− mice .
Neurofilament (NF) accumulations in somatodendritic compartment in Sacs R272C Purkinje cells
Rearrangement of the intermediate filament network in sacsin-deficient cells is also observed in ARSACS patient dermal fibroblasts . These cells display abnormal perinuclear accumulation of vimentin filaments . To verify if the R272C mutation had similar effect on the IF network, we labeled vimentin in CRISPR/Cas9 genetically engineered SACS knock-out fibroblasts (SACSem1KO), in which there if no sacsin expression, and our fibroblast line derived from a patient with two distinct SACS mutations (Fig. 6h-j). On one allele, this patient bears the common French-Canadian c.8844del mutation (del) and on the other, the c.816C > T (R272C) mutation. As expected, our SACSem1KO fibroblasts demonstrate perinuclear accumulations of vimentin often forming a ball-like shape (Fig. 6j). The vimentin network in SACSdel/R272C fibroblasts is also perturbed, with bundles of IF filaments observed surrounding the nucleus (Fig. 6i). Both these phenotypes are distinctively different from the vimentin network observed in control patient fibroblasts, however the IF bundling is less important in the SACSdel/R272C fibroblasts compared to the one in the SACSem1KO (Fig. 6h-j).
Mutated sacsin expression in the cerebellum and isocortex of Sacs R272C mice
Here we report that expression of mutated R272C sacsin protein in mice leads to a similar and potentially milder phenotype than that previously characterized in the Sacs−/− animals . However, a comparative parallel study of age-matched SacsR272C and Sacs−/− mice would require too many resources for the limited insight we expect such a study would provide, considering the large spectrum of clinical severity observed in human patients, even between those carrying the same mutations. The R272C mutation was previously shown to affect proper protein fold and/or protein stability of the sr1 domain . Although we did not investigate R272C mutant sacsin protein folding, we did identify a significant decrease in mutant protein levels on Western blot and immunohistochemical labelings from animal brains. SacsR272C mice exhibit significant balance deficit and muscle weakness detectable as early as 45 days of age. These balance difficulties preceded extensive neuronal loss, suggesting that the ataxic phenotype most likely corresponds to Purkinje cell dysfunction prior to degeneration. The progressive Purkinje cell loss was largely localized to the most anterior cerebellar lobules, just as observed in ARSACS patients. Our sacsin immunolabeling demonstrate that sacsin expression is detected in cerebellar PC across all vermal cerebellar lobules and therefore cannot account for a greater vulnerability of anterior PC in ARSACS. Other cellular or physiological properties of these cells must account for this greater vulnerability in ARSACS. We recently reported changes in synaptic input and intrinsic firing of cerebellar PC, as well as synaptic output to the DCN in Sacs−/− mice prior to their motor coordination deficit . These changes were only observed in anterior cerebellar lobules, but not in non-degenerating posterior lobules. These results support the idea that cerebellar PC across the cerebellum have distinct properties that could render certain populations more vulnerable to the absence or to deficient sacsin function. In our SacsR272C mouse model, we also observed Purkinje cell firing rate deficits in anterior lobules that are similar to those detected in our Sacs−/− mouse model , supporting the hypothesis that Purkinje cell firing deficits contribute to motor coordination deficits [23, 36].
The IF cytoskeletal rearrangement observed in numerous neuronal populations in the brains of SacsR272C and Sacs−/− mice, in Sacs−/− primary neuronal cultures, in ARSACS patient-derived fibroblasts and in genetically engineered Sacs−/− cell lines is the most striking cellular change observed to date in the ARSACS pathology [22, 34]. This phenotype appears to occur prior to motor coordination deficit and other cellular features of ARSACS, such as mitochondrial elongation and impaired transport, at least in Sacs−/− mice . We do not fully understand what causes the accumulations of non-phosphorylated NFH proteins in the somatodendritic compartment of Purkinje cells, but one explanation is that sacsin directly acts on NF assembly and/or turnover. Our recent results suggest a direct interaction of distinct sacsin domains in the regulation of IF assembly and dynamics, with certain domains, namely the SIRPT1 and DNAj domains, being capable of dismantling NF bundles in cultured Sacs−/− neurons . These results argue that sacsin could serve as an important IF protein co-chaperone.
Another possible explanation for the accumulation of NFH in the somatodendritic compartment of SacsR272C and Sacs−/− neurons could be a mis-targeting or mis-sorting of proteins. Neurons are highly polarized cells exhibiting axonal and somatodendritic domains with distinct complements of cytoplasmic organelles and cytoskeletal proteins. Polarized sorting is thought to depend mainly on selective association of these cytoskeletal organelles or proteins with different microtubule motors in the pre-axonal exclusion zone (PAEZ), a specialized area within the axon hillock and the axon initial segment (AIS) . Defects in this polarization could impede axonal proteins from entering the axon and stall them in the somatodendritic compartment causing neuronal defect. For example, Purkinje cell-specific knock-down of microtubule cross-linking factor 1 (Mtcl1) causes AIS disorganization by impairing ankyrin G localization, and loss of axonal polarity . In mice, genetic disruption of Mtcl1 results in abnormal motor coordination associated with Purkinje cell degeneration, arguing that Purkinje cells are susceptible to such deregulation of neuronal polarization . Furthermore, a point mutation in the C-terminal microtubule-binding domain of MTCL1 has been found to segregate in a Japanese dominant spinocerebellar ataxia family . The accumulation of NFH protein in the somatodendritic compartment of several neuronal populations in the SacsR272C and Sacs−/− mice raises the possibility that sorting of dendritic and axonal proteins might be perturbed in ARSACS. Understanding the potential role of sacsin in the establishment and/or maintenance of neuronal polarity will be an important area of future study. Further studies will also be needed to elucidate whether the bundles of IF cytoskeletal proteins in ARSACS are pathophysiological and directly lead to cellular death or are simply by-products. Although it is easy to conceptualize that large bundles of cytoskeletal proteins in the neuronal cell soma and dendritic branches would physically hinder organelle and cargo protein transport in ARSAC , future studies are needed to confirm its pathological role.
Our results demonstrate that expression of low levels of mutant R272C sacsin in mice leads to motor coordination deficit and muscle weakness reminiscent of the human ARSACS pathology, with similar cellular deficit previously observed in the Sacs−/− mouse model. The mutant R272C mouse demonstrate that missense SACS mutations are likely to interfere with sacsin function despite some low mutant protein levels, supporting that a loss of function most likely underlines its pathophysiology.
Materials & methods
Sacs R272C mice generation and analysis
SacsR272C mice were generated by Ozgene (Bentley, Australia) on a C57BL/6J background. Targeting vector was constructed by first cloning the gene segment which includes exons 6 through 8 into PelleR B00001F7_G01 Ozgene proprietary plasmid containing a PGK-neo cassette flanked by two FRT sites, followed by site-directed mutagenesis for introduction of the R272C mutation at the beginning of exon 7. Targeting vector was completed by incorporation of 6.3Kb 5′ and 3′ homology arms. Mice were genotyped by PCR using primers: 5′- AGCAACCTGCATCATTGTAGCAGAA -3′ and 5′- GGTTTCTGGTTTGAGGCAAT -3. Total RNA from mouse cerebella and cortex was extracted with the miRNeasy kit (Qiagen) and treated with DNAse I (Qiagen) according to the manufacturer’s instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer and RNA Integrity Numbers (RIN) were routinely above 9. For qRT-PCR, 1 μg of RNA was reversed transcribed using the High Capacity cDNA Reverse Transcriptase (ThermoFisher). The following primers were used to amplify Sacs: 5′-CGCTGAGACCAGCTTTCC-3′ and 5′-CCAATCTTGATCCAATCAGGTATC-3′. Real-time PCR was performed in technical duplicates using FastStart Universal SYBR Green Master (ROX) (Roche) on a ViiA™ 7 Real-Time PCR System (Applied Biosystems). The ΔΔCt method was used to calculate relative Sacs mRNA expression, with normalization to the endogenous genes Ppia and Hprt1. SacsR272C mice were maintained in the C57Bl/6J background and bred and maintained under standard conditions consistent with the Canadian Council on Animal Care and approved by the University Animal Care and MNI Animal Care committees.
Fibroblast cell lines
Control human-derived fibroblasts were obtained from the Repository for Mutant Human Cell Strains of the Montreal Children’s Hospital. SACSdel/R272C human-derived fibroblasts were obtained using the previously described protocol . Briefly, patient skin punch biopsies were minced in small pieces and put in 6-well plates in complete DMEM/20% FBS (Wisent) media. Media was changed every 2–3 days. Cells were trypsinized and passaged once they reached confluence. Fibroblasts were then frozen at 1 × 106 cells/ml per vial. Primary cultures were kept at low passage (p4–8). Cells were cultured in regular medium, DMEM (Wisent) with 10% FBS (Wisent) at 37 °C under 5% CO2 humidified atmosphere. Primary human-derived fibroblasts were immortalized at low passage as previously described  SACSem1KO CRISPR/Cas9 cell line was generated following manufacturer guidelines using sacsin double nickase plasmid (sc-404,592-NIC, SCBT). Briefly, cells were nucleofected with 2μg of vectors and positive clones were selected using 1μg.ul− 1 puromycin (ThermoFisher Scientific). Absence of sacsin was verified by Western blotting. Genomic DNA was extracted from clones of interest and Sanger sequenced using the following primers (Fwd: CACAGTAATCATGCAAAGTCTCTATGCCTG, Rev.: ACAGAGAAACTGGTGTTTAGAGTGACTTC). Our SACSem1KO Crispr/Cas9 cell line presents a 44pb duplication in exon 8 of the SACS gene (c.1668_1711dup) leading to insertion of a stop codon and total absence of protein (data not shown). Absence of off-target recombination was verified in silico (crispr.mit.edu). Studies using human cell lines were approved by the institutional review board of the Montreal Neurological Institute and with McGill University Research Ethics Board Committee.
For preparation of tissue sections, mice were anesthetized with mouse anesthetic cocktail (ketamine (100 mg/ml), xylazine (20 mg/ml) and acepromazine (10 mg/ml)), perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde. Brains were dissected and post-fixed for 2 h at 4 °C in the same fixative. Tissues were then equilibrated in 30% sucrose/PBS until sectioning. Sagittal sections (35 μm) were cut using a freezing sledge microtome. Free-floating sections were processed for immunofluorescence as previously described . Antibodies used were polyclonal anti-calbindin-D-28 k (Sigma, C2724), monoclonal anti-neurofilament-H (NFH) (Millipore, MAB5266), polyclonal anti-MAP2 (Abcam, ab5392).
For immunohistochemistry, mouse brains were dissected out, immersed in 4% paraformaldehyde and post-fixed for 48 h at 4 °C in the same fixative. Tissue were processed for paraffin embedding and sectioned at 4 μm in the parasagittal plan. For sacsin immunohistochemistry, sections were subjected to heat-mediated antigen retrieval in demasking solution (10 mM Tris-HCL, 1 mM EDTA, 0.05% Tween-20) at 95 °C for 35 min. Sections were allowed to cool down at room temperature for 30 min followed by inactivation of endogenous peroxidase. Sections were then incubated in blocking buffer (phosphate buffer 0.1 M; 10% normal goat serum; 0.25% TX-100) 1 h. Endogenous biotins were blocked with the avidin & biotin blocking kit (Vector Labs, SP-2001) according to the manufacturer’s protocol. Sections were then incubated with anti-sacsin (Abcam; ab181190) or anti-neurofilament-H (NFH) antibody (Millipore, MAB5266) diluted in phosphate buffer 0.1 M; 1% normal goat serum; 0.25% TX-100 overnight at 4 °C. Sections were then incubated with appropriate biotinylated secondary antibodies (Vectors Labs) followed by VECTASTAIN ABC reagent for 1 h, washed, and reacted with VECTOR DAB substrate. Sections were dehydrated in a graded series of ethanol dilutions, cleared in xylene, counterstained with cresyl-violet or not and coverslipped using Protocol mounting medium (Fisher Scientific). Immunolabeling was performed simultaneously in at least three aged-matched animal per group. Mean grey values were collected using ImageJ in three different fixed-sized regions of interest per mouse for Purkinje cell dendrites and axons, as well as for the corpus callosum. For Purkinje cell bodies, mean grey values were collected from 8 to 10 cell bodies with a fixed-sized region of interest.
Immunolabeling of human-derived fibroblasts was performed as followed. Cells plated onto 12 mm round glass coverslips were fixed in ice-cold methanol 7 min at -20 °C. Cells were then washed with phosphate buffered-saline (PBS) three times. Cells were incubated 30 min in PBS; 5% normal goat serum. Cells were then incubated in the presence of primary monoclonal anti-vimentin antibody (1/4000, clone V9, SIGMA-Aldrich) diluted in PBS; 1% normal goat serum for 2 h at room temperature. Secondary anti-mouse Alexa-Fluor 555 antibody (ThermoFisher Scientific) was applied for 45 min.
Imaging was performed using Zeiss Axiovert M2 microscope or an Olympus IX81 inverted microscope with appropriate lasers using an Andor/Yokogawa spinning disk system (CSU-X), with a sCMOS camera using a 20×, 60× or a 100× objective lenses (NA1.4).
Preparation of cerebellar tissue lysates and western blotting
We used our published protocols for the preparation of cerebellar protein extracts and western blot analysis . Immunoblots were probed with polyclonal anti-sacsin (Abcam, ab181207, 1:2000) and monoclonal anti-vinculin (SIGMA-Aldrich, V9131, 1:1000).
Mice were tested for motor balance, motor coordination and muscle strength using the balance beam, rotarod and inverted grid tests. Female and male cohorts were tested at 45, 90 and 180 days of age (females n ≥ 7 per groups, males n ≥ 6 per groups) (females n ≥ 9 per groups, males n ≥ 7 per groups). Behavioural testing was performed as previously described in Lariviere et al. .
Purkinje cell counts
Purkinje cell counts were performed as previously reported .
Acute slice preparation
Animals between the ages of P90–100 were deeply anaesthetized with isofluorane, rapidly sacrificed, and brains were removed into ice-cold low-Ca2+ artificial cerebrospinal fluid (ACSF) that was bubbled with an O2/CO2 (95% / 5%) mixture as previously described [23, 44]. Sagittal cerebellar vermis slices (250 μm) were cut using a VT1200S microtome (Leica Microsystems, Germany). Slicing ACSF contained (in mM): NaCl, 125; KCl, 2.5; MgCl2, 4; NaH2PO4, 1.25; KCl, 2.5; MgCl2, 4; NaH2PO4, 1.25; NaHCO3, 26; CaCl2, 2; dextrose, 25; with a final osmolality of ~ 320 mOsm and pH 7.4. Slices were then transferred to ACSF that contained 1 mM MgCl2 and 2 mM CaCl2 (incubation and recording ACSF), were incubated at 37 °C and then cooled to room temperature where they were incubated in bubbled ACSF for up to an additional 6 h.
Loose-cell attached recordings were made with a glass electrode pulled with a P-1000 puller (Sutter Instruments, Novato, CA, USA) filled with ACSF to record action potentials without dialyzing the intracellular solution and thereby altering intrinsic firing rates. Data was collected and analyzed off-line using custom acquisition and analysis routines with Igor Pro software (Wavemetrics, Portland, OR, USA). Statistical comparisons were made using Igor Pro or JMP (SAS, Cary, NC, USA) software. Data are represented as mean ± standard error of the mean (SEM), N = animal number, n = cell number.
Data for the behavioral phenotyping are shown as the mean ± standard error of the mean (SEM). Beam and rotarod analyses were done using GraphPad Prism7 software and significance level was set at 0.05. Two-way ANOVA with repeated-measures was performed to assess the effect of time and genotype followed by Tukey’s post hoc pairwise comparisons. For all other statistical analyses, comparisons were made using unpaired Student t-test with significance level of 0.05.
We would like to thank the Ataxia of Charlevoix-Saguenay foundation for their generous and constant support for this project. We thank Joseph Rocheford, Ph.D and Eve-Marie Charbonneau from the Neurophenotyping Centre at the Douglas Mental Health University Institute for conducting the study and for their technical expertise. We would also like to acknowledge the Genomics Platform of the Institute for Research in Immunology and Cancer for their qRT-PCR services.
This research was supported by Fondation de l’Ataxie de Charlevoix-Saguenay (www.arsacs.com) and the Canadian Institutes of Health Research Emerging team grant on rare diseases: Translating basic biology to enhanced patient care (126526). KC received a doctoral award from the Fonds de Recherche du Québec – Santé.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
RL, RAM, AJW and BB conceived the study and designed the experiments. RL managed and analyzed behavioral experiments. RL, NS and RG collected and/or processed tissues for genotyping or PCR for Sanger sequencing. KC performed qRT-PCR. NS performed PC quantification. RL and NS performed and analyzed histology and Western blots. BTM performed electrophysiology experiments and analyzed results. RL, AJW and BB wrote the manuscript. All authors read and approved the final manuscript.
All experiments were performed according to good practice of handling laboratory animals consistent with the Canadian Council on Animal Care and approved by the University Animal Care and MNI Animal Care committees. Studies using human cell lines were approved by the institutional review board of the Montreal Neurological Institute and with McGill University Research Ethics Board Committee.
Consent for publication
The authors declare that they have no competing interests.
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