Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a rare neurodegenerative disease caused by biallelic variants in the SACS gene encoding for sacsin. More than 200 pathogenic variants have been identified to date, most of which are missense. It is likely that the prevalence of autosomal recessive spastic ataxia of Charlevoix-Saguenay is underestimated due to the lack of an efficient diagnostic tool able to validate variants of uncertain significance. We have previously shown that sacsin is almost absent in fibroblasts of patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay regardless of the type of SACS variant, because sacsin carrying missense variants is cotranslationally degraded. In this work, we aimed to establish the pathogenicity of SACS variants by quantifying sacsin protein in blood samples, with relevant implications for autosomal recessive spastic ataxia of Charlevoix-Saguenay diagnosis. We developed a protocol to assess sacsin protein levels by western blot using small amounts of peripheral blood mononuclear cells, which can be propagated in culture and cryopreserved. The study involves eight patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (including a novel case) carrying variants of different types and positions along the SACS gene and two parents who are carriers of heterozygous missense variants. We show that patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (carrying either missense or truncating variants) almost completely lacked sacsin in peripheral blood mononuclear cells. Moreover, both carriers of a SACS missense variant showed 50% reduction in sacsin protein levels compared to controls. We also describe a patient with uniparental isodisomy carrying a homozygous nonsense variant near the 3\' end of the SACS gene. This resulted in a stable sacsin protein lacking the last 202 amino acids, probably due to escape of nonsense-mediated decay of mRNA. In conclusion, we have optimized a minimally invasive diagnostic tool for autosomal recessive spastic ataxia of Charlevoix-Saguenay in blood samples based on sacsin protein level assessment. Indeed, our results provide definite evidence that sacsin carrying missense pathogenic variants undergoes cotranslational degradation. The quantitative reduction in sacsin levels in the case of missense variants of uncertain significance allows defining them as pathogenic variants, something which cannot be predicted bioinformatically with high certainty.

BACKGROUND: Autosomal recessive spastic ataxia ofCharlevoix-Saguenay (ARSACS) is a rare neurodegenerative disorder characterizedby early-onset cerebellar ataxia, peripheral sensorimotor neuropathy, and lowerlimb spasticity. We present clinical andgenetic data of the first Bulgarian patients diagnosed with ARSACS by wholeexome sequencing (WES).
METHODS: Variant filtering was performed usinglocally established pipeline and the selected variants were analysed by Sangersequencing. All patients underwent clinical examination and testingincluding the standard rating scales for spastic paraplegia and ataxia.
RESULTS: Five different SACS gene variants, three of which novel, have been identified inpatients from three different ethnic groups. In addition to the classicalclinical triad, brain MRI revealed cerebellar atrophy, linear pontineT2-hypointensities, and hyperintense rim lateral tothalamus combined with retinal nerve fiber layer thickening on opticcoherence tomography (OCT).
CONCLUSIONS: We expand the mutation, geographic, and phenotypic spectrum of ARSACS, adding Bulgaria to the world map of the disease, and drawing attention to the fact that it is still misdiagnosed. We demonstrated that brain MRI and OCT are necessary clinical tests for ARSACS diagnosis, even if one of the cardinal clinical features is lacking.

UNASSIGNED: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare incurable neurodegenerative disease caused by mutations in the SACS gene, which codes for sacsin, a large protein involved in protein homeostasis, mitochondrial function, cytoskeletal dynamics, autophagy, cell adhesion and vesicle trafficking. However, the pathogenic mechanisms underlying sacsin dysfunction are still largely uncharacterized, and so attempts to develop therapies are still in the early stages.
UNASSIGNED: To achieve further understanding of how processes are altered by loss of sacsin, we used untargeted proteomics to compare protein profiles in ARSACS fibroblasts versus controls.
UNASSIGNED: Our analyses confirmed the involvement of known biological pathways and also implicated calcium and lipid homeostasis in ARSACS skin fibroblasts, a finding further verified in SH-SY5Y SACS -/- cells. Validation through mass spectrometry-based analysis and comparative quantification of lipids by LC-MS in fibroblasts revealed increased levels of ceramides coupled with a reduction of diacylglycerols.
UNASSIGNED: In addition to confirming aberrant Ca2+ homeostasis in ARSACS, this study described abnormal lipid levels associated with loss of sacsin.

BACKGROUND: Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) and Spastic Paraplegia Type 7 (SPG7) are paradigmatic spastic ataxias (SPAX) with suggested white matter (WM) involvement. Aim of this work was to thoroughly disentangle the degree of WM involvement in these conditions, evaluating both macrostructure and microstructure via the analysis of diffusion MRI (dMRI) data.
METHODS: In this multi-center prospective study, ARSACS and SPG7 patients and Healthy Controls (HC) were enrolled, all undergoing a standardized dMRI protocol and a clinimetrics evaluation including the Scale for the Assessment and Rating of Ataxia (SARA). Differences in terms of WM volume or global microstructural WM metrics were probed, as well as the possible occurrence of a spatially defined microstructural WM involvement via voxel-wise analyses, and its correlation with patients\' clinical status.
RESULTS: Data of 37 ARSACS (M/F = 21/16; 33.4 ± 12.4 years), 37 SPG7 (M/F = 24/13; 55.7 ± 10.7 years), and 29 HC (M/F = 13/16; 42.1 ± 17.2 years) were analyzed. While in SPG7, only a mild mean microstructural damage was found compared to HC, ARSACS patients present a severe WM involvement, with a reduced global volume (p < 0.001), an alteration of all microstructural metrics (all with p < 0.001), without a spatially defined pattern of damage but with a prominent involvement of commissural fibers. Finally, in ARSACS, a correlation between microstructural damage and SARA scores was found (p = 0.004).
CONCLUSIONS: In ARSACS, but not SPG7 patients, we observed a complex and multi-faced involvement of brain WM, with a clinically meaningful widespread loss of axonal and dendritic integrity, secondary demyelination and, overall, a reduction in cellularity and volume.

Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by loss-of-function mutation in the SACS gene, which encodes sacsin, a putative HSP70-HSP90 co-chaperone. Previous studies with Sacs knock-out (KO) mice and patient-derived fibroblasts suggested that SACSIN mutations inhibit the function of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). This in turn resulted in mitochondrial hyperfusion and dysfunction. We experimentally tested this hypothesis by genetically manipulating the mitochondrial fission/fusion equilibrium, creating double KO (DKO) mice that also lack positive (PP2A/Bβ2) and negative (PKA/AKAP1) regulators of Drp1. Neither promoting mitochondrial fusion (Bβ2 KO) nor fission (Akap1 KO) influenced progression of motor symptoms in Sacs KO mice. However, our studies identified profound learning and memory deficits in aged Sacs KO mice. Moreover, this cognitive impairment was rescued in a gene dose-dependent manner by deletion of the Drp1 inhibitor PKA/Akap1. Our results are inconsistent with mitochondrial dysfunction as a primary pathogenic mechanism in ARSACS. Instead, they imply that promoting mitochondrial fission may be beneficial at later stages of the disease when pathology extends to brain regions subserving learning and memory.

BACKGROUND: To specify peripheral nerve affection in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) by correlating high-resolution nerve ultrasound and nerve conduction studies.
METHODS: We assessed a cohort of 11 ARSACS patients with standardized nerve conduction studies and high-resolution ultrasound of peripheral nerves and compared nerve ultrasound findings to a healthy control group matched for age, sex, size and weight.
RESULTS: Mean age of patients was 39.0 (± 14.1) years and disease duration at assessment 30.6 (± 12.5) years. All patients presented with a spasticity, ataxia and peripheral neuropathy. Neuropathy appeared to be primarily demyelinating in 9/11 cases and was not classifiable in 2/11 cases due to not evocable potentials. Nerve ultrasound revealed a normal ultrasound pattern sum score (UPSS) in each ARSACS patient and no significant nerve enlargement compared to the control group.
CONCLUSIONS: Peripheral neuropathy in ARSACS showed primarily demyelinating rather than axonal characteristics and presented without nerve enlargement. As demyelinating neuropathies do commonly present enlarged nerves we recommend further genetic testing of the SACS gene in patients who present with this combination of demyelinating neuropathy without nerve enlargement. ARSACS cases that initially presented only with neuropathy without spasticity or ataxia and therefore were misdiagnosed as Charcot-Marie-Tooth disease are supporting this suggestion.

Autosomal recessive spastic ataxia in Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder caused by mutations in the sacsin molecular chaperone protein (SACS) gene. Since the first report from Quebec in 1978, many pathogenic ARSACS variants with significantly reduced chaperone activities have been reported worldwide in adolescents, with presumably altered protein folding. In this study, a novel SACS mutation (p.Val1335IIe, Heterozygous) was identified in a Korean patient in their 50s with late-onset ARSACS characterized by cerebellar ataxia and spasticity without peripheral neuropathy. The mutation was confirmed via whole exome sequencing and Sanger sequencing and was predicted to likely cause disease using prediction software. RT-PCR and ELISA showed decreased SACS mRNA expression and sacsin protein concentrations in the proband, supporting its implications in diseases with pathogenicity and reduced chaperone function from haploinsufficiency. Our results revealed the pathogenicity of the SACS Val1335IIe mutation in the proband patient\'s disease manifestation, even though the symptoms had a limited correlation with the typical ARSACS clinical triad, which could be due to the reduced chaperon function from haploinsufficiency. Furthermore, our study suggests that variants of SACS heterozygosity may have diverse symptoms, with a wide range of disease onsets for late-onset sacsinopathy.

The ataxia-linked protein sacsin has three regions of partial homology to Hsp90\'s N-terminal ATP binding domain. Although a crystal structure for this Hsp90-like domain has been reported the precise molecular interactions required for ATP-binding and hydrolysis are unclear and it is debatable whether ATP biding is compatible with these domains. Furthermore, the Identification of a sacsin domain(s) equivalent to the middle domain of Hsp90 has been elusive. Here we present the superimposition of an AlphaFold structure of sacsin with yeast Hsp90, which provides novel insights into sacsin\'s structure. We identify residues within the sacsin Hsp90-like domains that are required for ATP binding and hydrolysis, including the putative catalytic arginine residues equivalent to that of the Hsp90 middle domain. Importantly, our analysis allows comparison of the Hsp90 middle domain with corresponding sacsin regions and identifies a shorter lid segment, in the sacsin ATP-binding domains, than the one found in the N-terminal domain of Hsp90. Our results show how a realignment of residues in the lid segment of sacsin that are involved in ATP binding can better match equivalent residues seen in Hsp90, which we then corroborated using molecular dynamic simulations. We speculate, from a structural viewpoint, why some ATP competitive inhibitors of Hsp90 may not bind sacsin, while others would. Together our analysis supports the hypothesis that sacsin\'s function is ATP-driven and would be consistent with it having a role as a super molecular chaperone. We propose that the SR1 regions of sacsin be renamed as HSP-NRD (Hsp90 N-Terminal Repeat Domain; residues 84-324) and the fragment immediately after as HSP-MRD (Hsp90 Middle Repeat Domain; residues 325-518).

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is characterized by triad of progressive cerebellar ataxia, progressive spasticity, and axonal/demyelinating peripheral neuropathy. Other manifestations include dysarthria, weakness in lower extremities and distal muscle wasting, foot deformities, retinal striation, prolapse of the mitral valve and rarely intellectual disability, hearing loss, and myoclonic epilepsy. We describe a patient who developed peripheral sensorimotor neuropathy in the absence of spasticity on initial presentation. He had nerve root enhancement on magnetic resonance imaging (MRI) lumbar spine, and nerve conduction studies were suggestive of demyelinating polyneuropathy. Patient had mild cerebellar atrophy on MRI and some delay of motor milestones. Over the course of several months, he developed spasticity, and genetic analysis together with clinical presentation was consistent with ARSACS. He was noted to have a pathogenic mutation c.8108G>A (p. Arg2703His) inherited from mother and a variant of uncertain significance c.7216T>C (p. Ser2406Pro) inherited from his father in SACS gene. Atypical cases may present later in life or in absence of one of the classical features at the time of presentation, which may make diagnosis difficult. Our patient had such an atypical presentation of ARSACS. Young patients with neuropathy and concomitant cerebellar atrophy on MRI should raise suspicion for hereditary spastic ataxia syndrome. Follow-up examination can often reveal additional findings to aid the diagnosis.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset cerebellar ataxia caused by mutations in SACS, which encodes the protein sacsin. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament disorganization, and progressive death of cerebellar Purkinje neurons. It is unclear why the loss of sacsin causes these deficits or why they manifest as cerebellar ataxia. Here, we perform multi-omic profiling in sacsin knockout (KO) cells and identify alterations in microtubule dynamics and mislocalization of focal adhesion (FA) proteins, including multiple integrins. Deficits in FA structure, signaling, and function can be rescued by targeting PTEN, a negative regulator of FA signaling. ARSACS mice possess mislocalization of ITGA1 in Purkinje neurons and synaptic disorganization in the deep cerebellar nucleus (DCN). The sacsin interactome reveals that sacsin regulates interactions between cytoskeletal and synaptic adhesion proteins. Our findings suggest that disrupted trafficking of synaptic adhesion proteins is a causal molecular deficit in ARSACS.