Driving mitochondrial fission improves cognitive, but not motor deficits in a mouse model of Ataxia of Charlevoix-Saguenay

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Merrill, Chian Ju Jong, Stefan Strack This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4178088/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract 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 ( B β 2 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. ARSACS ataxia mitochondrial dynamics dynamin-related protein 1 protein phosphatase 2A A kinase anchoring protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction First identified in the Charlevoix-Saguenay region of Quebec, where it was maintained due to founder effects, ARSACS is now recognized as one of the most common recessive spastic ataxia worldwide (1, 2). ARSACS is caused by loss-of-function variants in the SACS gene and strikes homozygous carriers in early life. The Sacs knockout (KO) mouse recapitulates many of the cardinal features of ARSACS, including gait abnormalities and loss of cerebellar Purkinje cells (3). Sacsin, the protein encoded by the SACS gene, is a large (~520 kD), multi-domain protein with a predicted function as a HSP70/90 co-chaperone (1). An early report demonstrated mitochondrial localization of sacsin, as well as an interaction with the mitochondrial fission enzyme Drp1 (4). Mitochondria in neurons with absent or reduced expression of sacsin were abnormally elongated, suggesting that the protein is necessary for proper assembly of the mitochondrial fission machinery. Studies with patient-derived fibroblasts confirmed this conclusion (4, 5). Intermediate filament phenotypes have also been reported, including abnormal bundling of neurofilaments in the soma and dendrites, hypophosphorylation of neurofilament heavy polypeptide (NFH), as well as vimentin cages in patient fibroblasts. In fibroblasts, alterations in the autophagy-lysosomal system were seen as evidence that sacsin has an important role in proteostasis (3, 6). With this report, we asked whether dysregulation of Drp1-mediated mitochondrial fission underlies ARSACS pathology, or whether other molecular events, such as intermediate filament aggregation may be primary disease drivers. To this end, we took a genetic approach, crossing Sacs KO mice with germline KOs of two well-established regulators of Drp1, PP2A/Bβ2 and PKA/Akap1. Mitochondrial fission is regulated by reversible phosphorylation of Drp1 at a highly conserved Ser residue that is phosphorylated by protein kinase A (PKA) and dephosphorylated by two phosphatases, PP2A and PP2B (7). Phosphorylation of Ser637 inhibits Drp1-dependent mitochondrial fission leading to mitochondrial elongation by unopposed fusion, while dephosphorylation activates the fission enzyme, shortening mitochondria (8, 9). We previously reported on mice that lack a regulatory subunit of PP2A (Bβ2), which targets the phosphatase to mitochondria because it includes an alternatively spliced mitochondrial localization sequence (10, 11). Bβ2 is expressed throughout the central and peripheral nervous system, including the cerebellum, but undetectable in non-neuronal cells (Fig. 1). Intriguingly, a non-coding CAG repeat expansion in a promoter region of the gene encoding Bβ2 ( PPP2R2B ) causes spinocerebellar ataxia type-12 (SCA12) (12, 13). In brains of Bβ2 KO mice, Drp1 is hyperphosphorylated at Ser637, and, consistent with Drp1 inactivation, mitochondria are elongated. Bβ2 KO mice are also protected from cerebral ischemic stroke, likely as a consequence of increased bioenergetic reserves (spare respiratory capacity) (11). We further reported that A-kinase anchoring protein 1 (AKAP1) recruits PKA to the outer mitochondrial membrane to phosphorylate and inactivate Drp1 (14). AKAP1 is tethered to the outer-mitochondrial membrane (15) and ubiquitously expressed (Fig. 1). Akap1 KO mice exhibit smaller mitochondria in neurons and glia, along with exacerbated stroke outcomes (16). AKAP1/Bβ2 double KO (DKO) mice show normal Drp1 regulation, mitochondrial morphology, and stroke sensitivity, indicating that PKA and PP2A exert their effects via a shared effector, Drp1 (11). We generated Sacs/Bβ2 and Sacs/Akap1 DKO mice and tested for ARSACS disease modification at the behavioral level. We reasoned that the Akap1 KO could reverse mitochondrial elongation reported in Sacs KO neurons (4). The Bβ2 KO, on the other hand, might attenuate neurodegeneration (as it does in ischemic stroke), if the mitochondrial elongation observed in Sacs KO mice is an adaptive, rather than a disease-driving mechanism. More precipitous cerebellar decline in either Sacs/Bβ2 or Sacs/Akap1 DKO model would also be informative, as it would support the notion of ARSACS as a mitochondrial disease. To our surprise, neither DKO influenced deterioration of motor performance in ARSACS-model mice with age. However, we uncovered a striking decline in cognitive function in older Sacs KO mice. Equally striking, this decline was rescued by promoting mitochondrial fission by deleting the Drp1 inhibitor PKA/Akap1. Results Human whole-tissue mRNA sequencing data retrieved from the Broad Institute (gtexportal.org) indicates wide-spread expression of SACS and AKAP1 , whereas Bβ2 ( PPP2R2B ) expression is largely confined to the brain. Within brain regions, SACS expression is uniform, while Bβ2 expression is relatively low and AKAP1 expression is relatively high in the cerebellum (Fig. 1). The three gene products are therefore in the right place to functionally interact. We initially examined DKOs of Sacs and the fission driver Bβ2 (Fig. 2A), both of which cause mitochondrial elongation when deleted alone (4, 11). We set up crosses to yield Sacs wild-type and disease-relevant, homozygous null (-/-) mice combined with Bβ2 alleles of all three genotypes (+/+, +/-, -/-). Heterozygous Bβ2 KOs were included because they afford partial protection from ischemic stroke (11). The same cohorts of mice were analyzed at 3 and 6 months of age using the three-day accelerating Rotarod test, which measures motor coordination and motor learning. As reported before (3), Sacs deletion by itself significantly impaired performance at both ages. We included both male and female mice in these and subsequent experiments but detected no sex differences. Data from both sexes were therefore pooled for statistical analysis, with data points for male and female mice differentiated by symbol outline colors in each bar graph for transparency. At three months of age, all six mouse genotypes learned similarly to stay on the accelerating rod (similar slope of day-to-day performance increase), and heterozygous and homozygous deletion of PP2A/Bβ2 did not improve the rotarod performance in Sacs -/- mice (Fig. 2B, 2C). At six months of age, the same cohort of mice did not show a clear learning pattern (Fig 2D). Heterozygous and homozygous deletion of PP2A/Bβ2 did not improve the Rotarod performance in Sacs -/- mice at this age either (Fig 2E). We then investigated Sacs and Akap1 double knockout (DKO) mice to determine if the mitochondrial hyperfusion induced by Sacs knockout (KO) could be reversed by eliminating a restraint on Drp1 fission activity (Fig. 3A). Again, Sacs +/+ and Sacs -/- alleles were paired with all three Akap1 alleles (+/+, +/-, -/-). Mice with Akap1 +/- genotype were included because Akap1 heterozygosity improves neuroanatomical and metabolic symptoms in a mouse model of Bardet-Biedl syndrome (17). 3 months old mice were examined for motor-coordination and -learning using the 3-day accelerating Rotarod test. Motor-performance of mice of all genotypes improved over time, but Sacs -/- mice performed consistently worse than Sacs +/+ mice (Fig. 3B). Notably, while homozygous Akap1 deletion did not improve motor function in Sacs -/- mice, mice carrying one copy of the Akap1 gene performed at a level not significantly different from Sacs +/+ mice (Fig. 3C). Encouraged by the finding that Akap1 heterozygosity might alleviate motor deficits, we examined aged (13-16 months old) Sacs / Akap1 DKO mice, when Sacs KO symptoms are more pronounced. At this age, Sacs KO mice displayed severe impairments on the Rotarod. However, latency to fall was unaffected by the Akap1 genotype (Fig. 4A, B). Time crossing the balance beam, an indicator of motor coordination, was increased in Sacs -/- mice, with no significant effect of the Akap1 genotype (Fig. 4C). Distance traveled in the open field test was reduced in Sacs KO mice; again, without apparent influence of Akap1 gene dose (Fig. 4D). Likewise, the wire hang test indicated severely impaired grip strength in Sacs -/- mice, but no improvement when one or both Akap1 alleles had been deleted (Fig. 4E). Next, the same cohort of aged mice were subjected to an associative learning and memory paradigm, contextual fear conditioning. In this test, mice are placed in a context with novel visual, odor, and tactile cues and then subjected to a foot shock. 24 h later, mice are re-introduced into the same context and the time anticipating the foot shock (“freezing”) is recorded. Compared to Sacs +/+ , Sacs -/- mice displayed a highly significant deficit in associating the context with the foot shock they received the day prior. Remarkably, deletion of Akap1 improved the learning and memory performance in a gene-dose-dependent manner in Sac -/- mice, with homozygous Akap1 deletion resulting in near normal contextual condition (Fig. 4F). Discussion This study confirms a previous report that Sacs KO mice faithfully replicate the natural history of ARSACS (3). By analyzing DKOs with established regulators of Drp1, we also provide evidence against dysregulation of the mitochondrial fission enzyme as a primary disease driver in ARSACS. As in other neurodegenerative diseases, mitochondrial dysfunction is increasingly implicated in ARSACS pathology (18-20). For instance, MitoQ, a mitochondria-targeted antioxidant, was recently shown to improve motor coordination and delay Purkinje cell death in Sacs KO mice (21). In light of the present results, mitochondrial dysfunction in ARSACS is unlikely due to an imbalance of mitochondrial fission and fusion, but rather a secondary consequence of improper folding and aggregation of one or more of the critical clients of the sacsin co-chaperone complex. In a speculative scenario, aggregated neurofilaments in Sacs KO Purkinje neurons interfere with trafficking of mitochondria along neurites and recycling of dysfunctional mitochondria by mitophagy. We also report for the first time that loss of sacsin is associated with profound impairments in learning and memory in older mice. Cerebellar ataxias, including ARSACS, manifest not only with motor symptoms, but also with a spectrum of neuropsychiatric and learning disorders including dyslexia, attention deficit hyperactivity disorder, autism spectrum disorders, panic disorder, schizophrenia, and intellectual disabilities. Coined as “cognitive dysmetria” or “cerebellar cognitive affective syndrome”, this was recognized independently in the late 1990’s by Nancy Andreasen (22-24), Jeremy Schmahmann (25), and others (26). Case studies of ARSACS, specifically, listed a variety of non-motor symptoms, such as low motivation (apathy), dysphoria, but also paranoid ideation, irritability, and marked cognitive dysfunction, including anosognosia (27, 28). Whereas fMRI studies indicate that the cerebellum participates in the retrieval of episodic memory and other cognitive tasks (24), there remain questions how cerebellar disorders impair cognition. On the one hand, cerebellar nuclei project, directly or indirectly, to various brain areas involved in higher-order cognition, including the prefrontal cortex. Also, cerebellar lesions due to accidents or surgical resections can present with non-motor symptoms similar to cerebellar disorders. On the other hand, most cerebellar ataxia disease genes, including SACS , are ubiquitously expressed (Fig. 1), and cerebral atrophy commonly follows cerebellar atrophy in hereditary cerebellar ataxias. Further studies using conditional Sacs KO mice are needed to pinpoint the cellular and anatomical origins of non-motor symptoms of ARSACS. Also, future studies should address the temporal relationship between cognitive and motor symptoms in ARSACS and the mechanism by which Akap1 deletion improves cognitive decline in Sacs KO mice. Materials and methods Mice Mouse work was performed in accordance with the guidelines of the animal ethics committee of the University of Iowa. Mice were group-housed in a colony maintained with a standard 12 h light/dark cycle and given food and water ad libitum. Experiments were performed on age-matched mice of both sexes as indicated in bar graphs. Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals, as adopted by the National Institutes of Health, and with approval of the University of Iowa AAALC-accredited Institutional Animal Care and Use Committee. The Sacs -/- mice were a kind gift of Bernard Brais, McGill (3). The AKAP1 -/- mouse line was kindly provided by Dr. Stanley McKnight at University of Washington (29), and Bβ2 -/- mice were generated at the U. Iowa Mouse KO Core Facility (11). We generated mice with heterozygous or homozygous deletion of PP2A/Bβ2 or Akap1 that were either wild-type or null at the Sacs locus. Mice were in the C57BL/6J background and were backcrossed to C57BL/6J mice imported from the Jackson Laboratory (Bar Harbor, ME) every 6-10 generations to prevent genetic drift. Mice of all genotypes were born in expected Mendelian ratios and were fertile, except for Akap1 -/- females who are infertile (29). All mice achieved a normal lifespan, with many individuals surviving beyond two years. However, Sacs -/- mice with or without deletion of Bβ2 or Akap1 displayed progressive gait abnormality as documented (3). Behavioral Testing General. 5-7 days of habituation and handling was done prior to behavioral assessment. Mice were allowed to acclimatize to the testing environment for 30 minutes prior to the starting of the experiment trials on the day of testing. All genotypes were tested on the same day in a randomized order with experimenters blinded to genotype and sex. Accelerating rotarod. Mice were placed on a rotating rod (IITC Life Woodland Hills, CA) with gradual speed increase from 4 to 40 rpm over 5 minutes, in which the latency to fall was recorded. Testing consisted of four trials on each day for three consecutive days, with 5-10 minutes between each trial. The average of four daily trials was recorded for each mouse. Balance beam. The balance beam apparatus was acquired from MazeEngineers Inc (Skokie, IL). The balance beam test was performed as previously described (30). A 12 mm-wide beam was used in our tests. Mice were placed at one end of the beam and the latency for crossing the beam to the other end was recorded by MediaRecorder software (Noldus). Mice were trained for three times in each day for two consecutive days and tested for three times on day 3. The best performance (minimum latency to cross beam) was recorded on the testing day. Wire hang test. The test began with the mice placed on an elevated wire cage top, which was then inverted and suspended above the home cage. The time it took for the animal to fall was recorded. This test was conducted two times on one day, with 5-10 minutes between each trial. The best performance (longer hang time) was recorded. Contextual Fear Conditioning. The testing apparatus for 24-hour contextual recall was acquired from CleverSys Inc (Reston, VA). Conditioning was assessed using FreezeScan V.2 software, which measures the "freezing" behavior of the mouse. On the training day, the animals underwent a 3-minute trial, with an electric shock of 1.5 mA delivered at 2.5 min and lasting for two seconds. On the testing day, the animals were returned to the exact same environment and underwent a 5-minute trial without any shocks. Data were presented as percentage of time freezing on the testing day. Statistical Analysis Data were obtained and analyzed with experimenters blinded to genotype and sex. Statistics were analyzed, and plots were generated using GraphPad Prism software (version 10.2). All data were first analyzed by D’Agostino-Pearson test to determine the normality and then analyzed by two-way ANOVA with Dunnett’s multiple comparison tests. Motor learning was analyzed by linear regression. The false positive rate (a) was set at 0.05. Declarations Funding This work was supported by the Ataxia of Charlevoix-Saguenay Foundation. Additional support was provided by R21 AG080472-01 and the Simons Foundation Autism Research Initiative (SFARI). Author Contributions RAM and SS conceptualized the study. CC, RAM, and CJJ performed experiments, and CC and SS prepared the figures and wrote the manuscript. All authors read, edited, and approved the final manuscript before submission. Competing Interests The authors declare no competing interests. Data availability Gene expression data (Figure 1) are available from the Genotype-Tissue Expression (GTEx) Portal at gtexportal.org. All other data (Excel spreadsheets, Prism files, movies) can be obtained from the corresponding author upon request. Acknowledgements We acknowledge technical assistance by Yufang Kong, Kathleen Kelley, Gail Healy, and Marisol Lauffer. Many of the experiments were conducted at the Neural Circuits and Behavior Core of the Iowa Neuroscience Institute. Metabolomics data were obtained with the assistance of the U. Iowa Metabolomics Core Facility. Sacs KO mice were a kind gift of Bernard Brais, McGill (3), Akap1 KO mice were donated by G. Stanley McKnight, U. Washington (29), and Bβ2 KO mice were generated at the U. Iowa Mouse KO Core Facility. The data used for Figure 1 in this manuscript were obtained from the GTEx Portal on 3/8/24. The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. References Aly KA, Moutaoufik MT, Zilocchi M, Phanse S, Babu M. Insights into SACS pathological attributes in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)☆. Curr Opin Chem Biol. 2022;71:102211. Bagaria J, Bagyinszky E, An SSA. Genetics of Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) and Role of Sacsin in Neurodegeneration. Int J Mol Sci. 2022;23(1). Lariviere R, Gaudet R, Gentil BJ, Girard M, Conte TC, Minotti S, et al. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Apr, 2024 Reviews received at journal 17 Apr, 2024 Reviewers agreed at journal 07 Apr, 2024 Reviewers invited by journal 05 Apr, 2024 Editor assigned by journal 05 Apr, 2024 Submission checks completed at journal 05 Apr, 2024 First submitted to journal 27 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4178088","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288536420,"identity":"3b0be97e-705c-41d3-ae87-bf01a8f4f7ed","order_by":0,"name":"Chunling Chen","email":"","orcid":"","institution":"University of Iowa, Carver College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chunling","middleName":"","lastName":"Chen","suffix":""},{"id":288536422,"identity":"450d1106-2db5-4f3e-b1f4-4f0c223cf7c8","order_by":1,"name":"Ronald A. Merrill","email":"","orcid":"","institution":"University of Iowa, Carver College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"A.","lastName":"Merrill","suffix":""},{"id":288536423,"identity":"697dd519-e374-431d-a1ad-2646e0a222cd","order_by":2,"name":"Chian Ju Jong","email":"","orcid":"","institution":"University of Iowa, Carver College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chian","middleName":"Ju","lastName":"Jong","suffix":""},{"id":288536425,"identity":"b7aae842-fdc3-4364-af27-9bc6a77dea3f","order_by":3,"name":"Stefan Strack","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYJCCDzAGY0MFAw+EyYZHPRsD4wwQzQPWcoZkLY1tCGGcQH5+88GGnzsY7O2lDz/8OHPeYRl+ieQHDB/KDuPUYnCMLbGx9wxDYg9fmrHkxm2HeSRnpBkwzjiHRwsbj/kD3rb/CTw8DGaMD4FaDG4nGDDztuHWIt/G/7HxbxuDPQ8P+zfGh3NAWtI/MP/Fo4XhGA9jM28bA2MPD48Z48YGkJYcA2ZGPFoMjqUZNsu2Af1yhqdYcsaxdB7J+W8KDvacS8ftsObDDxvfAh3G3sO+8WNPjbU9P8/xjQ9+lFnjdhhWcIBE9aNgFIyCUTAK0AAA8iZSdIAFsUAAAAAASUVORK5CYII=","orcid":"","institution":"University of Iowa, Carver College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Strack","suffix":""}],"badges":[],"createdAt":"2024-03-27 19:44:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4178088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4178088/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54453582,"identity":"1c016148-32bf-46b9-940c-a491705ecd51","added_by":"auto","created_at":"2024-04-10 18:37:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":194719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue expression profiles of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSACS\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAKAP1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and PP2A/Bβ2 (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePPP2R2B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e).\u003c/strong\u003e Transcript abundance was obtained by human whole-tissue mRNA sequencing and is expressed as transcripts per million. Metadata were procured from the Genotype-Tissue Expression (GTEx) Portal (gtexportal.org) and are displayed with a focus on brain regions.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4178088/v1/d49572823dbfa4d3dcba45fd.png"},{"id":54452834,"identity":"603dbdbe-c0bc-45d6-a778-207c9a39ccd5","added_by":"auto","created_at":"2024-04-10 18:29:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":218589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePromoting mitochondrial fusion by deletion of the Drp1 activator PP2A/Bb2 does not improve \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSacs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e KO motor performance.\u003c/strong\u003e (A) Schematic showing that deletion of PP2A/Bβ2 promotes mitochondrial elongation by inhibiting the inhibitory dephosphorylation of the mitochondrial fission enzyme Drp1 at Ser637. (B-E) Motor coordination and motor learning were tested with the accelerating Rotarod test (4–40 rpm over 5 min) at three months (B, C) and six months of age (D, E). Graphs were plotted as the time (second) that mice remained on the rod before falling. (B) At 3 months of age, the linear regression analysis demonstrates significantly different slopes from 0 for all genotypes, indicating motor learning. (C) At the same age, \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice performed worse than \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice regardless of PP2A/Bβ2 genotype. (D) At 6 months, only SACS\u003csup\u003e-/-\u003c/sup\u003e Bβ2\u003csup\u003e+/-\u003c/sup\u003e mice showed motor learning (linear regression slope different from 0). (E) At the same age, \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice performed worse than \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice, again, independent of Bβ2 genotype. Plotted are means ± SD with data points representing individual mice. Black outlines represent male and gray outlines represent female mice. Data were analyzed by linear regression (B, D) or 2-way ANOVA with Dunnett’s post hoc test (C, E); *,p\u0026lt;0.05; **,p\u0026lt;0.01; ***,P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4178088/v1/ca8b486b227e95fd7caba487.png"},{"id":54452832,"identity":"f365de5a-74a6-4646-b175-f937232317c3","added_by":"auto","created_at":"2024-04-10 18:29:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterozygous \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAkap1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e deletion improves motor performance in young \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSacs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e KO mice.\u003c/strong\u003e (A) Schematic showing that deletion of \u003cem\u003eAkap1\u003c/em\u003e promotes shortening mitochondria by decreasing the inhibitory phosphorylation of the mitochondrial fission enzyme Drp1 at Ser637. (B, C) Motor coordination and motor learning were tested with the accelerating Rotarod test (4–40 rpm over 5 min) at 3 months of age. (B) Regression analysis demonstrates significant motor learning (slopes different from 0) of all genotypes. (C) \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice performed consistently worse than Sacs\u003csup\u003e+/+\u003c/sup\u003e mice, with no effect by homozygous deletion of \u003cem\u003eAkap1\u003c/em\u003e. However, at day 2 and 3, heterozygous deletion of \u003cem\u003eAkap1\u003c/em\u003e in Sacs\u003csup\u003e-/-\u003c/sup\u003e mice performed at a level not significantly different from Sacs\u003csup\u003e+/+\u003c/sup\u003e mice. See Fig. 2 for data presentation and analysis.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4178088/v1/d6fb11905c1c581e99196fab.png"},{"id":54452836,"identity":"8c4b59b6-c8d0-466c-8c51-4deff13b27db","added_by":"auto","created_at":"2024-04-10 18:29:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAkap1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eKO rescues cognitive, but not motor function in aged \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSacs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-/- mice.\u003c/strong\u003e (A, B) According to the 3-day accelerating Rotarod test, aged (13-16 months old) \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice performed worse than Sacs\u003csup\u003e+/+\u003c/sup\u003e mice regardless of \u003cem\u003eAkap1\u003c/em\u003e genotype. Motor learning was not evident from linear regression analysis. (C) Sacs\u003csup\u003e-/- \u003c/sup\u003emice took significantly more time to cross the balance beam and homozygous deletion of \u003cem\u003eAkap1\u003c/em\u003e did not improve their performance. However, \u003cem\u003eAkap1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/-\u003c/em\u003e\u003c/sup\u003e mice showed partial improvement. (D) Significant muscle weakness in \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice compared to \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e mice was detected by the wire hang test; however, hetero- or homozygous loss of \u003cem\u003eAkap1\u003c/em\u003e had no effect. (E) In the 10-min open field test, deletion of \u003cem\u003eAkap1\u003c/em\u003e did not improve the distance-traveled deficit of\u003cem\u003e Sacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice.\u0026nbsp; (F) \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e displayed weaker recall of contextual fear memory. Deletion of \u003cem\u003eAkap1\u003c/em\u003e improved the learning and memory performance in a gene dose-dependent manner. See Fig. 2 for data presentation and analysis. \u003cem\u003eAkap1\u003c/em\u003e genotype data in (D, E) was pooled and analyzed by Student’s T-test because there was no significant \u003cem\u003eSacs/Akap1\u003c/em\u003e genotype interaction.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4178088/v1/ab844c7cba2316208b6fbe9e.png"},{"id":54454165,"identity":"9e9e2b97-a39f-4ee3-861b-8247a51ee4c3","added_by":"auto","created_at":"2024-04-10 18:45:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1129580,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4178088/v1/4d892917-a0d7-4758-ba2f-0da15b1b4230.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Driving mitochondrial fission improves cognitive, but not motor deficits in a mouse model of Ataxia of Charlevoix-Saguenay","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFirst identified in the Charlevoix-Saguenay region of Quebec, where it was maintained due to founder effects, ARSACS is now recognized as one of the most common recessive spastic ataxia worldwide (1, 2). ARSACS is caused by loss-of-function variants in the \u003cem\u003eSACS\u003c/em\u003e gene and strikes homozygous carriers in early life. The \u003cem\u003eSacs\u003c/em\u003e knockout (KO) mouse recapitulates many of the cardinal features of ARSACS, including gait abnormalities and loss of cerebellar Purkinje cells (3). Sacsin, the protein encoded by the \u003cem\u003eSACS\u003c/em\u003e gene, is a large (~520 kD), multi-domain protein with a predicted function as a HSP70/90 co-chaperone (1).\u003c/p\u003e\n\n\u003cp\u003eAn early report demonstrated mitochondrial localization of sacsin, as well as an interaction with the mitochondrial fission enzyme Drp1 (4). Mitochondria in neurons with absent or reduced expression of sacsin were abnormally elongated, suggesting that the protein is necessary for proper assembly of the mitochondrial fission machinery. Studies with patient-derived fibroblasts confirmed this conclusion (4, 5). Intermediate filament phenotypes have also been reported, including abnormal bundling of neurofilaments in the soma and dendrites, hypophosphorylation of neurofilament heavy polypeptide (NFH), as well as vimentin cages in patient fibroblasts. In fibroblasts, alterations in the autophagy-lysosomal system were seen as evidence that sacsin has an important role in proteostasis (3, 6).\u003c/p\u003e\n\n\u003cp\u003eWith this report, we asked whether dysregulation of Drp1-mediated mitochondrial fission underlies ARSACS pathology, or whether other molecular events, such as intermediate filament aggregation may be primary disease drivers. To this end, we took a genetic approach, crossing \u003cem\u003eSacs\u003c/em\u003e KO mice with germline KOs of two well-established regulators of Drp1, PP2A/B\u0026beta;2 and PKA/Akap1. \u003c/p\u003e\n\n\u003cp\u003eMitochondrial fission is regulated by reversible phosphorylation of Drp1 at a highly conserved Ser residue that is phosphorylated by protein kinase A (PKA) and dephosphorylated by two phosphatases, PP2A and PP2B (7). Phosphorylation of Ser637 inhibits Drp1-dependent mitochondrial fission leading to mitochondrial elongation by unopposed fusion, while dephosphorylation activates the fission enzyme, shortening mitochondria (8, 9). We previously reported on mice that lack a regulatory subunit of PP2A (Bβ2), which targets the phosphatase to mitochondria because it includes an alternatively spliced mitochondrial localization sequence (10, 11). Bβ2 is expressed throughout the central and peripheral nervous system, including the cerebellum, but undetectable in non-neuronal cells (Fig. 1). Intriguingly, a non-coding CAG repeat expansion in a promoter region of the gene encoding Bβ2 (\u003cem\u003ePPP2R2B\u003c/em\u003e) causes spinocerebellar ataxia type-12 (SCA12) (12, 13). In brains of Bβ2 KO mice, Drp1 is hyperphosphorylated at Ser637, and, consistent with Drp1 inactivation, mitochondria are elongated. Bβ2 KO mice are also protected from cerebral ischemic stroke, likely as a consequence of increased bioenergetic reserves (spare respiratory capacity) (11). We further reported that A-kinase anchoring protein 1 (AKAP1) recruits PKA to the outer mitochondrial membrane to phosphorylate and inactivate Drp1 (14). AKAP1 is tethered to the outer-mitochondrial membrane (15) and ubiquitously expressed (Fig. 1). Akap1 KO mice exhibit smaller mitochondria in neurons and glia, along with exacerbated stroke outcomes (16). AKAP1/Bβ2 double KO (DKO) mice show normal Drp1 regulation, mitochondrial morphology, and stroke sensitivity, indicating that PKA and PP2A exert their effects via a shared effector, Drp1 (11). \u003c/p\u003e\n\n\u003cp\u003eWe generated Sacs/Bβ2 and Sacs/Akap1 DKO mice and tested for ARSACS disease modification at the behavioral level. We reasoned that the Akap1 KO could reverse mitochondrial elongation reported in \u003cem\u003eSacs\u003c/em\u003e KO neurons (4). The Bβ2 KO, on the other hand, might attenuate neurodegeneration (as it does in ischemic stroke), if the mitochondrial elongation observed in Sacs KO mice is an adaptive, rather than a disease-driving mechanism. More precipitous cerebellar decline in either Sacs/Bβ2 or Sacs/Akap1 DKO model would also be informative, as it would support the notion of ARSACS as a mitochondrial disease. To our surprise, neither DKO influenced deterioration of motor performance in ARSACS-model mice with age. However, we uncovered a striking decline in cognitive function in older \u003cem\u003eSacs\u003c/em\u003e KO mice. Equally striking, this decline was rescued by promoting mitochondrial fission by deleting the Drp1 inhibitor PKA/Akap1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eHuman whole-tissue mRNA sequencing data retrieved from the Broad Institute (gtexportal.org) indicates wide-spread expression of \u003cem\u003eSACS\u003c/em\u003e and \u003cem\u003eAKAP1\u003c/em\u003e, whereas Bβ2 (\u003cem\u003ePPP2R2B\u003c/em\u003e) expression is largely confined to the brain. Within brain regions, \u003cem\u003eSACS\u003c/em\u003e expression is uniform, while Bβ2 expression is relatively low and \u003cem\u003eAKAP1\u003c/em\u003e expression is relatively high in the cerebellum (Fig. 1). The three gene products are therefore in the right place to functionally interact. \u003c/p\u003e\n\n\u003cp\u003eWe initially examined DKOs of \u003cem\u003eSacs\u003c/em\u003e and the fission driver Bβ2 (Fig. 2A), both of which cause mitochondrial elongation when deleted alone (4, 11). We set up crosses to yield \u003cem\u003eSacs\u003c/em\u003e wild-type and disease-relevant, homozygous null (-/-) mice combined with Bβ2 alleles of all three genotypes (+/+, +/-, -/-). Heterozygous Bβ2 KOs were included because they afford partial protection from ischemic stroke (11). The same cohorts of mice were analyzed at 3 and 6 months of age using the three-day accelerating Rotarod test, which measures motor coordination and motor learning. As reported before (3), \u003cem\u003eSacs\u003c/em\u003e deletion by itself significantly impaired performance at both ages. We included both male and female mice in these and subsequent experiments but detected no sex differences. Data from both sexes were therefore pooled for statistical analysis, with data points for male and female mice differentiated by symbol outline colors in each bar graph for transparency.\u003c/p\u003e\n\n\u003cp\u003eAt three months of age, all six mouse genotypes learned similarly to stay on the accelerating rod (similar slope of day-to-day performance increase), and heterozygous and homozygous deletion of PP2A/B\u0026beta;2 did not improve the rotarod performance in Sacs\u003csup\u003e-/-\u003c/sup\u003e mice (Fig. 2B, 2C). At six months of age, the same cohort of mice did not show a clear learning pattern (Fig 2D). Heterozygous and homozygous deletion of PP2A/B\u0026beta;2 did not improve the Rotarod performance in Sacs\u003csup\u003e-/-\u003c/sup\u003e mice at this age either (Fig 2E).\u003c/p\u003e\n\n\u003cp\u003eWe then investigated Sacs and Akap1 double knockout (DKO) mice to determine if the mitochondrial hyperfusion induced by Sacs knockout (KO) could be reversed by eliminating a restraint on Drp1 fission activity (Fig. 3A). Again, \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e alleles were paired with all three \u003cem\u003eAkap1\u003c/em\u003e alleles (+/+, +/-, -/-). Mice with\u003cem\u003e Akap1\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e genotype were included because \u003cem\u003eAkap1\u003c/em\u003e heterozygosity improves neuroanatomical and metabolic symptoms in a mouse model of Bardet-Biedl syndrome (17). 3 months old mice were examined for motor-coordination and -learning using the 3-day accelerating Rotarod test. Motor-performance of mice of all genotypes improved over time, but \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice performed consistently worse than \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e mice (Fig. 3B). Notably, while homozygous \u003cem\u003eAkap1\u003c/em\u003e deletion did not improve motor function in \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, mice carrying one copy of the \u003cem\u003eAkap1\u003c/em\u003e gene performed at a level not significantly different from Sacs\u003csup\u003e+/+\u003c/sup\u003e mice (Fig. 3C). \u003c/p\u003e\n\n\u003cp\u003eEncouraged by the finding that Akap1 heterozygosity might alleviate motor deficits, we examined aged (13-16 months old) \u003cem\u003eSacs\u003c/em\u003e/\u003cem\u003eAkap1\u003c/em\u003e DKO mice, when \u003cem\u003eSacs\u003c/em\u003e KO symptoms are more pronounced. At this age, \u003cem\u003eSacs\u003c/em\u003e KO mice displayed severe impairments on the Rotarod. However, latency to fall was unaffected by the \u003cem\u003eAkap1\u003c/em\u003e genotype (Fig. 4A, B). Time crossing the balance beam, an indicator of motor coordination, was increased in \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, with no significant effect of the \u003cem\u003eAkap1\u003c/em\u003e genotype (Fig. 4C). Distance traveled in the open field test was reduced in \u003cem\u003eSacs\u003c/em\u003e KO mice; again, without apparent influence of \u003cem\u003eAkap1\u003c/em\u003e gene dose (Fig. 4D). Likewise, the wire hang test indicated severely impaired grip strength in \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice, but no improvement when one or both \u003cem\u003eAkap1\u003c/em\u003e alleles had been deleted (Fig. 4E). \u003c/p\u003e\n\n\u003cp\u003eNext, the same cohort of aged mice were subjected to an associative learning and memory paradigm, contextual fear conditioning. In this test, mice are placed in a context with novel visual, odor, and tactile cues and then subjected to a foot shock. 24 h later, mice are re-introduced into the same context and the time anticipating the foot shock (\u0026ldquo;freezing\u0026rdquo;) is recorded. Compared to \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, \u003cem\u003eSacs\u003c/em\u003e \u003csup\u003e-/-\u003c/sup\u003e mice displayed a highly significant deficit in associating the context with the foot shock they received the day prior. Remarkably, deletion of Akap1 improved the learning and memory performance in a gene-dose-dependent manner in Sac\u003csup\u003e-/- \u003c/sup\u003emice, with homozygous Akap1 deletion resulting in near normal contextual condition (Fig. 4F).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study confirms a previous report that \u003cem\u003eSacs\u003c/em\u003e KO mice faithfully replicate the natural history of ARSACS (3). By analyzing DKOs with established regulators of Drp1, we also provide evidence against dysregulation of the mitochondrial fission enzyme as a primary disease driver in ARSACS. As in other neurodegenerative diseases, mitochondrial dysfunction is increasingly implicated in ARSACS pathology (18-20). For instance, MitoQ, a mitochondria-targeted antioxidant, was recently shown to improve motor coordination and delay Purkinje cell death in \u003cem\u003eSacs\u003c/em\u003e KO mice (21). In light of the present results, mitochondrial dysfunction in ARSACS is unlikely due to an imbalance of mitochondrial fission and fusion, but rather a secondary consequence of improper folding and aggregation of one or more of the critical clients of the sacsin co-chaperone complex. In a speculative scenario, aggregated neurofilaments in Sacs KO Purkinje neurons interfere with trafficking of mitochondria along neurites and recycling of dysfunctional mitochondria by mitophagy.\u003c/p\u003e\n\n\u003cp\u003eWe also report for the first time that loss of sacsin is associated with profound impairments in learning and memory in older mice. Cerebellar ataxias, including ARSACS, manifest not only with motor symptoms, but also with a spectrum of neuropsychiatric and learning disorders including dyslexia, attention deficit hyperactivity disorder, autism spectrum disorders, panic disorder, schizophrenia, and intellectual disabilities. Coined as \u0026ldquo;cognitive dysmetria\u0026rdquo; or \u0026ldquo;cerebellar cognitive affective syndrome\u0026rdquo;, this was recognized independently in the late 1990\u0026rsquo;s by Nancy Andreasen (22-24), Jeremy Schmahmann (25), and others (26). Case studies of ARSACS, specifically, listed a variety of non-motor symptoms, such as low motivation (apathy), dysphoria, but also paranoid ideation, irritability, and marked cognitive dysfunction, including anosognosia (27, 28). \u003c/p\u003e\n\n\u003cp\u003eWhereas fMRI studies indicate that the cerebellum participates in the retrieval of episodic memory and other cognitive tasks (24), there remain questions how cerebellar disorders impair cognition. On the one hand, cerebellar nuclei project, directly or indirectly, to various brain areas involved in higher-order cognition, including the prefrontal cortex. Also, cerebellar lesions due to accidents or surgical resections can present with non-motor symptoms similar to cerebellar disorders. On the other hand, most cerebellar ataxia disease genes, including \u003cem\u003eSACS\u003c/em\u003e, are ubiquitously expressed (Fig. 1), and cerebral atrophy commonly follows cerebellar atrophy in hereditary cerebellar ataxias. \u003c/p\u003e\n\n\u003cp\u003eFurther studies using conditional Sacs KO mice are needed to pinpoint the cellular and anatomical origins of non-motor symptoms of ARSACS. Also, future studies should address the temporal relationship between cognitive and motor symptoms in ARSACS and the mechanism by which \u003cem\u003eAkap1\u003c/em\u003e deletion improves cognitive decline in \u003cem\u003eSacs\u003c/em\u003e KO mice. \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003e\u003cu\u003eMice\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMouse work was performed in accordance with the guidelines of the animal ethics committee of the University of Iowa. Mice were group-housed in a colony maintained with a standard 12 h light/dark cycle and given food and water ad libitum. Experiments were performed on age-matched mice of both sexes as indicated in bar graphs. Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals, as adopted by the National Institutes of Health, and with approval of the University of Iowa AAALC-accredited Institutional Animal Care and Use Committee.\u003c/p\u003e\n\n\u003cp\u003eThe Sacs\u003csup\u003e-/-\u003c/sup\u003e mice were a kind gift of Bernard Brais, McGill (3). The AKAP1\u003csup\u003e -/-\u003c/sup\u003e mouse line was kindly provided by Dr. Stanley McKnight at University of Washington (29), and Bβ2\u003csup\u003e-/-\u003c/sup\u003emice were generated at the U. Iowa Mouse KO Core Facility (11). We generated mice with heterozygous or homozygous deletion of PP2A/B\u0026beta;2 or \u003cem\u003eAkap1\u003c/em\u003e that were either wild-type or null at the \u003cem\u003eSacs\u003c/em\u003e locus. Mice were in the C57BL/6J background and were backcrossed to C57BL/6J mice imported from the Jackson Laboratory (Bar Harbor, ME) every 6-10 generations to prevent genetic drift. Mice of all genotypes were born in expected Mendelian ratios and were fertile, except for \u003cem\u003eAkap1\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e females who are infertile (29). All mice achieved a normal lifespan, with many individuals surviving beyond two years. However, \u003cem\u003eSacs\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice with or without deletion of B\u0026beta;2 or \u003cem\u003eAkap1\u003c/em\u003e displayed progressive gait abnormality as documented (3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eBehavioral Testing\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGeneral.\u003c/em\u003e 5-7 days of habituation and handling was done prior to behavioral assessment. Mice were allowed to acclimatize to the testing environment for 30 minutes prior to the starting of the experiment trials on the day of testing. All genotypes were tested on the same day in a randomized order with experimenters blinded to genotype and sex. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAccelerating rotarod. \u003c/em\u003eMice were placed on a rotating rod (IITC Life Woodland Hills, CA) with gradual speed increase from 4 to 40 rpm over 5 minutes, in which the latency to fall was recorded. Testing consisted of four trials on each day for three consecutive days, with 5-10 minutes between each trial. The average of four daily trials was recorded for each mouse.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBalance beam. \u003c/em\u003eThe balance beam apparatus was acquired from MazeEngineers Inc (Skokie, IL). The balance beam test was performed as previously described (30). A 12 mm-wide beam was used in our tests. Mice were placed at one end of the beam and the latency for crossing the beam to the other end was recorded by MediaRecorder software (Noldus). Mice were trained for three times in each day for two consecutive days and tested for three times on day 3. The best performance (minimum latency to cross beam) was recorded on the testing day. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWire hang test. \u003c/em\u003eThe test began with the mice placed on an elevated wire cage top, which was then inverted and suspended above the home cage. The time it took for the animal to fall was recorded. This test was conducted two times on one day, with 5-10 minutes between each trial. The best performance (longer hang time) was recorded. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eContextual Fear Conditioning. \u003c/em\u003eThe testing apparatus for 24-hour contextual recall was acquired from CleverSys Inc (Reston, VA). Conditioning was assessed using FreezeScan V.2 software, which measures the \u0026quot;freezing\u0026quot; behavior of the mouse. On the training day, the animals underwent a 3-minute trial, with an electric shock of 1.5 mA delivered at 2.5 min and lasting for two seconds. On the testing day, the animals were returned to the exact same environment and underwent a 5-minute trial without any shocks. Data were presented as percentage of time freezing on the testing day. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData were obtained and analyzed with experimenters blinded to genotype and sex. Statistics were analyzed, and plots were generated using GraphPad Prism software (version 10.2). All data were first analyzed by D\u0026rsquo;Agostino-Pearson test to determine the normality and then analyzed by two-way ANOVA with Dunnett\u0026rsquo;s multiple comparison tests. Motor learning was analyzed by linear regression. The false positive rate (a) was set at 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Ataxia of Charlevoix-Saguenay Foundation. Additional support was provided by R21 AG080472-01 and the Simons Foundation Autism Research Initiative (SFARI).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eRAM and SS conceptualized the study. CC, RAM, and CJJ performed experiments, and CC and SS prepared the figures and wrote the manuscript. All authors read, edited, and approved the final manuscript before submission. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe authors declare no competing interests. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression data (Figure 1) are available from the Genotype-Tissue Expression (GTEx) Portal at gtexportal.org. All other data (Excel spreadsheets, Prism files, movies) can be obtained from the corresponding author upon request. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge technical assistance by Yufang Kong, Kathleen Kelley, Gail Healy, and Marisol Lauffer. Many of the experiments were conducted at the Neural Circuits and Behavior Core of the Iowa Neuroscience Institute. Metabolomics data were obtained with the assistance of the U. Iowa Metabolomics Core Facility. \u003cem\u003eSacs\u003c/em\u003e KO mice were a kind gift of Bernard Brais, McGill (3), \u003cem\u003eAkap1\u003c/em\u003e KO mice were donated by G. Stanley McKnight, U. Washington (29), and Bβ2 KO mice were generated at the U. Iowa Mouse KO Core Facility. The data used for Figure 1 in this manuscript were obtained from the GTEx Portal on 3/8/24. The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAly KA, Moutaoufik MT, Zilocchi M, Phanse S, Babu M. Insights into SACS pathological attributes in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)☆. Curr Opin Chem Biol. 2022;71:102211.\u003c/li\u003e\n\u003cli\u003eBagaria J, Bagyinszky E, An SSA. Genetics of Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) and Role of Sacsin in Neurodegeneration. Int J Mol Sci. 2022;23(1).\u003c/li\u003e\n\u003cli\u003eLariviere R, Gaudet R, Gentil BJ, Girard M, Conte TC, Minotti S, et al. Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay. Human molecular genetics. 2015;24(3):727-39.\u003c/li\u003e\n\u003cli\u003eGirard M, Lariviere R, Parfitt DA, Deane EC, Gaudet R, Nossova N, et al. Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). Proceedings of the National Academy of Sciences of the United States of America. 2012;109(5):1661-6.\u003c/li\u003e\n\u003cli\u003eBradshaw TY, Romano LE, Duncan EJ, Nethisinghe S, Abeti R, Michael GJ, et al. A reduction in Drp1 mediated fission compromises mitochondrial health in autosomal recessive spastic ataxia of Charlevoix Saguenay. Human molecular genetics. 2016.\u003c/li\u003e\n\u003cli\u003eDuncan EJ, Lariviere R, Bradshaw TY, Longo F, Sgarioto N, Hayes MJ, et al. Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin. Human molecular genetics. 2017;26(16):3130-43.\u003c/li\u003e\n\u003cli\u003eFlippo KH, Strack S. 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Deletion of a Neuronal Drp1 Activator Protects against Cerebral Ischemia. J Neurosci. 2020;40(15):3119-29.\u003c/li\u003e\n\u003cli\u003eHolmes SE, O\u0026apos;Hearn E, Cortez-Apreza N, Hwang HS, Ross CA, Strack S, et al. Spinocerebellar ataxia 12 (SCA12). In: Wells R, Ashizawa T, editors. Genetic Instabilities and Neurologic Diseases: Academic Press; 2006. p. 461-73.\u003c/li\u003e\n\u003cli\u003eMerrill MA, Slupe AM, Strack S. Spinocerebellar Ataxia type 12 (SCA12): clinical features and pathogenic mechanisms. In: Storey E, editor. Spinocerebellar Ataxias: InTech (Rijeka, Croatia); 2011.\u003c/li\u003e\n\u003cli\u003eMerrill RA, Dagda RK, Dickey AS, Cribbs JT, Green SH, Usachev YM, et al. Mechanism of Neuroprotective Mitochondrial Remodeling by PKA/AKAP1. PLoS Biology. 2011;9(4):e1000612.\u003c/li\u003e\n\u003cli\u003eMerrill RA, Strack S. Mitochondria: A kinase anchoring protein 1, a signaling platform for mitochondrial form and function. Int J Biochem Cell Biol. 2014;48:92-6.\u003c/li\u003e\n\u003cli\u003eFlippo KH, Gnanasekaran A, Perkins GA, Ajmal A, Merrill RA, Dickey AS, et al. AKAP1 Protects from Cerebral Ischemic Stroke by Inhibiting Drp1-Dependent Mitochondrial Fission. J Neurosci. 2018;38(38):8233-42.\u003c/li\u003e\n\u003cli\u003eGuo DF, Merrill RA, Qian L, Hsu Y, Zhang Q, Lin Z, et al. The BBSome regulates mitochondria dynamics and function. Mol Metab. 2023;67:101654.\u003c/li\u003e\n\u003cli\u003eBattaglini M, Carmignani A, Martinelli C, Colica J, Marino A, Doccini S, et al. In vitro study of polydopamine nanoparticles as protective antioxidant agents in fibroblasts derived from ARSACS patients. Biomater Sci. 2022;10(14):3770-92.\u003c/li\u003e\n\u003cli\u003eMartinelli C, Battaglini M, Pucci C, Gioi S, Caracci C, Macaluso G, et al. Development of Nanostructured Lipid Carriers for the Delivery of Idebenone in Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay. ACS Omega. 2020;5(21):12451-66.\u003c/li\u003e\n\u003cli\u003eCriscuolo C, Procaccini C, Meschini MC, Cianflone A, Carbone R, Doccini S, et al. Powerhouse failure and oxidative damage in autosomal recessive spastic ataxia of Charlevoix-Saguenay. J Neurol. 2015;262(12):2755-63.\u003c/li\u003e\n\u003cli\u003eMarquez BT, Leung TCS, Hui J, Charron F, McKinney RA, Watt AJ. A mitochondrial-targeted antioxidant (MitoQ) improves motor coordination and reduces Purkinje cell death in a mouse model of ARSACS. Neurobiol Dis. 2023;183:106157.\u003c/li\u003e\n\u003cli\u003eNopoulos PC, Ceilley JW, Gailis EA, Andreasen NC. An MRI study of cerebellar vermis morphology in patients with schizophrenia: evidence in support of the cognitive dysmetria concept. Biol Psychiatry. 1999;46(5):703-11.\u003c/li\u003e\n\u003cli\u003eKim JJ, Andreasen NC, O\u0026apos;Leary DS, Wiser AK, Ponto LL, Watkins GL, et al. Direct comparison of the neural substrates of recognition memory for words and faces. Brain. 1999;122 ( Pt 6):1069-83.\u003c/li\u003e\n\u003cli\u003eAndreasen NC, O\u0026apos;Leary DS, Paradiso S, Cizadlo T, Arndt S, Watkins GL, et al. The cerebellum plays a role in conscious episodic memory retrieval. Hum Brain Mapp. 1999;8(4):226-34.\u003c/li\u003e\n\u003cli\u003eSchmahmann JD, Sherman JC. Cerebellar cognitive affective syndrome. Int Rev Neurobiol. 1997;41:433-40.\u003c/li\u003e\n\u003cli\u003eTavano A, Grasso R, Gagliardi C, Triulzi F, Bresolin N, Fabbro F, et al. Disorders of cognitive and affective development in cerebellar malformations. Brain. 2007;130(Pt 10):2646-60.\u003c/li\u003e\n\u003cli\u003eVerhoeven WM, Egger JI, Ahmed AI, Kremer BP, Vermeer S, van de Warrenburg BP. Cerebellar cognitive affective syndrome and autosomal recessive spastic ataxia of charlevoix-saguenay: a report of two male sibs. Psychopathology. 2012;45(3):193-9.\u003c/li\u003e\n\u003cli\u003eTremblay M, Girard-Cote L, Brais B, Gagnon C. Documenting manifestations and impacts of autosomal recessive spastic ataxia of Charlevoix-Saguenay to develop patient-reported outcome. Orphanet J Rare Dis. 2022;17(1):369.\u003c/li\u003e\n\u003cli\u003eNewhall KJ, Criniti AR, Cheah CS, Smith KC, Kafer KE, Burkart AD, et al. Dynamic anchoring of PKA is essential during oocyte maturation. Curr Biol. 2006;16(3):a321-7.\u003c/li\u003e\n\u003cli\u003eLuong TN, Carlisle HJ, Southwell A, Patterson PH. Assessment of motor balance and coordination in mice using the balance beam. J Vis Exp. 2011(49).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-cerebellum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cere","sideBox":"Learn more about [The Cerebellum](http://link.springer.com/journal/12311)","snPcode":"12311","submissionUrl":"https://submission.nature.com/new-submission/12311/3","title":"The Cerebellum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ARSACS, ataxia, mitochondrial dynamics, dynamin-related protein 1, protein phosphatase 2A, A kinase anchoring protein","lastPublishedDoi":"10.21203/rs.3.rs-4178088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4178088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by loss-of-function mutation in the \u003cem\u003eSACS\u003c/em\u003e gene, which encodes sacsin, a putative HSP70-HSP90 co-chaperone. Previous studies with \u003cem\u003eSacs\u003c/em\u003e knock-out (KO) mice and patient-derived fibroblasts suggested that \u003cem\u003eSACSIN\u003c/em\u003e 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 (\u003cem\u003eB\u003c/em\u003eβ\u003cem\u003e2\u003c/em\u003e KO) nor fission (\u003cem\u003eAkap1\u003c/em\u003e KO) influenced progression of motor symptoms in \u003cem\u003eSacs\u003c/em\u003e KO mice. However, our studies identified profound learning and memory deficits in aged \u003cem\u003eSacs\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Driving mitochondrial fission improves cognitive, but not motor deficits in a mouse model of Ataxia of Charlevoix-Saguenay","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 18:29:03","doi":"10.21203/rs.3.rs-4178088/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-18T16:23:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-18T01:19:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c1cc91b6-9162-4428-a247-3c8010dcf6c2","date":"2024-04-07T21:31:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-05T17:21:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-05T13:25:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-05T13:25:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Cerebellum","date":"2024-03-27T19:43:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-cerebellum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cere","sideBox":"Learn more about [The Cerebellum](http://link.springer.com/journal/12311)","snPcode":"12311","submissionUrl":"https://submission.nature.com/new-submission/12311/3","title":"The Cerebellum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"26da2481-6ed0-412a-acba-9703c2638ab9","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T17:43:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 18:29:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4178088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4178088","identity":"rs-4178088","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}