Long-Term Benefits of TUDCA Supplement in ARSACS Zebrafish Model

Long-Term Benefits of TUDCA Supplement in ARSACS Zebrafish Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Long-Term Benefits of TUDCA Supplement in ARSACS Zebrafish Model Valentina Naef, Stefania Della Vecchia, Michela Giacich, Rosario Licitra, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5957432/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS ) is an early-onset neurodevelopmental and neurodegenerative disorder characterized by ataxia, spasticity, and peripheral neuropathy. However, several studies have highlighted that some patients also experience cognitive, emotional and social deficits, suggesting a more complex clinical picture that extends beyond motor symptoms. Building on these findings, this study aimed to: i) investigate locomotor, social and cognitive deficits in adult sacs -/- zebrafish versus wild-type (WT) controls through behavioural tests; ii) identify molecular patterns associated with the adult disease phenotype using transcriptomic and proteomic analyses of sacs -/- and WT brains; iii) evaluate the effectiveness of long-term treatment with tauroursodeoxycholic acid (TUDCA) on behavioural outcomes and omics profiles in the zebrafish sacs -/- model. Our findings indicate impairments in cognitive, social, and emotional behaviors in aged sacs -/- zebrafish, which resemble some deficits observed in human patients. Transcriptomic and proteomic analyses of adult brains identified alterations in genes related to circadian rhythms and neuroinflammation. Notably, disruptions in sleep and circadian rhythms are frequently reported in individuals with cerebellar ataxia and may contribute to cognitive and behavioral changes. Long-term treatment with TUDCA, a neuroprotective molecule, was associated with partial improvements in social and cognitive behaviors and modifications in omics profiles in the zebrafish model. These results support the potential of further exploring TUDCA in future preclinical and clinical studies, while also emphasizing the need for additional investigations to better understand its mechanisms of action. Biological sciences/Drug discovery Biological sciences/Molecular biology Biological sciences/Neuroscience Health sciences/Diseases ARSACS Ataxia Zebrafish Neurodegeneration TUDCA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Autosomal recessive spastic ataxia of Charlevoix–Saguenay (ARSACS) is a distinct form of hereditary early-onset spastic ataxia characterized by progressive degeneration of the cerebellum and spinal cord due to mutations in the SACS gene 1 The SACS gene encodes sacsin, a large multidomain protein with a molecular weight of 520 kDa 2 . Sacsin is among the largest proteins encoded by the human genome, though its complex multidomain structure has only been partially characterized 3,4 . It is believed that sacsin plays a role in protein quality control, which may influence both neurodevelopment and neurodegeneration. However, despite the identification of several structural domains, the precise function of sacsin and the pathophysiological consequences of its dysfunction remain largely uncharacterized. Alongside the motor symptoms, which typically include the triad of ataxia, spasticity and peripheral neuropathy, some individuals with ARSACS exhibit intellectual disabilities and behavioural abnormalities 5 . Cerebellar defects resemble the features of cerebellar cognitive-affective syndrome (CCAS), a condition characterized by deficits in executive functioning, spatial cognition, language abilities and emotional regulation 1,6,7 . Mouse models of ARSACS have been developed, exhibiting progressive deficits in motor coordination that closely mirror the ataxia-like symptoms of the disease 8 . However, data on cognitive and behavioural aspects remain scarce. Recently, Chen and colleagues generated double knockout mice for Sacs /Bβ2 and Sacs /Akap1, to test behavioural modifications related to ARSACS, and observed a significant decline in cognitive abilities in older Sacs knock-out mice 9 . Their study provided the first experimental evidence linking ablation of the SACS gene not only to motor impairments but also to learning and memory deficits, laying the foundations for further investigation of this area 9 . Zebrafish possess a range of neurobehavioral traits that are translationally relevant. Zebrafish, are also increasingly recognized as suitable organisms for modelling neurodegenerative diseases, and thus as a valuable platform for exploring disease mechanisms and testing potential treatments 10 . Despite comprehensive definition of non-motor symptoms in patients with ARSACS, in model mice, and in the zebrafish R487Kfs*495 KO strain 11 , the pathophysiology and underlying mechanisms of the disease remain poorly understood. Although the cerebellum plays a role in the retrieval of episodic memory and in other cognitive tasks 12 , it is still unclear how it contributes to cognitive and affective deficits. Currently, there are no definitive treatments available for degenerative ataxia 13 , and options for addressing the progressive degeneration of Purkinje cells in patients are limited. The repurposing of FDA-approved drugs with the potential to act on multiple molecular targets is a promising avenue to explore at preclinical level. Aged mice treated with tauroursodeoxycholic acid (TUDCA) (300 mg/kg) exhibited enhanced energy expenditure, improved metabolic flexibility and better cognitive abilities 10 . Additionally, in a chronic Parkinson’s disease model exhibiting significant dopaminergic degeneration, pretreatment with TUDCA(500 mg/kg, intraperitoneally) protected against neuronal damage and mitigated the activation of microglia and astroglia 14 . On the basis of this previous evidence, we investigated whether dietary supplementation with TUDCA could improve the phenotype of our published zebrafish model of ARSACS 11,15 . We showed that TUDCA significantly improved motor coordination, social interaction and cognitive impairments in this model. We found that TUDCA influences omics profiles in our zebrafish model. Transcriptomic and proteomic analyses suggest that TUDCA may modulate key cellular processes, including stress response, protein quality control, membrane stability, inflammation, and cytoskeletal dynamics. TUDCA could have potential benefits in improving locomotor and cognitive functions while also affecting molecular pathways related to neuroinflammation and cellular homeostasis. However, further studies will be necessary to validate these findings and better characterize the metabolic and functional effects of TUDCA. Overall, this study supports the need for further investigation into TUDCA’s potential therapeutic effects. RESULTS 3.1 TUDCA improves early survival in sacs -/- zebrafish without affecting growth or morphology ARSACS is an incurable condition that requires ongoing research to identify potential treatments and interventions. As a proof of concept, we tested a diet supplemented with TUDCA in sacs -/- zebrafish ( Fig.1A ). This experimental diet was based on this compound’s previously documented neuroprotective and anti-inflammatory properties 16 . During the first month of life—a critical period typically characterized by high mortality in our ARSACS model—we observed a significant improvement in survival rates. Notably, after this critical window, survival stabilized over time ( Fig. 1B ). To exclude potential metabolic side effects, particularly since TUDCA had not previously been tested in zebrafish over such a long period before, we assessed the fish at three months of age. No significant morphological abnormalities or changes in body weight were observed when comparing the sacs -/- mutants to the TUDCA-treated sacs -/- group. TUDCA treatment seemed to ameliorate the growth of the untreated mutants. ( Fig. 1C ). 3.2 TUDCA treatment enhances locomotor activity and reduces anxiety in sacs -/- zebrafish At the larval stages, we found that the sacs -/- zebrafish exhibited symptoms resembling motor impairments observed in humans with ARSACS 15 . Using the novel tank diving test ( Fig. 2B )., we observed that one-year-old sacs -/- zebrafish, similar to Sacs -/- mice 17 , displayed reduced speed of movement and impaired motor control ( Fig. 2B ). Active behaviour driven by the innate motivation to explore unfamiliar surroundings constitutes a typical normal response to a novel environment 18 , and is a trait evolutionarily conserved across many species 19 . However, adult sacs -/- zebrafish exhibited anxiety, a passive response characterized by a prolonged time spent in the lower half of the tank, and thus reduced exploratory activity ( Fig. 2C ), which suggested a decline in their ability or motivation to explore new environments. To further assess anxiety-related behavior, we employed the open-field test ( Fig. 2D ), which provides complementary information to the novel tank diving test by specifically evaluating thigmotaxis (or “wall-hugging”) 19 . In this test, we analysed the exploratory behaviour of adult sacs -/- zebrafish observing that TUDCA-treated sacs -/- zebrafish showed a moderately enhanced exploratory and locomotor response compared with untreated sacs -/- specimens ( Fig. 2E). Furthermore, we compared the time spent in the inner zone (centre) versus the outer zone of the tank 19 ( Fig. 2F) . Wild-type fish habituated more quickly to the environment and exhibited reduced thigmotaxis, indicative of lower anxiety levels. In contrast, adult sacs -/- zebrafish were less inclined to venture away from the safety of the edges of the tank and demonstrated heightened anxiety ( Fig. 2F ). Notably, while TUDCA supplementation seemed to partially rescued the anxiety phenotype, its effects appeared stronger in the novel tank diving test than in the open-field test. This discrepancy may stem from differences in the nature of the two behavioral paradigms. Indeed, the novel tank diving test primarily assesses the acute stress response to a novel environment, with zebrafish typically exhibiting an initial preference for the bottom of the tank before gradually exploring the upper zones. In contrast, the open-field test evaluates thigmotaxis, a behavior driven by long-term anxiety levels and risk assessment, where increased center avoidance indicates heightened anxiety. These findings suggest that TUDCA treatment may potentially lead to partial recovery of both motor and non-motor symptoms in the sacs -/- model. It is speculative to hypothesize that the stronger effect observed in the novel tank diving test may indicate that TUDCA primarily improves stress reactivity and exploratory drive rather than directly modulating chronic anxiety-related behaviors. 3.3 TUDCA ameliorates social deficits in sacs -/- zebrafish Beyond its role in motor symptoms, recent studies have also highlighted involvement of the cerebellum in cognitive, emotional and social functions 6 . Patients with cerebellar ataxia may exhibit difficulties in social interactions and in understanding others’ emotions, which can impact personal relationships and overall quality of life 20 . Direct studies linking cerebellar degeneration to changes in social and cognitive behaviour in ARSACS patients are limited, although impairment of social skills and severe psychiatric symptoms have been reported in these patients 21,22 . Zebrafish are a highly social species that prefer to spend time in proximity to conspecifics 23 . Their shoaling behaviour serves several adaptive functions, providing protection from predators for example, as well as increasing foraging efficiency and mating success 23 . Interestingly, cerebellar circuits in zebrafish also play an important role in social orienting behaviour 24 . To explore the effect of loss of sacsin on social behaviours, we performed a shoaling test to compare social behaviour between homogeneous groups of zebrafish 25 . In a novel tank, stressed fish tend to swim closer together, maintaining smaller inter-fish distances than non-stressed fish 25 . Indeed, tighter shoals indicate higher anxiety 26 . We measured the average inter-fish distance and found that adult sacs -/- zebrafish, compared with WT fish, appeared more stressed, swimming closer together (inter-fish distance of < 6 cm), a trait that reduced their exploratory behaviour ( Fig. 3A-B) . However, TUDCA-treated sacs -/- fish showed greater inter-fish distances than their untreated sacs -/- counterparts, demonstrating less anxiety. The social preference and interaction tests were subsequently performed as already described 27 ( Fig. 3C-D ). During the habituation phase, we continued to observe increased exploratory behaviour in TUDCA-treated versus untreated sacs -/- fish. In the test phase, a group of four conspecific zebrafish was placed in the right side of the tank, and a single fish per experimental group was placed in the left side as reported in a previous study 20 . We observed that adult WT zebrafish generally contacted the group on the right side and spent more time in the conspecific sector than the empty sector, showing a strong group-forming tendency 27 . In contrast, sacs -/- zebrafish spent their time evenly throughout the tank, exhibiting reduced social contact with the peer group and a lower proportion of time in the conspecific sector ( Fig. 3C-D ). When treated with TUDCA, however, sacs -/- fish showed improved sociability ( Fig. 3C-D ). 3.4 TUDCA improves cognitive performance in the ARSACS zebrafish model Cognitive symptoms have been reported in some cases of ARSACS 5,28 and in the Sacs -KO mouse model 9 . Although fMRI studies indicate that the cerebellum participates in the recovery of episodic memory and other cognitive tasks, and CCAS 12 has been described in ARSACS 6,7,21 , it is still not clear how cerebellar disorders impair cognition. Furthermore, most of the genes associated with cerebellar ataxia diseases, including SACS , are ubiquitously expressed, and brain atrophy commonly follows cerebellar atrophy in hereditary ataxias 29 . Since zebrafish can be used to model complex human behavioural traits such as reward responsiveness, learning, and memory 30 , we took advantage of this characteristic to investigate the potential presence of cognitive impairments in sacs -/- mutants . We performed the novel object recognition (NOR) test and analysed the time spent by the animals exploring objects 30 in the open-field test apparatus. The NOR test leverages animals’ naturally greater tendency to explore novel objects over familiar ones, and thus explores their curiosity and memory capabilities 31 . Zebrafish possess recognition memory for simple 2- and 3-dimensional geometrical shapes 32 . We placed adult fish in a tank containing two identical blue cubes ( Fig. 4A ) and allowed them to explore freely for a set amount of time, becoming familiar with the objects present (phase 1, Fig. 4A-B ). After training, the animals were submitted to a retention interval of 1 h, then, we put the fish back in same tank, in which we had placed one of the familiar blue objects and a novel object (red cube), and evaluated the amount of time they spent exploring the novel object compared with the familiar one (phase 2, Fig. 4A-4B ), which is an indicator of their recognition memory 30,31 . In the training session (phase 1), no preference between the two identical blue cubes was observed. In the test session (phase 2) we found that adult WT fish spent more time exploring the novel object, a result indicating memory retention as previously reported 32 , whereas adult sacs -/- fish did not show a preference between the familiar and novel object (Fig. 4B-C) . However, when treated with TUDCA, adult sacs -/- zebrafish showed a significant preference for the new object compared with the familiar object (****p < 0.0001), which suggests an improvement in their cognitive performance. 3.5 Key insights from multiomics analyses in the zebrafish ARSACS model Understanding early cellular stresses and altered pathways in the brain that may contribute to the onset of ARSACS is crucial for developing preventative treatments. However, obtaining these insights through studies in living humans is challenging. We performed transcriptome analysis comparing whole brains from 12-month-old WT and homozygous sacs -mutant fish. 527 genes were differentially expressed in the entire brains of the sacs -/- group compared with the WT group ( Supplementary File 1 ). 213 genes showed increased expression and 314 showed decreased expression. Gene ontology (GO) and protein-protein interaction (PPI) analyses revealed enrichment of genes associated with circadian rhythms that have not been reported in other ARSACS models ( Fig. 5A-B ). Therefore, we performed gene set enrichment analyses (GSEA) to predict which cellular processes were affected. These analyses identified several pathways related to biological processes such as neuroinflammation and response to oxidative stress (Fig. 6A) . PPI analyses revealed downregulation of several proteins related to mitochondrial activity and oxidative phosphorylation ( Fig. 6B ), consistent with mitochondrial dysfunction as reported in the literature 33–35 . The overlap between our results and those reported in the literature further supports the potential of our model in modelling ARSACS pathology. To gain a more comprehensive understanding of the molecular alterations in our model and evaluate potential downstream effects at the protein level, we performed proteomic analysis on adult brains. This analysis revealed a complex network of upregulated and downregulated proteins, which could indicate potential neurodegenerative molecular changes in sacs -/- adult zebrafish, which could be further explored in future studies. In Tables 1-2, we highlighted significant proteins associated with biological processes that are altered in ARSACS pathology in others model, as previously described in 36 . In particular, we found an imbalance in key processes such as Mitochondrial Function , Oxidative Stress , Neuroinflammation , ER Stress and Synaptic Signalling (Tables 1-2) that, in combination with alterations in circadian patterns, could exacerbate neuronal damage, leading to a cycle of chronic stress and inflammation in the brain 37 . Notably, these same pathways have also emerged in previous in vitro studies in ARSACS primary cells, further supporting their relevance to disease pathophysiology 38 . Table 1: List of upregulated proteins identified through proteomics analysis in the brains of adult sacs -/- zebrafish compared with wild-type (WT) counterparts. GENE CATEGORY FUNCTION REF MT2 Oxidative Stress Anti-inflammatory, and anti-apoptotic agent 39,40 FLAD1 Oxidative Stress Cofactor in redox reactions 41,42 UOX Oxidative Stress Involved in the urate catabolic process, an antioxidant pathway reducing oxidative damage 43 MRPS15 Oxidative Stress Involved in pathways related to mitochondrial translation and protein metabolism 44,45 VPS18 ER Stress Involved in protein trafficking and lysosomal degradation 46 CYP22K6 Oxidative Stress Involved in oxidative metabolism 47 TSR2 ER Stress Involved in apoptosis 48 TRNT1 ER Stress Linked to ER stress and increased oxidative stress 49,50 TBCE ER Stress Involved in microtubule cytoskeleton organization 51 CRP Neuroinflammation Marker of neuroinflammation and peripheral inflammation 52 FABP10A Neuroinflammation Involved in fatty acid transport 53,54 Table 2 . List of downregulated proteins identified through proteomic analysis in the brains of adult sacs -/- zebrafish compared with wild-type (WT) counterparts. GENE CATEGORY FUNCTION REF MT-ND2 Mitochondrial Function Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) 55 MT-CO1 Mitochondrial Function Part of respiratory chain complex IV, involved in ATP synthesis coupled electron transport 56 MCTS1 Cell Cycle Regulation and Apoptosis Involved in the regulation of various processes, including cell cycle modulation and apoptosis 57 B2M Immune Regulation Subunit of major histocompatibility complex (MHC) class I 58,59 OPTN Neuroinflammation & Cell Death Involved in various vesicular trafficking pathways 60 HPX Oxidative Stress Involved in protecting cells from oxidative stress 61 TRAPPC2 Intracellular Vesicle Trafficking Involved in the targeting and fusion of endoplasmic reticulum-to-Golgi transport vesicles 62,63 DYNC1I2A Retrograde Transport Acts as a retrograde microtubule motor to transport organelles and vesicles 64 3.6. TUDCA modulates gene expression and protein regulation in the ARSACS zebrafish model To gain a better understanding of diet-induced changes in whole-brain gene expression, we performed RNA-seq analysis. A comparative analysis between sacs -/- and TUDCA-treated sacs -/- zebrafish indicated that dietary supplementation with TUDCA may be associated with modifications in gene expression signatures related to circadian rhythm and neuroinflammatory pathways. As reported in the literature, we found that TUDCA may play a multifaceted role in supporting metabolic health. In particular, as previously reported, it appears to influence cholesterol and fat metabolism, reducing the ER Stress 65 that occurs when misfolded proteins accumulate. Of 817 genes differentially expressed in the whole brains of the TUDCA-treated sacs -/- group compared with the untreated sacs -/- fish, 422 showed increased expression, while 395 exhibited decreased expression ( Supplementary file 2 ). GO analysis revealed differentially expressed genes mainly related to Developmental Growth, Lipid Metabolism and Intermediate Filament Cytoskeleton Organization ( Fig. 7A ) . GSEA revealed an enrichment of genes related to categories such as Lipid Metabolism and Oxidative Phosphorylation ( Fig. 7B ) . PPI analysis revealed 150 upregulated genes that are functionally related to Lipid Metabolism (highlighted in red, Fig. 8A ), Oxidative Phosphorylation (highlighted in green, Fig. 8A ) or Neurotransmitter and Synaptic Transmission (highlighted in blue, Fig. 8A ). Oxidative activity/removal of superoxide radical (highlighted in green, Fig. 8B ), cell redox homeostasis (highlighted in pink/yellow, Fig. 8B ), mitochondrion organization (highlighted in red, Fig. 8B ), which may indicate a protective and adaptive effect aimed at maintaining redox homeostasis and preventing cellular damage after TUDCA treatment. These results could support the ability of TUDCA to mitigate oxidative stress in our ARSACS model, as previously described 16 . We also compared the transcriptomic profile of TUDCA-treated sacs -/- zebrafish with that of WT fish ( Supplementary File 3 ). This analysis indicated that the transcriptomic profile of treated mutants did not fully overlap with that of WT fish, suggesting that while TUDCA may ameliorate some molecular alterations, it does not completely restore the WT-like state. Specifically, GSEA analysis identified enrichment in pathways related to biological processes such as developmental growth , connective tissue development , tissue regeneration , and cytoskeletal organization ( Supplementary Figure 1A ). PPI analysis further revealed an upregulation of genes associated with cell cycle regulation , cytoskeletal organization , chromatin remodelling , and neurodevelopmental processes . These changes could reflect a compensatory response aimed at mitigating aspects of neurodegeneration ( Supplementary Figure 1B ). Additionally, we observed higher expression of genes related to mitophagy , which aligns with the proposed role of TUDCA in promoting mitochondrial quality control ( Supplementary Figure 1B ). In contrast, immune-related pathways, including cytokine production and immune response signaling , were downregulated, suggesting a potential anti-inflammatory effect of TUDCA ( Supplementary Figure 2A ). Interestingly, we also identified alterations in RNA processing and splicing pathways , which may indicate that certain gene regulatory mechanisms remain dysregulated despite TUDCA treatment ( Supplementary Figure 2B). These data could suggest that, while TUDCA treatment ameliorates some molecular deficits, it does not fully restore the WT-like transcriptional state, highlighting both its therapeutic potential and its limitations in completely reversing ARSACS-associated dysregulations. Additionally, to evaluate downstream effects at the protein level we analysed proteomic data. We identified differentially expressed proteins related to several processes such as Autophagy and Lysosomal Function , Protein Degradation , Oxidative Stress Response , Lipid Transport and Metabolism. The upregulation of proteins related to processes like Synaptic Integrity and Synaptic Plasticity , such as SCRIB and CNTN5, is intriguing ( Table 3 ). Table 3 . List of upregulated proteins identified through proteomic analysis in the brains of TUDCA-treated vs. untreated sacs -/- zebrafish. GENE CATEGORY FUNCTION REF LAMP5 Protein Trafficking Involved in establishment of protein localization to organelle 66 USP46 Protein Degradation Involved in protein deubiquitination and regulation of GABAergic synaptic transmission 67 SCRIB Synaptic Stability Involved in neuronal stability and synaptic integrity, maintaining neural connections even under neurodegenerative stress 68,69 CNTN5 Synaptic Plasticity Involved in the formation of axon connections in the developing nervous system 70,71 HYCC1 Myelination Involved in neuron-to-glia signalling to initiate or maintain myelination 72 CLPTM1L Lipid Metabolism A scramblase that moves GlcN-PI across the ER membrane, aiding glycosylphosphatidylinositol (GPI) biosynthesis, which is essential for protein post-translational modification 73 TMEM11 Mitochondrial Function Localizes to the outer mitochondrial membrane where it regulates BNIP3/BNIP3L-dependent receptor-mediated mitophagy 74 NXN Antioxidant Defence Member of the thioredoxin superfamily, a group of small, multifunctional redox-active proteins 75 PTDSS1 Cell Signalling A phospholipid found in membranes that plays a role in various cellular functions, including development, cell communication, programmed cell death 76 These changes suggest a putative improvement in brain function in our ARSACS model, leading to an enhancement of behaviours related to learning, memory and social interaction. Downregulation of immune-related proteins like B2M and IFI30 suggest a possible dampening of inflammatory responses, in accordance with TUDCA’s known anti-inflammatory properties 16 . Additionally, the level of reduction of proteins related to the ubiquitin-proteasome system, such as MARCHF2, demonstrates the potential of TUDCA to reduce proteotoxic stress, likely by stabilizing protein folding and minimizing protein accumulation 77,78 ( Table 4 ). These results suggest that TUDCA could exert neuroprotective effects through multiple cellular pathways, potentially slowing down disease progression and improving motor and cognitive function in our model. However, to confirm these speculative findings, further studies will be necessary to validate the obtained results. Table 4 . List of downregulated proteins identified through proteomic analysis in the brains of TUDCA-treated vs. untreated adult sacs -/- zebrafish. GENE CATEGORY FUNCTION REF B2M Immune Regulation An important subunit of major histocompatibility complex (MHC) class I Downregulation may impair immune response. 79 AGR2 Protein Folding A member of the disulfide isomerase (PDI) family of proteins found in the endoplasmic reticulum 80 MTTP Lipid Metabolism This protein is crucial for lipoprotein formation. Putative inhibitor of ferroptosis 81 VPS25 Protein Sorting Component of ESCRT-I. Selectively modulates FGF signalling by directing receptor sorting through endosomes 82 MARCHF2 Ubiquitination Member of the MARCH family of membrane-bound E3 ubiquitin ligases 83,84 ARPIN Cytoskeleton and Cell Motility Involved in directional locomotion, it regulates actin filament dynamics. 85 FABP10A Lipid Transporter Involved in intracellular binding and trafficking of long fatty acids in the liver 53,54 IFI30 Inflammation Involved in antigen processing 86 DISCUSSION Beyond the motor dysfunction caused by the progressive cerebellar degeneration, some patients with ARSACS disease exhibit cognitive impairment and behavioural problems 5 including apathy, dysphoria, paranoid thoughts, irritability, and significant cognitive impairment 87 . Several research has demonstrated that cerebellar damage often leads to cognitive deficits and to affective problems, thereby highlighting the cerebellum’s significant role in cognitive and emotional processes 88 , so that the concept of CCAS-like has been proposed to describe cognitive and emotional alterations linked to cerebellar dysfunction in some instances 6,89 .Although the cognitive and affective phenotype observed in ARSACS patients is complex and goes beyond the definition of CCAS, cerebellar dysfunction may contribute to its development 21,22 . In line with findings in Sacs -KO mice 9 , we observed that loss of sacsin in zebrafish not only affects locomotor activity but also leads to anxiety-like behaviour, social impairment and deficits in object recognition memory. Our findings further strengthen the evidence that loss of sacsin is linked to significant cognitive deficits that become apparent in adulthood. These results reflect the multifaceted role of sacsin in maintaining neural function and suggest that its absence may disrupt critical pathways involved in cognitive processing. The cognitive impairments observed fit into the broader spectrum of neurological abnormalities associated with sacsin deficiency, highlighting the need for continued research to unravel the underlying mechanisms. Our second focus, through transcriptomic and proteomic analyses, was to identify molecular pathways that may be altered in adult sacs -/- fish compared with their WT counterparts. This approach aimed to provide deeper insights into the neurobiology of neurodegeneration in our ARSACS model. Dysregulation of the mitochondrial fission enzyme is recognized as a primary driver of the disease; however, since this is a neurodegenerative disorder, other molecular pathways and mechanisms likely contribute simultaneously, worsening both motor and behavioural symptoms and further aggravating the overall progression. In this study, we provide novel insights into the molecular mechanisms underlying ARSACS and their contribution to disease progression. Through RNA transcriptomic analysis of 1-year-old sacs-/- zebrafish brains, we identified significant alterations in the expression of circadian rhythm-related factors, such as PER and CRY family genes, as well as other genes related to neuroinflammation. Circadian rhythms direct a wide range of physiological functions, and alterations of them directly affect human health 90 . On the other hand, neuroinflammation could be a key player in the progression of ARSACS. Notably, several studies have found that alteration of the circadian cycle is closely related to the body’s inflammatory response, and in the context of neurodegenerative disorders creates a feedback loop that may exacerbate neurodegenerative progression 91 . Immune cells in humans and animals, including microglia, neutrophils, monocytes and lymphocytes, could express clock genes. Thus, circadian cycle alterations may be an important factor mediating central and peripheral inflammatory responses 91 . Disruption of circadian rhythms and neuroinflammation might contribute to neurodegeneration and to clinical manifestations like cognitive impairment 92,93 as observed in our sacs -/- zebrafish model. Proteomic analyses highlighted the interplay occurring between upregulated and downregulated proteins in the brains of 1-year-old sacs -/- zebrafish compared with those of WT specimens, which could suggests a dynamic attempt by the neurons to modulate oxidative stress and neurodegeneration, acting on neuroinflammation and synaptic communication. Downregulation of key proteins such as MT-CO1 and HPX worsens the neurons’ ability to manage damage. For instance, low levels of HPX, which plays a critical role in iron homeostasis 61 , can exacerbate oxidative stress and worsen mitochondrial dysfunction. Furthermore, downregulation of TRAPPC2 may lead to Golgi fragmentation and arrest of anterograde trafficking, thereby impairing Golgi function 63 . The increased presence of pro-inflammatory proteins like CRP and OPTN could suggests a chronic inflammatory state. Although the proteomic analyses did not identify proteins directly involved in the regulation of circadian rhythms such as the core clock players (e.g., CLOCK, BMAL1, PER and CRY), several proteins may indirectly influence circadian rhythms. Circadian rhythms and mitochondrial proteins are known to be closely interconnected 94 , influencing protein levels and acetylation of mitochondrial genes, as well as mitochondrial morphology and oxidative phosphorylation 94 . Downregulation of mitochondrial-related genes such as MCTS1, MT-ND2 and MT-CO1 significantly impairs mitochondrial function leading to inefficient mitochondrial respiration, a decline in ATP production, and increased reactive oxygen species (ROS) generation, in turn leading to neurodegeneration 95–97 . Indeed, reduced energy production and accumulation of ROS can further alter circadian rhythms 98 . In this scenario, our study provides preliminary evidence suggesting a potential beneficial effect of long-term treatment with TUDCA, which has previously shown efficacy in other models of neurodegeneration such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease, acting as a mitochondrial stabilizer, anti-apoptotic agent, and anti-inflammatory compound 99–101 . One of the key mechanisms by which TUDCA works is by reducing ER stress 65 . In a model of chronic unpredictable stress-induced depression, administration of TUDCA was able to improve the anxiety-like and depressive-like behaviour driven by neuroinflammation, oxido-nitrosative stress and ER stress 102 . We administered a TUDCA-enriched diet to sacs -/- zebrafish from embryonic development to one year of age. Behavioural tests revealed partial but significant improvements in locomotor activity, social behaviour, and cognitive performance, highlighting TUDCA’s capacity to mitigate both motor and non-motor symptoms of ARSACS. Using a multi-omics approach, TUDCA appears to modulate key molecular pathways involved in the progression of ARSACS, particularly those related to circadian rhythms and neuroinflammation. Transcriptomic and proteomic analyses suggest potential neuroprotective effects of TUDCA, with proteomic data indicating its involvement in stress response, protein quality control, membrane stability, inflammation, and cytoskeletal dynamics. Notably, decreased levels of immune-related proteins like B2M and IFI30 suggest a dampening of inflammatory responses, consistent with TUDCA’s known anti-inflammatory properties 103 . In addition, the fatty acid-binding protein FABP10A, which was upregulated in sacs adult brains, is downregulated following TUDCA treatment. Given the role of FABPs in lipid metabolism and inflammatory processes 104 , this finding suggests that TUDCA may contribute to restoring lipid homeostasis, alleviating metabolic stress in sacs mutants. Considering the strong link between lipid dysregulation and neuroinflammation, the reduction in FABP10A expression may indicate a decrease in inflammatory signalling, highlighting a broader impact of TUDCA treatment on neuroinflammatory pathways. Furthermore, the downregulation of ubiquitin-proteasome-related proteins such as SSUH2 and MARCHF2 highlights its potential to alleviate proteotoxic stress by stabilizing protein folding and reducing aberrant protein accumulation. The increased expression of USP46, linked to reduced anxiety and depressive-like behaviours in mouse models 105,106 , further suggests that TUDCA may influence proteostasis, and could potentially ameliorate stress responses and cognitive functions. However, when comparing the transcriptomic profile of TUDCA-treated mutants with that of WT fish, we observed that it does not fully overlap with the WT-like transcriptional state. This suggests that while TUDCA ameliorates some molecular deficits, it does not completely restore gene expression patterns to WT levels. This aligns with our behavioral findings, where TUDCA treatment led to partial improvements in locomotor and anxiety-related phenotypes rather than complete normalization. The partial recovery may indicate that TUDCA effectively targets specific pathological mechanisms, such as neuroinflammation and mitochondrial dysfunction, while other molecular disruptions caused by sacsin loss remain unaddressed. Further studies are needed to validate these findings and to better understand the precise mechanisms underlying TUDCA’s effects. While the transcriptomic and proteomic analyses conducted in this study provide valuable new insights into the molecular alterations occurring in ARSACS, they do not directly establish causality between the observed changes and disease symptoms and additional functional studies will be required to confirm the specific roles of these altered pathways. In particular, the transcriptomic changes related to circadian rhythms and neuroinflammation should be validated using independent experimental approaches. Moreover, further investigations are necessary to elucidate the cellular and anatomical origins of the non-motor symptoms observed in ARSACS, which remain less well characterized compared to motor deficits. Gaining deeper insight into how these symptoms interact and evolve over time could help identify critical windows for therapeutic intervention and contribute to the development of more targeted treatment strategies. METHODS 3.1 Fish husbandry and feeding The adult sacs -/- zebrafish used in this study were generated through CRISPR/Cas9 genome editing in the wild-type AB background. These mutants carry a 10-bp deletion in exon 7 of the sacs gene, leading to a frameshift mutation and a premature stop codon at residue 495 (R487Kfs*495). This ensures that both WT and sacs -/- zebrafish share the same genetic lineage, minimizing the risk of background-related variability. The wild-type AB strain was obtained from the Department of Veterinary Sciences at the University of Pisa. The homozygous sacs -/- zebrafish mutant line was crossed, and the F2 progeny were used to investigate the adult phenotype. The control animals used in the study were from the same batch of offspring as the sacs -/- mutants and were genetically similar, except for the loss of function in the sacs gene. Animals were kept in automated re-circulating systems (Zebtec, Tecniplast, Italy) with reverse osmosis filtered water equilibrated to reach the species-recommended temperature (28° C ± 2° C), pH (7.0 and 7.5), conductivity and ammonia, nitrite, nitrate and chloride levels. Animals were subjected to a light/dark cycle of 14/10 hours. All experiments were conducted in accordance with the European Union (EU) Directive 2010/63/EU on the protection of animals used for scientific purposes, under the supervision of the Institutional Animal Care and Use Committee (IACUC) of the University of Pisa, and in compliance with the 3R principles. TUDCA (TUDCA, Millipore, Darmstadt, Germany ) supplementation was carried out by including the compound in the pelleted feed supplementation was carried out by including the compound in the pelleted feed (inclusion rate 0,1% on a fed basis).The purified drug was added to self-made experimental zebrafish feed according to published indications 107 . In brief, the dry feed was finely ground, mixed with osmosis water containing dissolved trehalose to form a dough, then re-pelleted, dried, and stored in the refrigerator until use 107 . In total, two different diets were prepared: 1 control diet and 1 experimental diet, the latter containing TUDCA acid and sodium salt. The decision to use a 0.1% concentration was based on preliminary dosage/effect experiments on mortality and growth in both WT fish and sacs mutant fish, evaluated within the first 30 days of life. The chosen dosage was found to be the most effective. The experimental diet was administered twice daily from 5 dpf until adulthood (12 months). Food was provided every day at the same time, with quantities adjusted according to the fish's age and body weight. Specifically, larvae were fed an amount equivalent to 9–10% of their body weight, juveniles received 6–8%, and adults were fed 5%, following recommendations for dry feed with appropriate protein and energy content, as described in 108 . Control fish were maintained under the same conditions as the treated fish but without exposure to any treatment. To exclude potential metabolic side effects, particularly since TUDCA had not previously been tested in zebrafish, we evaluated the fish at three months of age, as they are already considered adults at this stage. Welfare assessment was conducted following the guidelines outlined in the University of Queensland's “Score Sheet for Scoring Endpoints in Zebrafish ” (https://research-support.uq.edu.au/resources-and-support/ethics-integrity-and-compliance/animal-ethics/monitoring-animals), evaluating several aspects such as free swimming behavior in the tank, body weight, and abdominal swelling. The overall welfare status of the analyzed subjects was determined based on the final score. After completing the behavioral experiments, the fish were sacrificed, and organ dissection was performed on the same day. The extracted brains were preserved under identical conditions and subsequently processed as described in the following section. RNA-sequencing For transcriptomic analysis, 4 brains were isolated from adult animals per experimental group. RNA was extracted from individual samples to maintain biological variability according to standard procedures. All animals from each experimental group were sacrificed and harvested on the same day to ensure consistency and avoid batch effects. RNA integrity and concentration were assessed using the RNA 6000 Pico Kit on a Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA integrity number was between 5.6 and 8.1 for all samples. Due to partial sample degradation, a ribodepletion approach for RNA-seq analysis was employed. 50ng total-RNA was depleted with Danio rerio -specific rRNA probes using the dedicated ribo-Pool panel DP-R024-101 (SiTOOLS Biotech, Planegg, Germany). The depleted RNA was used to prepare the library using the SMARTer Stranded Total RNA-Seq kit v3 (Pico Input-Mammalian, Takara Bio, San Jose, CA). Quality and size of RNA-seq libraries were assessed by capillary electrophoretic analysis with an Agilent 4150 Tape station (Agilent Technologies). Libraries were quantified by real-time PCR against a standard curve with the KAPA Library Quantification Kit (KapaBiosystems, Wilmington, MA). Libraries were pooled at equimolar concentration and sequenced in 150PE on a NovaSeq6000 (Illumina, San Diego, CA), generating an average of 48.3 million fragments per sample. GO and KEGG pathway enrichment analysis of differentially expressed genes with padj < 0.05 were performed using ClusterProfiler (v3.18.1) [101]. Subsequently, the top 20 enriched GO biological process categories were plotted. GSEA were performed to determine the enrichment of specific gene sets retrieved from the Molecular Signatures Database (https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). 3.3 Proteomic analysis 3.3.1 Protein isolation For proteomic analysis, 4 brains were isolated from adult animals per experimental group. RNA was extracted from individual samples to maintain biological variability according to standard procedures. All animals from each experimental group were sacrificed and harvested on the same day to ensure consistency and avoid batch effects. Zebrafish brains were pottered and passed through a 70-μm diameter filter before undergoing a wash with PBS supplemented with protease inhibitors (20 g/ml leupeptin, 25 g/ml aprotinin, 10 g/ml pepstatin, 0.5 mM benzamide) and phosphatase inhibitor (1 mM Na3VO4, Merck, Darmstadt, Germany, Cat. No. 13721-39-6) followed by centrifugation at 4°C, 1500 rpm for 15 min. The pellet was then disrupted by RIPA buffer, incubated at 4°C for 60 min, vortexed four times (once every 15 min), and then sonicated for 30 s. Samples were centrifuged at 13,850 rcf for 10 min and supernatants were collected, measured, and supplemented with the same amount of 20% SDS-6% DTT. Subsequently, they were incubated at 95 °C for 5 min. Once cooled, five volumes of MATF (methanol, acetone, and tributyl phosphate, 1:12:1) were added, and samples were incubated for 1 h with agitation. The total proteins were finally pelleted by centrifugation at 12,000 rcf for 15 min, the supernatants were removed, and the protein precipitates dried for about 30 min at RT. The dried pellets were resuspended in 250 μL of 5% SDS in 50 mM ammonium bicarbonate. After quantification, proteins underwent reduction and alkylation followed by trypsin digestion by using Preomics iST (PreOmics, Billerica, MA). 3.3.2 Mass spectrometry Tryptic digests were analysed using an Ultimate 3000 chromatography system (Thermo Scientific Instruments, Waltham, MA, USA) equipped with a PepMap RSLC C18 EASY spray column (Thermo Scientific Instruments, Cat. No. 13294749) at a flow rate of 250 nl/min and a temperature of 60°C. Eluate was analysed by mass spectrometry (MS) using a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer equipped with an Easyspray source (both Thermo Scientific Instruments). The data were acquired, in the mass range of 200–2,000 m/z, in data-dependent (DDA) mode alternating between MS and MS/MS scans, using the software Xcalibur (version 4.1, Thermo Scientific Instruments). To perform MS analysis, the desiccated tryptic digests were resuspended with 1.5 mL of the LC-LOAD component of the iST-BCT kit (PreOmics), and analysed by nano-UHPLC-MS/MS using an Ultimate 3000 chromatography system equipped with a PepMap RSLC C18 EASY spray column (75 mm, 50 cm, 2 mm particle size) (both Thermo Fisher Scientific) at a flow rate of 250 nL/min and a temperature of 60°C. The mobile phases were: (A) 0.1% v/v formic acid in water, and (B) 80% acetonitrile, 20% water and 0.08% v/v formic acid. A 105-min gradient was selected: 0.0–3.0 min isocratic 2% B; 3.0–7.0 min 7% B; 7.0–65.0 min 30% B; 65.0–78.0 min 45% B; 78.0–83.0 min 80% B; 83.0–85.0 isocratic 80% B; 85.0–85.1 2% B; and finally, 85.1–105.0 isocratic 2% B. After separation, the eluate was sent directly to an Easyspray source connected to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Fisher Scientific). The data were acquired in DDA mode, alternating between MS and MS/MS scans. The software Xcalibur (version 4.1, Thermo Fisher Scientific) was used for operating the UHPLC/HR-MS. MS scans were acquired at a resolution of 70,000 between 200 and 2000 m/z, with an automatic gain control (AGC) target of 3.0 x 106 and a maximum injection time (maxIT) of 100 ms. MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 1.0 x 105 and a maxIT of 50 ms. A quadrupole isolation window of 2.0 m/z was used, and HCD was performed using 30% normalized collision energy. 3.3.4 Mass spectrometry data analysis Mass spectrometry data were processed with ProteomeDiscoverer® (v. 2.4.1.15) (Thermo Scientific Instrument, Waltham, MA) using a workflow adapted for LTQ ORBITRAP label-free quantification. Briefly, in the processing step we used the Danio rerio - tr_canonical v2022-03-02 database for identifying peptide spectral matches in MS/MS spectra and concatenated decoy (strict target false discovery rate = 0.01, relaxed target false discovery rate = 0.05 for proteins, peptides and peptide spectral matches). Protein quantification was calculated by summed abundances of peptides, and the differential analysis was performed pairwise ratio-based and t-test background-based using the IMP-apQuant node. After conducting a proteome differential analysis, differentially expressed proteins were identified for each experimental point. These were then used to perform further gene ontology analyses. Data were deposited in the PRIDE repository (PXD058522). 3.4 Behavioural analysis 3.4.1 Novel tank Zebrafish were exposed to the experimental challenge in a pre-treatment beaker before being transferred (via a net) to the novel tank for behavioural observation and phenotyping as previously reported 109,110 . After pre-treatment, zebrafish were placed individually in a 1.5-L trapezoidal tank maximally filled with aquarium water. In these experiments, behavioural activity was recorded for 5 min using a webcam set up in front of the tank to analyse the diving response. The tank was virtually divided into two areas (bottom and top) and time spent in the bottom part was used to assess anxiety-like phenotypes. Zebrafish behavioral activity was recorded (1920 × 1080 px). The videos were analysed using Noldus EthoVision® XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA). Open-field test and thigmotaxis Behavioural experiments were conducted at between 10 a.m. and 4 p.m. in a 30 × 30 × 30 cm tank with opaque walls and partitions. A video camera was suspended above the tank. Adult zebrafish were allowed to swim for 5 min inside the tank, and 5-min videos were recorded. Thigmotaxis was evaluated as reported elsewhere 111 . 3.4.2 Shoaling test Behavioural experiments were conducted at between 10 a.m. and 4 p.m. in a 30 × 30 × 30 cm tank with opaque walls and partitions. Adult zebrafish were acclimated to the novel tank apparatus for 5 min before the test, after which 5-min videos were recorded. The shoaling assessment was performed by measuring the inter-fish distance (the average of all distances between each zebrafish in a shoal). The method for measuring inter-fish distance was also based on the analysis provided by Noldus EthoVision® XT17 software. This software tracks the precise position of each fish in the arena over time, allowing for accurate inter-fish distance measurements. 3.4.3 Social preference test Social preference testing was performed with three chambers as already described 23 . In general, social behaviour is assessed by observing how an individual respond to, or interacts with, a social stimulus. Social preference tests are composed of two operational phases. The first is the habituation phase, during which the tested zebrafish is left alone in a chamber of the test tank to explore the novel environment. The second is the interaction phase, which starts with the introduction of the social stimulus consisting of one, or usually two, small groups of live conspecifics 23 . The zebrafish behaviours were quantified as distance distribution or as presence in a zone adjacent to the group of conspecifics. The time ratio [Time ratio %=Time spent in conspecific sector/ total time observed)×100]. The distance ratio [(Distance ratio %=Time spent in conspecific sector/ total time observed) ×100]. The videos were analysed using Noldus EthoVision® XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA). 3.4.4 Novel object recognition task Behavioural experiments were conducted at between 10 a.m. and 4 p.m. in a 30 × 30 × 30 cm tank with opaque walls and partitions. Before training, each animal was habituated to the experimental apparatus (open field) in the absence of objects for 5 min. In the training phase (phase 1), animals were exposed to two identical cubes (of the same colour) for 10 min. After training, the animals were submitted to a retention interval of 1 h. This separation period is a standard methodological approach used to assess short-term memory, allowing for memory consolidation and preventing immediate recognition effects that could bias the results. In the test (phase 2), a new object (of a different colour) replaced one of the copies of the familiar object and the time spent exploring each object during a period of 10 minutes was evaluated. The familiar blue objects used in phase 2 were exactly the same ones from phase 1, ensuring continuity in the experimental setup. To avoid a thigmotaxis effect, the distances between the objects and the walls were kept the same. We calculated the exploration time of each object (%). This measurement reflects proximity rather than active exploration based on head orientation. The exploration area was defined as an 8 × 8 cm area centred on the object and preference times were calculated as reported in 30 . The videos were analysed using Noldus EthoVision® XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA). 3.4. Statistical analysis The videos were analysed using Noldus EthoVision® XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA). Data analysis was performed using GraphPad Prism software. ANOVA followed by Tukey’s multiple comparison test was used to assess significance. Statistical significance is reported as follows: * p < 0.05, ** p < 0.01, *** p <0.001, or **** p < 0.0001. Graphs were generated using GraphPad Prism 9 software. Data are presented as mean ± S.E.M Declarations Data availability Proteomics data that support the findings of this study have been deposited in the PRIDE repository (PXD058522). Transcriptomics data have been deposited in the ZENODO repository (https://zenodo.org/records/14809944) and (https://zenodo.org/records/15123596). Funding This work was supported by Fondation de l’Ataxie Charlevoix-Saguenay (seed grant 2022 to V.N.) and in part by Fondazione Telethon (grants GSA21G006 and GSA24A001 to V.N.) FMS is partially supported by Ricerca Corrente 2024-25 (RC 5X1000). Ethics approval This study does not include human samples. All animal procedures were authorized by the University of Pisa Animal Ethics Committee and the Italian Ministry of Health (n° 620/2024-PR). The study is reported in accordance with ARRIVE guidelines. CRediT authorship contribution statement Valentina Naef : Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Stefania Della Vecchia : review & editing. Michela Giacich: Visualization & Data curation Rosario Licitra : Methodology. Tiziana Bachetti: Visualization, Formal analysis & Data curation. Gabriela Coronel Vargas : Methodology . Marco Ponassi: Methodology. Filippo M. Santorelli : Writing – review & editing, Supervision & Funding acquisition. Declaration of competing interest The authors declare that they have no conflicts of interest. 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Supplementary Files Supplementaryfile1.xlsx Supplementaryfile2.xlsx SupplementaryFile3.xlsx SupplementaryFigure1and2.docx Cite Share Download PDF Status: Published Journal Publication published 14 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviews received at journal 18 May, 2025 Reviewers agreed at journal 05 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 02 May, 2025 Submission checks completed at journal 22 Apr, 2025 First submitted to journal 07 Apr, 2025 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. 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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-5957432","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":439637271,"identity":"2112e4c7-8b33-496d-b811-e7d6561afd21","order_by":0,"name":"Valentina Naef","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYFAC5gYgIQHl2NgACcbGA/i1MAK1JMC0pKWBRYjRAuOkHQZTeLUYHD/Y+Ljwh0UefwPvwQ8fEs7brW0/DLSlxiYap5Yzic3GMxIkiiUO8CVLzki4nbztTCJQy7G03AYcWiQbEtukeRIkgMp4DKR5f9xONjsAZDM2HMatpf9h+2+QlvkHeIx//0k4l2x2/iF+LfwSiW3MIC0bDvCYSTMkHLAzu0HAFn6Jh83SPGkSiRsP85hZ9iQkJ5jdANqSgMcvbPzJBz/z2NQlzjveY3zjR4Kdvdn59IcPPtTY4NSCAMwQKhGsMoGgciRgT4riUTAKRsEoGBkAAFYJYX9ZP5BrAAAAAElFTkSuQmCC","orcid":"","institution":"Neurobiology and Molecular Medicine, IRCCS Fondazione Stella Maris","correspondingAuthor":true,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Naef","suffix":""},{"id":439637272,"identity":"9d3bce7d-c998-41be-b320-a2c1bd50a966","order_by":1,"name":"Stefania Della Vecchia","email":"","orcid":"","institution":"Neurobiology and Molecular Medicine, IRCCS Fondazione Stella Maris","correspondingAuthor":false,"prefix":"","firstName":"Stefania","middleName":"Della","lastName":"Vecchia","suffix":""},{"id":439637273,"identity":"7ab249b6-06e1-4d98-8371-41242408c9ed","order_by":2,"name":"Michela Giacich","email":"","orcid":"","institution":"Neurobiology and Molecular Medicine, IRCCS Fondazione Stella Maris","correspondingAuthor":false,"prefix":"","firstName":"Michela","middleName":"","lastName":"Giacich","suffix":""},{"id":439637274,"identity":"0640ecfa-cbc0-43e3-bfd2-461879a4acb0","order_by":3,"name":"Rosario Licitra","email":"","orcid":"","institution":"Department of Veterinary Sciences, University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Rosario","middleName":"","lastName":"Licitra","suffix":""},{"id":439637275,"identity":"3366b250-2188-47f5-90f6-2f7637e998a5","order_by":4,"name":"Tiziana Bachetti","email":"","orcid":"","institution":"UO Proteomics and Mass Spectrometry, IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Tiziana","middleName":"","lastName":"Bachetti","suffix":""},{"id":439637277,"identity":"74886da5-5e99-4528-a02b-ddb0b19e27bf","order_by":5,"name":"Gabriela Coronel Vargas","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Coronel","lastName":"Vargas","suffix":""},{"id":439637278,"identity":"3dcf6320-d10b-4f4c-a1d9-5884c9fcbfe2","order_by":6,"name":"Marco Ponassi","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Ponassi","suffix":""},{"id":439637279,"identity":"aa8f973b-98a9-42e9-9640-1f0642a20e85","order_by":7,"name":"Filippo Maria Santorelli","email":"","orcid":"","institution":"Neurobiology and Molecular Medicine, IRCCS Fondazione Stella Maris","correspondingAuthor":false,"prefix":"","firstName":"Filippo","middleName":"Maria","lastName":"Santorelli","suffix":""}],"badges":[],"createdAt":"2025-02-04 10:53:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5957432/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5957432/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-10850-0","type":"published","date":"2025-07-14T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80725197,"identity":"47f63e0a-f37a-4cbb-b402-c17edb65dc20","added_by":"auto","created_at":"2025-04-16 11:39:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":180800,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Experimental timeline. Images were created using BioRender, a scientific image and illustration software. Effects of the TUDCA-supplemented diet treatment on the survival rate and growth of juvenile and adult zebrafish modelling ARSACS. (B) Kaplan-Meier survival comparison among groups, showing a significant effect of TUDCA in sacs-/- treated fish (log-rank (Mantel-Cox) test) ***p \u0026lt; 0.001. (C) Data are presented as mean ± S.E.M. (N = 5 males and 5 females per experimental group). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test: ns (not significant), *p \u0026lt; 0.05, ***p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/0400ffdce8cf50c7e409a3eb.png"},{"id":80725199,"identity":"5510380a-7990-4e8b-afb0-ac0cf82c01af","added_by":"auto","created_at":"2025-04-16 11:39:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":321987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Zebrafish locomotor activity and anxiety-like behaviour during a 5-minute novel tank test. \u003cstrong\u003e(B)\u003c/strong\u003e Total distance travelled (cm) and average speed while moving (cm/s). \u003cstrong\u003e(C)\u003c/strong\u003e Time (s) spent in the bottom half of the tank (below dotted line). \u003cstrong\u003e(D)\u003c/strong\u003e Schematic diagram of the open-field test and thigmotaxis test in adult zebrafish and heat maps. In the analysis of thigmotaxis test, the area of the peripheral zone is equal to the central zone. Single adult fish were placed in an open-field apparatus for 5 min, to quantify their exploration and analyse its pattern. \u003cstrong\u003e(E)\u003c/strong\u003eTotal distance travelled (cm) and average speed while moving (cm/s). Data are presented as box plots, where the central line represents the median, the box indicates the interquartile range (IQR), and the whiskers extend to 1.5 times the IQR. (\u003cstrong\u003eF\u003c/strong\u003e) Average time spent in zones. Data are presented as mean ± S.E.M. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, and ****p \u0026lt; 0.0001 were calculated by ANOVA followed by Tukey’s multiple comparison test. ns, no significant difference. Images were created using BioRender, a scientific image and illustration software. (N= 5 males and 5 females per experimental group)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/1b4b04e938f605d2eae8f47e.png"},{"id":80727773,"identity":"b1a0b082-5098-456a-a270-954ac30a5428","added_by":"auto","created_at":"2025-04-16 12:03:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":271601,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Shoaling test. The test involves placing a group of conspecific fish in a novel tank for 5 min, after an acclimatization time. (B) The shoal is video recorded for behavioural analysis, and to quantify social cohesion, which is measured by the average mean distance between members of the group. (C) The heat maps show that control zebrafish exhibited a significantly higher frequency of proximity to a group of zebrafish compared with sacs-/- zebrafish. Furthermore, pretreatment with TUDCA improved the sociability of sacs-deficient fish compared with untreated mutant fish. (D) [(Distance Ratio% = distance travelled in the conspecific sector /by the total distance travelled) ×100] and [(Time Ratio %=Time spent in conspecific sector/ total time observed)\u003c/p\u003e\n\u003cp\u003e×100]. Data are presented as box plots, where the central line represents the median, the box indicates the interquartile range (IQR), and the whiskers extend to 1.5 times the IQR. (N= 5 males and 5 females per experimental group). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 and ****p \u0026lt; 0.0001 were calculated by ANOVA followed by Tukey’s multiple comparison test. ns, no significant difference. Images were created using BioRender, a scientific image and illustration software.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/805d489a687ae1c4894531d5.png"},{"id":80725198,"identity":"1c0bc0fe-4897-4f62-957f-5c706ec17ccf","added_by":"auto","created_at":"2025-04-16 11:39:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":314353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagram of the novel object recognition test. \u003cstrong\u003e(B)\u003c/strong\u003e The heat map shows that adult \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish fed with TUDCA exhibited a significant preference for exploration of the novel object, similar to adult controls. Instead, untreated adult \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish did not show a preference between the familiar and novel object. \u003cstrong\u003e(C)\u003c/strong\u003e The exploration time of each object (%) was analyzed during training between two identical objects 1 and 2 and between the new object (\u003cstrong\u003eNO\u003c/strong\u003e) and the familiar object (\u003cstrong\u003eFO\u003c/strong\u003e) in the test session. Data are presented as box plots, where the central line represents the median, the box indicates the interquartile range (IQR), and the whiskers extend to 1.5 times the IQR. (N= 5 males and 5 females per experimental group). ns, no significant difference, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 and ****p \u0026lt; 0.0001 were calculated by ANOVA followed by Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/5d75212c0e5f765231c2e53d.png"},{"id":80726203,"identity":"eae27443-3e37-4b93-9287-1b68a5ee565f","added_by":"auto","created_at":"2025-04-16 11:47:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":291937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The top 20 enriched GO biological process categories were plotted. \u003cstrong\u003e(B)\u0026nbsp;\u003c/strong\u003eProtein-protein interaction analysis using the STRING bioinformatic suite (https://string-db.org/) revealed an enrichment of genes associated with circadian rhythms. Network nodes represent proteins involved in rhythmic processes (blue) and circadian rhythms (red). The edges represent protein-protein associations, with thickness indicating the strength of data support. PPI enrichment p-value:\u0026lt; 1.0e-16.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/226c2bbe7312b324514bc0c9.png"},{"id":80725208,"identity":"5b530c7f-f488-4560-9bb2-454616d687d5","added_by":"auto","created_at":"2025-04-16 11:39:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":603650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e. GSEA in adult\u0026nbsp;\u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u0026nbsp;zebrafish brains compared with WT.\u003cstrong\u003e\u0026nbsp;(B)\u0026nbsp;\u003c/strong\u003eProtein-protein interaction analysis using the STRING bioinformatic suite (https://string-db.org/) revealed an enrichment of down-regulated genes associated with mitochondrial function. Network nodes represent proteins and each node represents all the proteins produced by a single, protein-coding gene locus. The edges represent protein-protein associations, with thickness and colors indicating the strength of data support. PPI enrichment p-value:\u0026lt; 1.0e-16.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/4d1121d1c65375119243baba.png"},{"id":80727098,"identity":"ca7570af-f942-44ec-a50e-b9e2c4731d96","added_by":"auto","created_at":"2025-04-16 11:55:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":367902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The top 20 enriched GO biological process categories were plotted. \u003cstrong\u003e(B)\u003c/strong\u003e. GSEA in the brains of TUDCA-treated \u003cem\u003esacs \u003c/em\u003evs WT adult \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/725ad2160d1d3ae59997dc7d.png"},{"id":80725209,"identity":"f630e63c-2242-4661-9113-0e1709a27162","added_by":"auto","created_at":"2025-04-16 11:39:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":840427,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-protein interaction analysis using the STRING bioinformatic suite (https://string-db.org/) in the brains of treated vs. untreated adult\u0026nbsp;\u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u0026nbsp;zebrafish.\u0026nbsp;\u003cstrong\u003e(A)\u003c/strong\u003e\u0026nbsp;\u003cem\u003eLipid Metabolism\u003c/em\u003e\u0026nbsp;(highlighted in red),\u0026nbsp;\u003cem\u003eOxidative Phosphorylation\u003c/em\u003e\u0026nbsp;(highlighted in green) and\u0026nbsp;\u003cem\u003eNeurotransmitter and Synaptic Transmission\u003c/em\u003e\u0026nbsp;(highlighted in blue).\u0026nbsp;\u003cstrong\u003e(B)\u003c/strong\u003e\u0026nbsp;\u003cem\u003eOxidative\u003c/em\u003e\u0026nbsp;\u003cem\u003eactivity/removal of superoxide radical\u003c/em\u003e\u0026nbsp;(highlighted in green),\u0026nbsp;\u003cem\u003ecell redox homeostasis\u003c/em\u003e\u0026nbsp;(highlighted in pink/yellow),\u0026nbsp;\u003cem\u003emitochondrion organization\u003c/em\u003e\u0026nbsp;(highlighted in red). Network nodes represent proteins and each node represents all the proteins produced by a single, protein-coding gene locus. The edges represent protein-protein associations, with thickness indicating the strength of data support. PPI enrichment p-value:\u0026lt; 1.0e-16.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/0628fb5eb7b0585c4f2c395e.png"},{"id":87219335,"identity":"e46400e7-697a-43de-acf2-f59d62840474","added_by":"auto","created_at":"2025-07-21 16:03:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4784495,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/4eee8698-a2e8-449b-b947-cf48f3c5b7c9.pdf"},{"id":80727095,"identity":"2d180fdc-fb64-42a3-908c-af413d46501e","added_by":"auto","created_at":"2025-04-16 11:55:59","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":52588,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/ee9a07eddaff604692b76182.xlsx"},{"id":80725206,"identity":"85b86abf-88ec-48e5-bd37-696ed94ed237","added_by":"auto","created_at":"2025-04-16 11:39:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":75840,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/e5a8a3546065612e45e5b7ff.xlsx"},{"id":80725237,"identity":"462aea69-ac3f-49a9-a45b-0c8c799eeb79","added_by":"auto","created_at":"2025-04-16 11:39:59","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5700343,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/e71c4cc5a53fdad1743b15a3.xlsx"},{"id":80726208,"identity":"76ebfe50-c607-4c3e-bda0-ca8c1eab97a7","added_by":"auto","created_at":"2025-04-16 11:47:59","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":686477,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1and2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5957432/v1/00fcce1d18ce2d350ca3299c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Long-Term Benefits of TUDCA Supplement in ARSACS Zebrafish Model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAutosomal recessive spastic ataxia of Charlevoix\u0026ndash;Saguenay (ARSACS) is a distinct form of hereditary early-onset spastic ataxia characterized by progressive degeneration of the cerebellum and spinal cord due to mutations in the \u003cem\u003eSACS\u003c/em\u003e gene\u003csup\u003e1\u003c/sup\u003e The \u003cem\u003eSACS\u003c/em\u003e gene encodes sacsin, a large multidomain protein with a molecular weight of 520 kDa\u003csup\u003e2\u003c/sup\u003e. Sacsin is among the largest proteins encoded by the human genome, though its complex multidomain structure has only been partially characterized\u003csup\u003e3,4\u003c/sup\u003e. It is believed that sacsin plays a role in protein quality control, which may influence both neurodevelopment and neurodegeneration. However, despite the identification of several structural domains, the precise function of sacsin and the pathophysiological consequences of its dysfunction remain largely uncharacterized. Alongside the motor symptoms, which typically include the triad of ataxia, spasticity and peripheral neuropathy, some individuals with ARSACS exhibit intellectual disabilities and behavioural abnormalities\u003csup\u003e5\u003c/sup\u003e.\u0026nbsp;Cerebellar defects resemble the features of cerebellar cognitive-affective syndrome (CCAS), a condition characterized by deficits in executive functioning, spatial cognition, language abilities and emotional regulation\u003csup\u003e1,6,7\u003c/sup\u003e. Mouse models of ARSACS have been developed, exhibiting progressive deficits in motor coordination that closely mirror the ataxia-like symptoms of the disease\u003csup\u003e8\u003c/sup\u003e. However, data on cognitive and behavioural aspects remain scarce. Recently, Chen and colleagues generated double knockout mice for \u003cem\u003eSacs\u003c/em\u003e/B\u0026beta;2 and \u003cem\u003eSacs\u003c/em\u003e/Akap1, to test behavioural modifications related to ARSACS, and observed a significant decline in cognitive abilities in older \u003cem\u003eSacs\u003c/em\u003e knock-out mice\u003csup\u003e9\u003c/sup\u003e. Their study provided the first experimental evidence linking ablation of the \u003cem\u003eSACS\u003c/em\u003e gene not only to motor impairments but also to learning and memory deficits, laying the foundations for further investigation of this area\u003csup\u003e9\u003c/sup\u003e. Zebrafish possess a range of neurobehavioral traits that are translationally relevant. Zebrafish, are also increasingly recognized as suitable organisms for modelling neurodegenerative diseases, and thus as a valuable platform for exploring disease mechanisms and testing potential treatments\u003csup\u003e10\u003c/sup\u003e. Despite comprehensive definition of non-motor symptoms in patients with ARSACS, in model mice, and in the zebrafish\u003csup\u003eR487Kfs*495\u003c/sup\u003e KO strain\u003csup\u003e11\u003c/sup\u003e, the pathophysiology and underlying mechanisms of the disease remain poorly understood. Although the cerebellum plays a role in the retrieval of episodic memory and in other cognitive tasks\u003csup\u003e12\u003c/sup\u003e, it is still unclear how it contributes to cognitive and affective deficits.\u0026nbsp;Currently, there are no definitive treatments available for degenerative ataxia\u003csup\u003e13\u003c/sup\u003e, and options for addressing the progressive degeneration of Purkinje cells in patients are limited. The repurposing of FDA-approved drugs with the potential to act on multiple molecular targets is a promising avenue to explore at preclinical level. Aged mice treated with tauroursodeoxycholic acid (TUDCA) (300 mg/kg) exhibited enhanced energy expenditure, improved metabolic flexibility and better cognitive abilities\u003csup\u003e10\u003c/sup\u003e. Additionally, in a chronic Parkinson\u0026rsquo;s disease model exhibiting significant dopaminergic degeneration, pretreatment with TUDCA(500 mg/kg, intraperitoneally) protected against neuronal damage and mitigated the activation of microglia and astroglia\u003csup\u003e14\u003c/sup\u003e. On the basis of this previous evidence, we investigated whether dietary supplementation with TUDCA could improve the phenotype of our published zebrafish model of ARSACS\u003csup\u003e11,15\u003c/sup\u003e. We showed that TUDCA significantly improved motor coordination, social interaction and cognitive impairments in this model. We found that TUDCA influences omics profiles in our zebrafish model. Transcriptomic and proteomic analyses suggest that TUDCA may modulate key cellular processes, including stress response, protein quality control, membrane stability, inflammation, and cytoskeletal dynamics. TUDCA could have potential benefits in improving locomotor and cognitive functions while also affecting molecular pathways related to neuroinflammation and cellular homeostasis. However, further studies will be necessary to validate these findings and better characterize the metabolic and functional effects of TUDCA. Overall, this study supports the need for further investigation into TUDCA\u0026rsquo;s potential therapeutic effects.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e \u003cstrong\u003eTUDCA improves early survival in \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish without affecting growth or morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eARSACS is an incurable condition that requires ongoing research to identify potential treatments and interventions. As a proof of concept, we tested a diet supplemented with TUDCA in \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish (\u003cstrong\u003eFig.1A\u003c/strong\u003e). This experimental diet was based on this compound\u0026rsquo;s previously documented neuroprotective and anti-inflammatory properties\u003csup\u003e16\u003c/sup\u003e. During the first month of life\u0026mdash;a critical period typically characterized by high mortality in our ARSACS model\u0026mdash;we observed a significant improvement in survival rates. Notably, after this critical window, survival stabilized over time (\u003cstrong\u003eFig. 1B\u003c/strong\u003e). To exclude potential metabolic side effects, particularly since TUDCA had not previously been tested in zebrafish over such a long period before, we assessed the fish at three months of age. No significant morphological abnormalities or changes in body weight were observed when comparing the \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutants to the TUDCA-treated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e group. TUDCA treatment seemed to ameliorate the growth of the untreated mutants. (\u003cstrong\u003eFig. 1C\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 TUDCA treatment enhances locomotor activity and reduces anxiety in \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the larval stages, we found that the \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish exhibited symptoms resembling motor impairments observed in humans with ARSACS\u003csup\u003e15\u003c/sup\u003e. Using the novel tank diving test (\u003cstrong\u003eFig. 2B\u003c/strong\u003e)., we observed that one-year-old\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish, similar to \u003cem\u003eSacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice\u003csup\u003e17\u003c/sup\u003e, displayed reduced speed of movement and impaired motor control (\u003cstrong\u003eFig. 2B\u003c/strong\u003e). Active behaviour driven by the innate motivation to explore unfamiliar surroundings constitutes a \u0026nbsp;typical normal response to a novel environment\u003csup\u003e18\u003c/sup\u003e, and is a trait evolutionarily conserved across many species\u003csup\u003e19\u003c/sup\u003e. However, adult \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e-/-\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/sup\u003ezebrafish exhibited anxiety, a passive response characterized by a prolonged time spent in the lower half of the tank, and thus reduced exploratory activity (\u003cstrong\u003eFig. 2C\u003c/strong\u003e), which suggested a decline in their ability or motivation to explore new environments. To further assess anxiety-related behavior, we employed the open-field test (\u003cstrong\u003eFig. 2D\u003c/strong\u003e), which provides complementary information to the novel tank diving test by specifically evaluating thigmotaxis (or \u0026ldquo;wall-hugging\u0026rdquo;)\u003csup\u003e19\u003c/sup\u003e. In this test, we analysed the exploratory behaviour of adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish observing that TUDCA-treated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish showed a moderately enhanced exploratory and locomotor response compared with untreated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e specimens \u0026nbsp;(\u003cstrong\u003eFig. 2E).\u0026nbsp;\u003c/strong\u003eFurthermore, we\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecompared the time spent in the inner zone (centre) versus the outer zone of the tank\u003csup\u003e19\u003c/sup\u003e (\u003cstrong\u003eFig. 2F)\u003c/strong\u003e. Wild-type fish habituated more quickly to the environment and exhibited reduced thigmotaxis, indicative of lower anxiety levels. In contrast, adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish were less inclined to venture away from the safety of the edges of the tank and demonstrated heightened anxiety (\u003cstrong\u003eFig. 2F\u003c/strong\u003e). Notably, while TUDCA supplementation seemed to partially rescued the anxiety phenotype, its effects appeared stronger in the novel tank diving test than in the open-field test. This discrepancy may stem from differences in the nature of the two behavioral paradigms. Indeed, the novel tank diving test primarily assesses the acute stress response to a novel environment, with zebrafish typically exhibiting an initial preference for the bottom of the tank before gradually exploring the upper zones. In contrast, the open-field test evaluates thigmotaxis, a behavior driven by long-term anxiety levels and risk assessment, where increased center avoidance indicates heightened anxiety. These findings suggest that TUDCA treatment may potentially lead to partial recovery of both motor and non-motor symptoms in the \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e model. It is speculative to hypothesize that the stronger effect observed in the novel tank diving test may indicate that TUDCA primarily improves stress reactivity and exploratory drive rather than directly modulating chronic anxiety-related behaviors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 TUDCA ameliorates \u0026nbsp;social deficits in \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeyond its role in motor symptoms, recent studies have also highlighted involvement of the cerebellum in cognitive, emotional and social functions\u003csup\u003e6\u003c/sup\u003e. Patients with cerebellar ataxia may exhibit difficulties in social interactions and in understanding others\u0026rsquo; emotions, which can impact personal relationships and overall quality of life\u003csup\u003e20\u003c/sup\u003e. Direct studies linking cerebellar degeneration to changes in social and cognitive behaviour in ARSACS patients are limited, although impairment of social skills and severe psychiatric symptoms have been reported in these patients\u003csup\u003e21,22\u003c/sup\u003e. Zebrafish are a highly social species that prefer to spend time in proximity to conspecifics\u003csup\u003e23\u003c/sup\u003e. Their shoaling behaviour serves several adaptive functions, providing protection from predators for example, as well as increasing foraging efficiency and mating success\u003csup\u003e23\u003c/sup\u003e.\u0026nbsp;Interestingly, cerebellar circuits in zebrafish also play an important role in social orienting behaviour\u003csup\u003e24\u003c/sup\u003e.\u0026nbsp;To explore the effect of loss of sacsin on social behaviours, we performed a shoaling test to compare social behaviour between homogeneous groups of zebrafish\u003csup\u003e25\u003c/sup\u003e. In a novel tank, stressed fish tend to swim closer together, maintaining smaller inter-fish distances than non-stressed fish\u003csup\u003e25\u003c/sup\u003e. Indeed, tighter shoals indicate higher anxiety\u003csup\u003e26\u003c/sup\u003e. We measured the average inter-fish distance and found that adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish, compared with WT fish, appeared more stressed, swimming closer together (inter-fish distance of \u0026lt; 6 cm), a trait that reduced their exploratory behaviour (\u003cstrong\u003eFig. 3A-B)\u003c/strong\u003e. However, TUDCA-treated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e fish showed greater inter-fish distances than their untreated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e counterparts, demonstrating less anxiety. The social preference and interaction tests were subsequently performed as already described\u003csup\u003e27\u003c/sup\u003e (\u003cstrong\u003eFig. 3C-D\u003c/strong\u003e). During the habituation phase, we continued to observe increased exploratory behaviour in TUDCA-treated versus untreated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003efish. In the test phase, a group of four conspecific zebrafish was placed in the right side of the tank, and a single fish per experimental group was placed in the left side as reported in a previous study\u003csup\u003e20\u003c/sup\u003e. We observed that adult WT zebrafish generally contacted the group on the right side and spent more time in the conspecific sector than the empty sector, showing a strong group-forming tendency\u003csup\u003e27\u003c/sup\u003e. In contrast, \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish spent their time evenly throughout the tank, exhibiting reduced social contact with the peer group and a lower proportion of time in the conspecific sector (\u003cstrong\u003eFig. 3C-D\u003c/strong\u003e). When treated with TUDCA, however, \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e fish showed improved sociability (\u003cstrong\u003eFig. 3C-D\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u003c/strong\u003e \u003cstrong\u003eTUDCA improves cognitive performance in the ARSACS zebrafish model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCognitive symptoms have been reported in some cases of ARSACS\u003csup\u003e5,28\u003c/sup\u003e and in the \u003cem\u003eSacs\u003c/em\u003e-KO mouse model\u003csup\u003e9\u003c/sup\u003e. Although fMRI studies indicate that the cerebellum participates in the recovery of episodic memory and other cognitive tasks, and CCAS\u003csup\u003e12\u003c/sup\u003e has been described in ARSACS\u003csup\u003e6,7,21\u003c/sup\u003e, it is still not clear how cerebellar disorders impair cognition. Furthermore, most of the genes associated with cerebellar ataxia diseases, including \u003cem\u003eSACS\u003c/em\u003e, are ubiquitously expressed, and brain atrophy commonly follows cerebellar atrophy in hereditary ataxias\u003csup\u003e29\u003c/sup\u003e. Since zebrafish can be used to model complex human behavioural traits such as reward responsiveness, learning, and memory\u003csup\u003e30\u003c/sup\u003e, we took advantage of this characteristic to investigate the potential presence of cognitive impairments in \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutants . We performed the novel object recognition (NOR) test and analysed the time spent by the animals exploring objects\u003csup\u003e30\u003c/sup\u003e in the open-field test apparatus. The NOR test leverages animals\u0026rsquo; naturally greater tendency to explore novel objects over familiar ones, and thus explores their curiosity and memory capabilities\u003csup\u003e31\u003c/sup\u003e. \u0026nbsp;Zebrafish possess recognition memory for simple 2- and 3-dimensional geometrical shapes\u003csup\u003e32\u003c/sup\u003e. We placed adult fish in a tank containing two identical blue cubes (\u003cstrong\u003eFig. 4A\u003c/strong\u003e) and allowed them to explore freely for a set amount of time, becoming familiar with the objects present (phase 1,\u003cstrong\u003e\u0026nbsp;Fig. 4A-B\u003c/strong\u003e). After training, the animals were submitted to a retention interval of 1 h, then, we put the fish back in same tank, in which we had placed one of the familiar blue objects and a novel object (red cube), and evaluated the amount of time they spent exploring the novel object compared with the familiar one (phase 2,\u003cstrong\u003e\u0026nbsp;Fig. 4A-4B\u003c/strong\u003e), which is an indicator of their recognition memory\u003csup\u003e30,31\u003c/sup\u003e. \u0026nbsp;In the training session (phase 1), no preference between the two identical blue cubes was observed. In the test session (phase 2) we found that adult WT fish spent more time exploring the novel object, a result indicating memory retention as previously reported\u003csup\u003e32\u003c/sup\u003e, whereas adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e fish did not show a preference between the familiar and novel object \u003cstrong\u003e(Fig. 4B-C)\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHowever, when treated with TUDCA, adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish showed a significant preference for the new object compared with the familiar object (****p \u0026lt; 0.0001), which suggests an improvement in their cognitive performance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Key insights from multiomics analyses in the zebrafish ARSACS model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnderstanding early cellular stresses and altered pathways in the brain that may contribute to the onset of ARSACS is crucial for developing preventative treatments. However, obtaining these insights through studies in living humans is challenging. We performed transcriptome analysis comparing whole brains from 12-month-old WT and homozygous \u003cem\u003esacs\u003c/em\u003e-mutant fish. 527 genes were differentially expressed in the entire brains of the \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e group compared with the WT group (\u003cstrong\u003eSupplementary File 1\u003c/strong\u003e). 213 genes showed increased expression and 314 showed decreased expression. Gene ontology\u0026nbsp;(GO) and protein-protein interaction (PPI) analyses revealed enrichment of genes associated with circadian rhythms that have not been reported in other ARSACS models (\u003cstrong\u003eFig. 5A-B\u003c/strong\u003e). Therefore, we performed gene set enrichment analyses (GSEA) to predict which cellular processes were affected. These analyses identified several pathways related to biological processes such as neuroinflammation and response to oxidative stress\u003cstrong\u003e\u0026nbsp;(Fig. 6A)\u003c/strong\u003e. PPI analyses revealed downregulation of several proteins related to mitochondrial activity and oxidative phosphorylation (\u003cstrong\u003eFig. 6B\u003c/strong\u003e), consistent with mitochondrial dysfunction as reported in the literature\u003csup\u003e33\u0026ndash;35\u003c/sup\u003e. The overlap between our results and those reported in the literature further supports the potential of our model in modelling ARSACS pathology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo gain a more comprehensive understanding of the molecular alterations in our model and evaluate potential downstream effects at the protein level, we performed proteomic analysis on adult brains. This analysis revealed a complex network of upregulated and downregulated proteins, which could indicate potential neurodegenerative molecular changes in \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eadult zebrafish, which could be further explored in future studies. In Tables 1-2, we highlighted significant proteins associated with biological processes that are altered in ARSACS pathology in others model, as previously described in\u003csup\u003e36\u003c/sup\u003e. In particular, we found an \u0026nbsp;imbalance in key processes such as \u003cem\u003eMitochondrial Function\u003c/em\u003e, \u003cem\u003eOxidative Stress\u003c/em\u003e, \u003cem\u003eNeuroinflammation\u003c/em\u003e, \u003cem\u003eER Stress\u003c/em\u003e and \u003cem\u003eSynaptic Signalling\u003c/em\u003e (Tables 1-2) that, in combination with alterations in circadian patterns, could exacerbate neuronal damage, leading to a cycle of chronic stress and inflammation in the brain\u003csup\u003e37\u003c/sup\u003e. \u0026nbsp;Notably, these same pathways have also emerged in previous \u003cem\u003ein vitro\u003c/em\u003e studies in ARSACS primary cells, further supporting their relevance to disease pathophysiology\u003csup\u003e38\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eTable 1:\u003c/strong\u003e List of upregulated proteins identified through proteomics analysis in the brains of adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish compared with wild-type (WT) counterparts.\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCATEGORY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFUNCTION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eREF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eMT2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eOxidative Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003eAnti-inflammatory, and anti-apoptotic agent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e39,40\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eFLAD1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eOxidative Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003eCofactor in redox reactions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e41,42\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eUOX\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eOxidative Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInvolved in the urate catabolic process, an antioxidant pathway reducing oxidative damage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e43\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eMRPS15\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eOxidative Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003eInvolved in pathways related to mitochondrial translation and protein metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e44,45\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eVPS18\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eER Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp;Involved in protein trafficking and lysosomal degradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e46\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eCYP22K6\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eOxidative Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003eInvolved in oxidative metabolism\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e47\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eTSR2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eER Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInvolved in apoptosis\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e48\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eTRNT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eER Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eLinked to ER stress and increased oxidative stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e49,50\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eTBCE\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eER Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInvolved in microtubule cytoskeleton organization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e51\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eCRP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eNeuroinflammation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMarker of neuroinflammation and peripheral inflammation\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e52\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cem\u003eFABP10A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.8824%;\"\u003e\n \u003cp\u003eNeuroinflammation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.9118%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInvolved in fatty acid transport\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e53,54\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. List of downregulated proteins identified through proteomic analysis in the brains of adult\u0026nbsp;\u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish compared with wild-type (WT) counterparts.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCATEGORY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFUNCTION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eREF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eMT-ND2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eMitochondrial Function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCore subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I)\u0026nbsp;\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e55\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eMT-CO1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eMitochondrial Function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003ePart of respiratory chain complex IV, involved in ATP synthesis coupled electron transport\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e56\u003c/sup\u003e\u003csup\u003e\u003cbr\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eMCTS1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eCell Cycle Regulation and Apoptosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003eInvolved in the regulation of various processes, including cell cycle modulation and apoptosis\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e57\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eImmune Regulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSubunit of major histocompatibility complex (MHC) class I\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e58,59\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eOPTN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eNeuroinflammation \u0026amp; Cell Death\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003eInvolved in various vesicular trafficking pathways\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e60\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eHPX\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eOxidative Stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003eInvolved in protecting cells from oxidative stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e61\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eTRAPPC2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eIntracellular Vesicle Trafficking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInvolved in the targeting and fusion of endoplasmic reticulum-to-Golgi transport vesicles\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e62,63\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2941%;\"\u003e\n \u003cp\u003e\u003cem\u003eDYNC1I2A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.2059%;\"\u003e\n \u003cp\u003eRetrograde Transport\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52.7941%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eActs as a retrograde microtubule motor to transport organelles and vesicles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.70588%;\"\u003e\n \u003cp\u003e\u003csup\u003e64\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. TUDCA modulates gene expression and protein regulation in the ARSACS zebrafish model\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo gain a better understanding of diet-induced changes in whole-brain gene expression, we performed RNA-seq analysis. A comparative analysis between \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e and TUDCA-treated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish indicated that dietary supplementation with TUDCA may be associated with modifications in gene expression signatures related to circadian rhythm and neuroinflammatory pathways. As reported in the literature, we found that TUDCA may play a multifaceted role in supporting metabolic health. In particular, as previously reported, it appears to influence cholesterol and fat metabolism, reducing the \u003cem\u003eER Stress\u003c/em\u003e\u003csup\u003e65\u003c/sup\u003e that occurs when misfolded proteins accumulate. Of 817 genes differentially expressed in the whole brains of the TUDCA-treated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e group compared with the untreated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e fish, 422 showed increased expression, while 395 exhibited decreased expression (\u003cstrong\u003eSupplementary file 2\u003c/strong\u003e). GO analysis revealed differentially expressed genes mainly related to \u003cem\u003eDevelopmental Growth, Lipid Metabolism\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Intermediate Filament Cytoskeleton Organization\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig. 7A\u003c/strong\u003e)\u003cem\u003e.\u003c/em\u003e GSEA revealed an enrichment of genes related to categories such as \u003cem\u003eLipid Metabolism\u003c/em\u003e and \u003cem\u003eOxidative Phosphorylation\u003c/em\u003e\u0026nbsp; (\u003cstrong\u003eFig. 7B\u003c/strong\u003e)\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePPI analysis revealed 150 upregulated genes that are functionally related to \u003cem\u003eLipid Metabolism\u003c/em\u003e (highlighted in red, \u003cstrong\u003eFig. 8A\u003c/strong\u003e), \u003cem\u003eOxidative Phosphorylation\u003c/em\u003e (highlighted in green, \u003cstrong\u003eFig. 8A\u003c/strong\u003e) or \u003cem\u003eNeurotransmitter and Synaptic Transmission\u003c/em\u003e (highlighted in blue, \u003cstrong\u003eFig. 8A\u003c/strong\u003e). \u0026nbsp;\u003cem\u003eOxidative\u003c/em\u003e \u003cem\u003eactivity/removal of superoxide radical\u003c/em\u003e (highlighted in green,\u003cstrong\u003e\u0026nbsp;Fig. 8B\u003c/strong\u003e), \u003cem\u003ecell redox homeostasis\u003c/em\u003e (highlighted in pink/yellow,\u003cstrong\u003e\u0026nbsp;Fig. 8B\u003c/strong\u003e), \u003cem\u003emitochondrion organization\u003c/em\u003e (highlighted in red,\u003cstrong\u003e\u0026nbsp;Fig. 8B\u003c/strong\u003e), which may indicate a protective and adaptive effect aimed at maintaining redox homeostasis and preventing cellular damage after TUDCA treatment. These results could support the ability of TUDCA to mitigate oxidative stress in our ARSACS model, as previously described\u003csup\u003e16\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;We also compared the transcriptomic profile of TUDCA-treated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish with that of WT fish (\u003cstrong\u003eSupplementary File 3\u003c/strong\u003e). This analysis indicated that the transcriptomic profile of treated mutants did not fully overlap with that of WT fish, suggesting that while TUDCA may ameliorate some molecular alterations, it does not completely restore the WT-like state. Specifically, GSEA analysis identified enrichment in pathways related to biological processes such as \u003cem\u003edevelopmental growth\u003c/em\u003e, \u003cem\u003econnective tissue development\u003c/em\u003e, \u003cem\u003etissue regeneration\u003c/em\u003e, and \u003cem\u003ecytoskeletal organization\u003c/em\u003e (\u003cstrong\u003eSupplementary Figure 1A\u003c/strong\u003e). PPI analysis further revealed an upregulation of genes associated with \u003cem\u003ecell cycle regulation\u003c/em\u003e, \u003cem\u003ecytoskeletal organization\u003c/em\u003e, \u003cem\u003echromatin remodelling\u003c/em\u003e, and \u003cem\u003eneurodevelopmental processes\u003c/em\u003e. These changes could reflect a compensatory response aimed at mitigating aspects of neurodegeneration (\u003cstrong\u003eSupplementary Figure 1B\u003c/strong\u003e). Additionally, we observed higher expression of genes related to \u003cem\u003emitophagy\u003c/em\u003e, which aligns with the proposed role of TUDCA in promoting mitochondrial quality control (\u003cstrong\u003eSupplementary Figure 1B\u003c/strong\u003e). In contrast, immune-related pathways, including \u003cem\u003ecytokine production\u003c/em\u003e and \u003cem\u003eimmune response signaling\u003c/em\u003e, were downregulated, suggesting a potential anti-inflammatory effect of TUDCA (\u003cstrong\u003eSupplementary Figure 2A\u003c/strong\u003e). Interestingly, we also identified alterations in \u003cem\u003eRNA processing\u003c/em\u003e and \u003cem\u003esplicing pathways\u003c/em\u003e, which may indicate that certain gene regulatory mechanisms remain dysregulated despite TUDCA treatment (\u003cstrong\u003eSupplementary Figure 2B).\u003c/strong\u003e These data could suggest that, while TUDCA treatment ameliorates some molecular deficits, it does not fully restore the WT-like transcriptional state, highlighting both its therapeutic potential and its limitations in completely reversing ARSACS-associated dysregulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, to evaluate downstream effects at the protein level we analysed proteomic data. We identified differentially expressed proteins related to several processes such as \u003cem\u003eAutophagy and Lysosomal Function\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Protein Degradation\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Oxidative Stress Response\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Lipid Transport and Metabolism.\u0026nbsp;\u003c/em\u003eThe upregulation of proteins related to processes like \u003cem\u003eSynaptic Integrity\u003c/em\u003e and \u003cem\u003eSynaptic\u003c/em\u003e \u003cem\u003ePlasticity\u003c/em\u003e, such as SCRIB and CNTN5, is intriguing (\u003cstrong\u003eTable 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e. List of upregulated proteins identified through proteomic analysis in the brains of TUDCA-treated vs. untreated \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"680\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCATEGORY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFUNCTION\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eREF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eLAMP5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eProtein Trafficking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eInvolved in establishment of protein localization to organelle\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003csup\u003e66\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eUSP46\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003cp\u003eDegradation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eInvolved in protein deubiquitination and regulation of GABAergic synaptic transmission\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e67\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eSCRIB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eSynaptic Stability\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eInvolved in neuronal stability and synaptic integrity, maintaining neural connections even under neurodegenerative stress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e68,69\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eCNTN5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eSynaptic Plasticity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eInvolved in the formation of axon connections in the developing nervous system\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003csup\u003e70,71\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eHYCC1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eMyelination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eInvolved in neuron-to-glia signalling to initiate or maintain myelination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003csup\u003e72\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eCLPTM1L\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eLipid Metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eA scramblase that moves GlcN-PI across the ER membrane, aiding glycosylphosphatidylinositol (GPI) biosynthesis, which is essential for protein post-translational modification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003csup\u003e73\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eTMEM11\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eMitochondrial Function\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eLocalizes to the outer mitochondrial membrane where it regulates BNIP3/BNIP3L-dependent receptor-mediated mitophagy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003csup\u003e74\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003eNXN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eAntioxidant Defence\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eMember of the thioredoxin superfamily, a group of small, multifunctional redox-active proteins\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e75\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 15.2717%;\"\u003e\n \u003cp\u003e\u003cem\u003ePTDSS1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.3833%;\"\u003e\n \u003cp\u003eCell Signalling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51.395%;\"\u003e\n \u003cp\u003eA phospholipid found in membranes that plays a role in various cellular functions, including development, cell communication, programmed cell death\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9501%;\"\u003e\n \u003cp\u003e\u003csup\u003e76\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThese changes suggest a putative improvement in brain function in our ARSACS model, leading to an enhancement of behaviours related to learning, memory and social interaction. Downregulation of immune-related proteins like B2M and IFI30 suggest a possible dampening of inflammatory responses, in accordance with TUDCA\u0026rsquo;s known anti-inflammatory properties\u003csup\u003e16\u003c/sup\u003e. Additionally, the level of reduction of proteins related to the ubiquitin-proteasome system, such as MARCHF2, demonstrates the potential of TUDCA to reduce proteotoxic stress, likely by stabilizing protein folding and minimizing protein accumulation\u003csup\u003e77,78\u003c/sup\u003e (\u003cstrong\u003eTable 4\u003c/strong\u003e). These results suggest that TUDCA could exert neuroprotective effects through multiple cellular pathways, potentially slowing down disease progression and improving motor and cognitive function in our model. However, to confirm these speculative findings, further studies will be necessary to validate the obtained results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e. List of downregulated proteins identified through proteomic analysis in the brains of TUDCA-treated vs. untreated adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGENE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCATEGORY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFUNCTION\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eREF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eB2M\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eImmune Regulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAn important subunit of major histocompatibility complex (MHC) class I\u003c/p\u003e\n \u003cp\u003eDownregulation may impair immune response.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e79\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eAGR2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eProtein Folding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003eA member of the disulfide isomerase (PDI) family of proteins found in the endoplasmic reticulum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e80\u003c/sup\u003e\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eMTTP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eLipid Metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003eThis protein is crucial for lipoprotein formation. Putative inhibitor of ferroptosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e81\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eVPS25\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eProtein Sorting\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003eComponent of ESCRT-I. Selectively modulates FGF signalling by directing receptor sorting through endosomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e82\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eMARCHF2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eUbiquitination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eMember of the MARCH family of membrane-bound E3 ubiquitin ligases\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e83,84\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eARPIN\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eCytoskeleton and Cell Motility\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003eInvolved in directional locomotion, it regulates actin filament dynamics.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e85\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eFABP10A\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eLipid Transporter\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003eInvolved in intracellular binding and trafficking of long fatty acids in the liver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e53,54\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 13.6232%;\"\u003e\n \u003cp\u003e\u003cem\u003eIFI30\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.2754%;\"\u003e\n \u003cp\u003eInflammation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50.7246%;\"\u003e\n \u003cp\u003eInvolved in antigen processing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.3768%;\"\u003e\n \u003cp\u003e\u003csup\u003e86\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBeyond the motor dysfunction caused by the progressive cerebellar degeneration, some patients with ARSACS disease exhibit cognitive impairment and behavioural problems\u003csup\u003e5\u003c/sup\u003e including apathy, dysphoria, paranoid thoughts, irritability, and significant cognitive impairment\u003csup\u003e87\u003c/sup\u003e. Several research has demonstrated that cerebellar damage often leads to cognitive deficits and to affective problems, thereby highlighting the cerebellum\u0026rsquo;s significant role in cognitive and emotional processes\u003csup\u003e88\u003c/sup\u003e, so that the concept of CCAS-like has been proposed to describe cognitive and emotional alterations linked to cerebellar dysfunction in some instances \u003csup\u003e6,89\u003c/sup\u003e.Although the cognitive and affective phenotype observed in ARSACS patients is complex and goes beyond the definition of CCAS, cerebellar dysfunction may contribute to its development\u003csup\u003e21,22\u003c/sup\u003e. In line with findings in \u003cem\u003eSacs\u003c/em\u003e-KO mice\u003csup\u003e9\u003c/sup\u003e, \u0026nbsp;we observed that loss of sacsin in zebrafish not only affects locomotor activity but also leads to anxiety-like behaviour, social impairment and deficits in object recognition memory. Our findings further strengthen the evidence that loss of sacsin is linked to significant cognitive deficits that become apparent in adulthood. These results reflect the multifaceted role of sacsin in maintaining neural function and suggest that its absence may disrupt critical pathways involved in cognitive processing. The cognitive impairments observed fit into the broader spectrum of neurological abnormalities associated with sacsin deficiency, highlighting the need for continued research to unravel the underlying mechanisms. Our second focus, through transcriptomic and proteomic analyses, was to identify molecular pathways that may be altered in adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e fish compared with their WT counterparts. This approach aimed to provide deeper insights into the neurobiology of neurodegeneration in our ARSACS model. Dysregulation of the mitochondrial fission enzyme is recognized as a primary driver of the disease; however, since this is a neurodegenerative disorder, other molecular pathways and mechanisms likely contribute simultaneously, worsening both motor and behavioural symptoms and further aggravating the overall progression. In this study, we provide novel insights into the molecular mechanisms underlying ARSACS and their contribution to disease progression. Through RNA transcriptomic analysis of 1-year-old \u003cem\u003esacs-/-\u003c/em\u003e zebrafish brains, we identified significant alterations in the expression of circadian rhythm-related factors, such as PER and CRY family genes, as well as other genes related to neuroinflammation. Circadian rhythms direct a wide range of physiological functions, and alterations of them directly affect human health\u003csup\u003e90\u003c/sup\u003e. On the other hand, neuroinflammation could be a key player in the progression of ARSACS. Notably, several studies have found that alteration of the circadian cycle is closely related to the body\u0026rsquo;s inflammatory response, and in the context of neurodegenerative disorders creates a feedback loop that may exacerbate neurodegenerative progression\u003csup\u003e91\u003c/sup\u003e. Immune cells in humans and animals, including microglia, neutrophils, monocytes and lymphocytes, could express clock genes. Thus, circadian cycle alterations may be an important factor mediating central and peripheral inflammatory responses\u003csup\u003e91\u003c/sup\u003e. Disruption of circadian rhythms and neuroinflammation might contribute to neurodegeneration and to clinical manifestations like cognitive impairment\u003csup\u003e92,93\u003c/sup\u003e as observed in our \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003ezebrafish model. Proteomic analyses highlighted the interplay occurring between upregulated and downregulated proteins in the brains of 1-year-old \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish compared with those of WT specimens, which could suggests a dynamic attempt by the neurons to modulate oxidative stress and neurodegeneration, acting on neuroinflammation and synaptic communication. Downregulation of key proteins such as MT-CO1 and HPX worsens the neurons\u0026rsquo; ability to manage damage.\u0026nbsp;For instance, low levels of HPX, which plays a critical role in iron homeostasis\u003csup\u003e61\u003c/sup\u003e, can exacerbate oxidative stress and worsen mitochondrial dysfunction. Furthermore, downregulation of TRAPPC2 may lead to Golgi fragmentation and arrest of anterograde trafficking, thereby impairing Golgi function\u003csup\u003e63\u003c/sup\u003e. The increased presence of pro-inflammatory proteins like CRP and OPTN could suggests a chronic inflammatory state. Although the proteomic analyses did not identify proteins directly involved in the regulation of circadian rhythms such as the core clock players (e.g., CLOCK, BMAL1, PER and CRY), several proteins may indirectly influence circadian rhythms. Circadian rhythms and mitochondrial proteins are known to be closely interconnected\u003csup\u003e94\u003c/sup\u003e, influencing protein levels and acetylation of mitochondrial genes, as well as mitochondrial morphology and oxidative phosphorylation\u003csup\u003e94\u003c/sup\u003e. Downregulation of mitochondrial-related genes such as MCTS1, MT-ND2 and MT-CO1 significantly impairs mitochondrial function leading to inefficient mitochondrial respiration, a decline in ATP production, and increased reactive oxygen species (ROS) generation, in turn leading to neurodegeneration\u003csup\u003e95\u0026ndash;97\u003c/sup\u003e. Indeed, reduced energy production and accumulation of ROS can further alter circadian rhythms\u003csup\u003e98\u003c/sup\u003e. In this scenario, our study provides preliminary evidence suggesting a potential beneficial effect of long-term treatment with TUDCA, which has previously shown efficacy in other models of neurodegeneration such as Parkinson\u0026rsquo;s disease, Alzheimer\u0026rsquo;s disease and\u0026nbsp;Huntington\u0026rsquo;s disease, acting as a mitochondrial stabilizer, anti-apoptotic agent, and anti-inflammatory compound\u003csup\u003e99\u0026ndash;101\u003c/sup\u003e. One of the key mechanisms by which TUDCA works is by reducing ER stress\u003csup\u003e65\u003c/sup\u003e. In a model of chronic unpredictable stress-induced depression, administration of TUDCA was able to improve the anxiety-like and depressive-like behaviour\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edriven by\u0026nbsp;neuroinflammation, oxido-nitrosative stress and ER stress\u003csup\u003e102\u003c/sup\u003e. We administered a TUDCA-enriched diet to \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish from embryonic development to one year of age. Behavioural tests revealed partial but significant improvements in locomotor activity, social behaviour, and cognitive performance, highlighting TUDCA\u0026rsquo;s capacity to mitigate both motor and non-motor symptoms of ARSACS.\u0026nbsp;Using a multi-omics approach, TUDCA appears to modulate key molecular pathways involved in the progression of ARSACS, particularly those related to circadian rhythms and neuroinflammation. Transcriptomic and proteomic analyses suggest potential neuroprotective effects of TUDCA, with proteomic data indicating its involvement in stress response, protein quality control, membrane stability, inflammation, and cytoskeletal dynamics. Notably, decreased levels of immune-related proteins like B2M and IFI30 suggest a dampening of inflammatory responses, consistent with TUDCA\u0026rsquo;s known anti-inflammatory properties\u003csup\u003e103\u003c/sup\u003e. In addition, the fatty acid-binding protein FABP10A, which was upregulated in \u003cem\u003esacs\u003c/em\u003e adult brains, is downregulated following TUDCA treatment. Given the role of FABPs in lipid metabolism and inflammatory processes\u003csup\u003e104\u003c/sup\u003e, this finding suggests that TUDCA may contribute to restoring lipid homeostasis, alleviating metabolic stress in \u003cem\u003esacs\u003c/em\u003e mutants. Considering the strong link between lipid dysregulation and neuroinflammation, the reduction in FABP10A expression may indicate a decrease in inflammatory signalling, highlighting a broader impact of TUDCA treatment on neuroinflammatory pathways. Furthermore, the downregulation of ubiquitin-proteasome-related proteins such as SSUH2 and MARCHF2 highlights its potential to alleviate proteotoxic stress by stabilizing protein folding and reducing aberrant protein accumulation. The increased expression of USP46, linked to reduced anxiety and depressive-like behaviours in mouse models\u003csup\u003e105,106\u003c/sup\u003e, further suggests that TUDCA may influence proteostasis, and could potentially ameliorate stress responses and cognitive functions. However, when comparing the transcriptomic profile of TUDCA-treated mutants with that of WT fish, we observed that it does not fully overlap with the WT-like transcriptional state. This suggests that while TUDCA ameliorates some molecular deficits, it does not completely restore gene expression patterns to WT levels. This aligns with our behavioral findings, where TUDCA treatment led to partial improvements in locomotor and anxiety-related phenotypes rather than complete normalization. The partial recovery may indicate that TUDCA effectively targets specific pathological mechanisms, such as neuroinflammation and mitochondrial dysfunction, while other molecular disruptions caused by sacsin loss remain unaddressed. Further studies are needed to validate these findings and to better understand the precise mechanisms underlying TUDCA\u0026rsquo;s effects. While the transcriptomic and proteomic analyses conducted in this study provide valuable new insights into the molecular alterations occurring in ARSACS, they do not directly establish causality between the observed changes and disease symptoms and additional functional studies will be required to confirm the specific roles of these altered pathways. In particular, the transcriptomic changes related to circadian rhythms and neuroinflammation should be validated using independent experimental approaches. Moreover, further investigations are necessary to elucidate the cellular and anatomical origins of the non-motor symptoms observed in ARSACS, which remain less well characterized compared to motor deficits. Gaining deeper insight into how these symptoms interact and evolve over time could help identify critical windows for therapeutic intervention and contribute to the development of more targeted treatment strategies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003e3.1 Fish husbandry and feeding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe adult \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish used in this study were generated through CRISPR/Cas9 genome editing in the wild-type AB background. These mutants carry a 10-bp deletion in exon 7 of the \u003cem\u003esacs\u003c/em\u003e gene, leading to a frameshift mutation and a premature stop codon at residue 495 (R487Kfs*495). This ensures that both WT and \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ezebrafish share the same genetic lineage, minimizing the risk of background-related variability. The wild-type AB strain was obtained from the Department of Veterinary Sciences at the University of Pisa. The homozygous \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish mutant line was crossed, and the F2 progeny were used to investigate the adult phenotype. The control animals used in the study were from the same batch of offspring as the \u003cem\u003esacs\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mutants and were genetically similar, except for the loss of function in the \u003cem\u003esacs\u003c/em\u003e gene. Animals were\u003csup\u003e\u0026nbsp;\u003c/sup\u003ekept in automated re-circulating systems\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(Zebtec, Tecniplast, Italy) with reverse osmosis filtered water equilibrated to reach the\u003csup\u003e\u0026nbsp;\u003c/sup\u003especies-recommended temperature (28\u0026deg; C \u0026plusmn; 2\u0026deg; C), pH (7.0 and 7.5), conductivity and\u003csup\u003e\u0026nbsp;\u003c/sup\u003eammonia, nitrite, nitrate and chloride levels. Animals were subjected to a light/dark\u003csup\u003e\u0026nbsp;\u003c/sup\u003ecycle of 14/10 hours. All experiments were conducted in accordance with the European Union (EU) Directive 2010/63/EU on the protection of animals used for scientific purposes, under the supervision of the Institutional Animal Care and Use Committee (IACUC) of the University of Pisa, and in compliance with the 3R principles. TUDCA (TUDCA, Millipore, Darmstadt, Germany ) supplementation was carried out by including the compound in the pelleted feed supplementation was carried out by including the compound in the pelleted feed (inclusion rate 0,1% on a fed basis).The purified drug was added to self-made experimental zebrafish feed according to published indications\u003csup\u003e107\u003c/sup\u003e. In brief, the dry feed was finely ground, mixed with osmosis water containing dissolved trehalose to form a dough, then re-pelleted, dried, and stored in the refrigerator until use\u003csup\u003e107\u003c/sup\u003e. In total, two different diets were prepared: \u0026nbsp; 1 control diet and 1 experimental diet, the latter containing TUDCA acid and sodium salt. The decision to use a 0.1% concentration was based on preliminary dosage/effect experiments on mortality and growth in both WT fish and \u003cem\u003esacs\u003c/em\u003e mutant fish, evaluated within the first 30 days of life. The chosen dosage was found to be the most effective. The experimental diet was administered twice daily from 5 dpf until adulthood (12 months). Food was provided every day at the same time, with quantities adjusted according to the fish\u0026apos;s age and body weight. Specifically, larvae were fed an amount equivalent to 9\u0026ndash;10% of their body weight, juveniles received 6\u0026ndash;8%, and adults were fed 5%, following recommendations for dry feed with appropriate protein and energy content, as described in\u003csup\u003e108\u003c/sup\u003e. Control fish were maintained under the same conditions as the treated fish but without exposure to any treatment. To exclude potential metabolic side effects, particularly since TUDCA had not previously been tested in zebrafish, we evaluated the fish at three months of age, as they are already considered adults at this stage. Welfare assessment was conducted following the guidelines outlined in the University of Queensland\u0026apos;s \u003cem\u003e\u0026ldquo;Score Sheet for Scoring Endpoints in Zebrafish\u003c/em\u003e\u0026rdquo; (https://research-support.uq.edu.au/resources-and-support/ethics-integrity-and-compliance/animal-ethics/monitoring-animals), evaluating several aspects such as free swimming behavior in the tank, body weight, and abdominal swelling. The overall welfare status of the analyzed subjects was determined based on the final score. \u0026nbsp;After completing the behavioral experiments, the fish were sacrificed, and organ dissection was performed on the same day. The extracted brains were preserved under identical conditions and subsequently processed as described in the following section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transcriptomic analysis, 4 brains were isolated from adult animals per experimental group. RNA was extracted from individual samples to maintain biological variability according to standard procedures. All animals from each experimental group were sacrificed and harvested on the same day to ensure consistency and avoid batch effects. RNA integrity and concentration were assessed using the RNA 6000 Pico Kit on a Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA integrity number was between 5.6 and 8.1 for all samples. Due to partial sample degradation, a ribodepletion approach for RNA-seq analysis was employed. 50ng total-RNA was depleted with \u003cem\u003eDanio rerio\u003c/em\u003e-specific rRNA probes using the dedicated ribo-Pool panel DP-R024-101 (SiTOOLS Biotech, Planegg, Germany). The depleted RNA was used to prepare the library using the SMARTer Stranded Total RNA-Seq kit v3 (Pico Input-Mammalian, Takara Bio, San Jose, CA). Quality and size of RNA-seq libraries were assessed by capillary electrophoretic analysis with an Agilent 4150 Tape station (Agilent Technologies). Libraries were quantified by real-time PCR against a standard curve with the KAPA Library Quantification Kit (KapaBiosystems, Wilmington, MA). Libraries were pooled at equimolar concentration and sequenced in 150PE on a NovaSeq6000 (Illumina, San Diego, CA), generating an average of 48.3 million fragments per sample. GO and KEGG pathway enrichment analysis of differentially expressed genes with padj \u0026lt; 0.05 were performed using ClusterProfiler (v3.18.1) [101]. Subsequently, the top 20 enriched GO biological process categories were plotted.\u0026nbsp;GSEA were performed to determine the enrichment of specific gene sets retrieved from the Molecular Signatures Database (https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Proteomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1 Protein isolation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor proteomic analysis, 4 brains were isolated from adult animals per experimental group. RNA was extracted from individual samples to maintain biological variability according to standard procedures. All animals from each experimental group were sacrificed and harvested on the same day to ensure consistency and avoid batch effects. Zebrafish brains were pottered and passed through a 70-\u0026mu;m diameter filter before undergoing a wash with PBS supplemented with protease inhibitors (20 g/ml leupeptin, 25 g/ml aprotinin, 10 g/ml pepstatin, 0.5 mM benzamide) and phosphatase inhibitor (1 mM Na3VO4, Merck, Darmstadt, Germany, Cat. No. 13721-39-6) followed by centrifugation at 4\u0026deg;C, 1500 rpm for 15 min. The pellet was then disrupted by RIPA buffer, incubated at 4\u0026deg;C for 60 min, vortexed four times (once every 15 min), and then sonicated for 30 s. Samples were centrifuged at 13,850 rcf for 10 min and supernatants were collected, measured, and supplemented with the same amount of 20% SDS-6% DTT. Subsequently, they were incubated at 95 \u0026deg;C for 5 min. Once cooled, five volumes of MATF (methanol, acetone, and tributyl phosphate, 1:12:1) were added, and samples were incubated for 1 h with agitation. The total proteins were finally pelleted by centrifugation at 12,000 rcf for 15 min, the supernatants were removed, and the protein precipitates dried for about 30 min at RT. The dried pellets were resuspended in 250 \u0026mu;L of 5% SDS in 50 mM ammonium bicarbonate. After quantification, proteins underwent reduction and alkylation followed by trypsin digestion by using Preomics iST (PreOmics, Billerica, MA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.2 Mass spectrometry\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTryptic digests were analysed using an Ultimate 3000 chromatography system (Thermo Scientific Instruments, Waltham, MA, USA) equipped with a PepMap RSLC C18 EASY spray column (Thermo Scientific Instruments, Cat. No. 13294749) at a flow rate of 250 nl/min and a temperature of 60\u0026deg;C. Eluate was analysed by mass spectrometry (MS) using a Q Exactive\u0026trade; Plus Hybrid Quadrupole-Orbitrap\u0026trade; mass spectrometer equipped with an Easyspray source (both Thermo Scientific Instruments). The data were acquired, in the mass range of 200\u0026ndash;2,000 m/z, in data-dependent (DDA) mode alternating between MS and MS/MS scans, using the software Xcalibur (version 4.1, Thermo Scientific Instruments). To perform MS analysis, the desiccated tryptic digests were resuspended with 1.5 mL of the LC-LOAD component of the iST-BCT kit (PreOmics), and analysed by nano-UHPLC-MS/MS using an Ultimate 3000 chromatography system equipped with a PepMap RSLC C18 EASY spray column (75 mm, 50 cm, 2 mm particle size) (both Thermo Fisher Scientific) at a flow rate of 250 nL/min and a temperature of 60\u0026deg;C. The mobile phases were: (A) 0.1% v/v formic acid in water, and (B) 80% acetonitrile, 20% water and 0.08% v/v formic acid. A 105-min gradient was selected: 0.0\u0026ndash;3.0 min isocratic 2% B; 3.0\u0026ndash;7.0 min 7% B; 7.0\u0026ndash;65.0 min 30% B; 65.0\u0026ndash;78.0 min 45% B; 78.0\u0026ndash;83.0 min 80% B; 83.0\u0026ndash;85.0 isocratic 80% B; 85.0\u0026ndash;85.1 2% B; and finally, 85.1\u0026ndash;105.0 isocratic 2% B. After separation, the eluate was sent directly to an Easyspray source connected to a Q Exactive\u0026trade; Plus Hybrid Quadrupole-Orbitrap\u0026trade; mass spectrometer (Thermo Fisher Scientific). The data were acquired in DDA mode, alternating between MS and MS/MS scans. The software Xcalibur (version 4.1, Thermo Fisher Scientific) was used for operating the UHPLC/HR-MS. MS scans were acquired at a resolution of 70,000 between 200 and 2000 m/z, with an automatic gain control (AGC) target of 3.0 x 106 and a maximum injection time (maxIT) of 100 ms. MS/MS spectra were acquired at a resolution of 17,500 with an AGC target of 1.0 x 105 and a maxIT of 50 ms. A quadrupole isolation window of 2.0 m/z was used, and HCD was performed using 30% normalized collision energy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.4 Mass spectrometry data analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMass spectrometry data were processed with ProteomeDiscoverer\u0026reg; (v. 2.4.1.15) (Thermo Scientific Instrument, Waltham, MA) using a workflow adapted for LTQ ORBITRAP label-free quantification. Briefly, in the processing step we used the \u003cem\u003eDanio rerio\u003c/em\u003e - tr_canonical v2022-03-02 database for identifying peptide spectral matches in MS/MS spectra and concatenated decoy (strict target false discovery rate = 0.01, relaxed target false discovery rate = 0.05 for proteins, peptides and peptide spectral matches). Protein quantification was calculated by summed abundances of peptides, and the differential analysis was performed pairwise ratio-based and t-test background-based using the IMP-apQuant node. After conducting a proteome differential analysis, differentially expressed proteins were identified for each experimental point. These were then used to perform further gene ontology analyses. Data were deposited in the PRIDE repository (PXD058522).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Behavioural analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNovel tank\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Zebrafish were exposed to the experimental challenge in a pre-treatment beaker before being transferred (via a net) to the novel tank for behavioural observation and phenotyping as previously reported\u003csup\u003e109,110\u003c/sup\u003e . After pre-treatment, zebrafish were placed individually in a 1.5-L trapezoidal tank maximally filled with aquarium water. In these experiments, behavioural activity was recorded for 5 min using a webcam set up in front of the tank to analyse the diving response. The tank was virtually divided into two areas (bottom and top) and time spent in the bottom part was used to assess anxiety-like phenotypes.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eZebrafish behavioral activity was recorded (1920 \u0026times; 1080 px). The videos were analysed using Noldus EthoVision\u0026reg; XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen-field test and thigmotaxis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBehavioural experiments were conducted at between 10 a.m. and 4 p.m. in a 30 \u0026times; 30 \u0026times; 30 cm tank with opaque walls and partitions. A video camera was suspended above the tank. Adult zebrafish were allowed to swim for 5 min inside the tank, and 5-min videos were recorded. Thigmotaxis was evaluated as reported elsewhere\u003csup\u003e111\u003c/sup\u003e .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 Shoaling test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBehavioural experiments were conducted at between 10 a.m. and 4 p.m. in a 30 \u0026times; 30 \u0026times; 30 cm tank with opaque walls and partitions. Adult zebrafish were acclimated to the novel tank apparatus for 5 min before the test, after which 5-min videos were recorded. The shoaling assessment was performed by measuring the inter-fish distance (the average of all distances between each zebrafish in a shoal). The method for measuring inter-fish distance was also based on the analysis provided by Noldus EthoVision\u0026reg; XT17 software. This software tracks the precise position of each fish in the arena over time, allowing for accurate inter-fish distance measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.3 Social preference test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSocial preference testing was performed with three chambers as already described\u003csup\u003e23\u003c/sup\u003e. In general, social behaviour is assessed by observing how an individual respond to, or interacts with, a social stimulus. Social preference tests are composed of two operational phases. The first is the habituation phase, during which the tested zebrafish is left alone in a chamber of the test tank to explore the novel environment. The second is the interaction phase, which starts with the introduction of the social stimulus consisting of one, or usually two, small groups of live conspecifics\u003csup\u003e23\u003c/sup\u003e. The zebrafish behaviours were quantified as distance distribution or as presence in a zone adjacent to the group of conspecifics. The time ratio [Time ratio %=Time spent in conspecific sector/ total time observed)\u0026times;100]. The distance ratio [(Distance ratio %=Time spent in conspecific sector/ total time observed) \u0026times;100]. The videos were analysed using Noldus EthoVision\u0026reg; XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.4 Novel object recognition task\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBehavioural experiments were conducted at between 10 a.m. and 4 p.m. in a 30 \u0026times; 30 \u0026times; 30 cm tank with opaque walls and partitions. Before training, each animal was habituated to the experimental apparatus (open field) in the absence of objects for 5 min. In the training phase (phase 1), animals were exposed to two identical cubes (of the same colour) for 10 min. After training, the animals were submitted to a retention interval of 1 h. This separation period is a standard methodological approach used to assess short-term memory, allowing for memory consolidation and preventing immediate recognition effects that could bias the results. In the test (phase 2), a new object (of a different colour) replaced one of the copies of the familiar object and the time spent exploring each object during a period of 10 minutes was evaluated. The familiar blue objects used in phase 2 were exactly the same ones from phase 1, ensuring continuity in the experimental setup. To avoid a thigmotaxis effect, the distances between the objects and the walls were kept the same. We calculated the exploration time of each object (%). This measurement reflects proximity rather than active exploration based on head orientation. The exploration area was defined as an 8 \u0026times; 8 cm area centred on the object and preference times were calculated as reported in\u003csup\u003e30\u003c/sup\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe videos were analysed using Noldus EthoVision\u0026reg; XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe videos were analysed using Noldus EthoVision\u0026reg; XT17 tracking software (Noldus Information Technologies, Inc., Leesburg, VA, USA).\u003csup\u003e\u0026nbsp;\u003c/sup\u003eData analysis was performed using GraphPad Prism software. ANOVA followed by Tukey\u0026rsquo;s multiple comparison test was used to assess significance. Statistical significance is reported as follows: * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt;0.001, or **** p \u0026lt; 0.0001. Graphs were generated using GraphPad Prism 9 software. Data are presented as mean \u0026plusmn; S.E.M\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomics data that support the findings of this study have been deposited in the PRIDE repository (PXD058522). Transcriptomics data have been deposited in the ZENODO repository (https://zenodo.org/records/14809944) and (https://zenodo.org/records/15123596).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Fondation de l\u0026rsquo;Ataxie Charlevoix-Saguenay (seed grant 2022 to V.N.) and in part by\u0026nbsp;Fondazione Telethon (grants\u0026nbsp;GSA21G006 and\u0026nbsp;GSA24A001 to V.N.) FMS is partially supported by Ricerca Corrente 2024-25 (RC 5X1000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not include human samples. All animal procedures were authorized by the University of Pisa Animal Ethics Committee and the Italian Ministry of Health (n\u0026deg; 620/2024-PR). The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValentina Naef\u003c/strong\u003e: Writing \u0026ndash; original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.\u0026nbsp;\u003cstrong\u003eStefania Della Vecchia\u003c/strong\u003e:\u0026nbsp;review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Michela Giacich:\u0026nbsp;\u003c/strong\u003eVisualization \u0026amp; Data curation\u0026nbsp;\u003cstrong\u003eRosario Licitra\u003c/strong\u003e: Methodology. \u0026nbsp;\u003cstrong\u003eTiziana Bachetti:\u003c/strong\u003e Visualization, Formal analysis \u0026amp; Data curation.\u0026nbsp;\u003cstrong\u003eGabriela Coronel Vargas\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eMethodology\u003cstrong\u003e. Marco Ponassi:\u003c/strong\u003e Methodology. \u0026nbsp;\u0026nbsp;\u003cstrong\u003eFilippo M. Santorelli\u003c/strong\u003e:\u0026nbsp;Writing \u0026ndash; review \u0026amp; editing, Supervision \u0026amp; Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Fondation de l\u0026rsquo;Ataxie Charlevoix-Saguenay and the the Italian patients\u0026apos; association (ARSACS OdV) for their constant encouragement and support. We acknowledge the editorial support of Dr. Catherine J. Wrenn for expert language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBouhlal, Y., Amouri, R., El Euch-Fayeche, G. \u0026amp; Hentati, F. 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Autism\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 23 (2018).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ARSACS, Ataxia, Zebrafish, Neurodegeneration, TUDCA","lastPublishedDoi":"10.21203/rs.3.rs-5957432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5957432/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\u003cb\u003e)\u003c/b\u003e is an early-onset neurodevelopmental and neurodegenerative disorder characterized by ataxia, spasticity, and peripheral neuropathy. However, several studies have highlighted that some patients also experience cognitive, emotional and social deficits, suggesting a more complex clinical picture that extends beyond motor symptoms. Building on these findings, this study aimed to: i) investigate locomotor, social and cognitive deficits in adult \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish versus wild-type (WT) controls through behavioural tests; ii) identify molecular patterns associated with the adult disease phenotype using transcriptomic and proteomic analyses of \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e and WT brains; iii) evaluate the effectiveness of long-term treatment with tauroursodeoxycholic acid (TUDCA) on behavioural outcomes and omics profiles in the zebrafish \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e model. Our findings indicate impairments in cognitive, social, and emotional behaviors in aged \u003cem\u003esacs\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e zebrafish, which resemble some deficits observed in human patients. Transcriptomic and proteomic analyses of adult brains identified alterations in genes related to circadian rhythms and neuroinflammation. Notably, disruptions in sleep and circadian rhythms are frequently reported in individuals with cerebellar ataxia and may contribute to cognitive and behavioral changes. Long-term treatment with TUDCA, a neuroprotective molecule, was associated with partial improvements in social and cognitive behaviors and modifications in omics profiles in the zebrafish model. These results support the potential of further exploring TUDCA in future preclinical and clinical studies, while also emphasizing the need for additional investigations to better understand its mechanisms of action.\u003c/p\u003e","manuscriptTitle":"Long-Term Benefits of TUDCA Supplement in ARSACS Zebrafish Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 11:39:54","doi":"10.21203/rs.3.rs-5957432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-19T08:18:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T23:45:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308677661970692851416279600964454022083","date":"2025-06-03T17:36:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-18T13:59:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302881794310633132477201745611536337482","date":"2025-05-05T13:28:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122504600442436240972387717652168760706","date":"2025-05-02T13:49:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-02T11:26:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-22T12:30:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-07T15:36:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2945ec02-9c97-44cf-9c83-dbc485f1d2b3","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46803230,"name":"Biological sciences/Drug discovery"},{"id":46803231,"name":"Biological sciences/Molecular biology"},{"id":46803232,"name":"Biological sciences/Neuroscience"},{"id":46803233,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2025-07-21T16:00:14+00:00","versionOfRecord":{"articleIdentity":"rs-5957432","link":"https://doi.org/10.1038/s41598-025-10850-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-14 15:57:18","publishedOnDateReadable":"July 14th, 2025"},"versionCreatedAt":"2025-04-16 11:39:54","video":"","vorDoi":"10.1038/s41598-025-10850-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-10850-0","workflowStages":[]},"version":"v1","identity":"rs-5957432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5957432","identity":"rs-5957432","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}