Biology subjects

Santana, M. M.

Publications and source records attributed to Santana, M. M..

4 recordsLinked to original sources

bioRxiv2025

rAAV-Delivered Bicistronic Artificial microRNAs for Allele-Specific Silencing Improve Motor and Molecular Outcomes in Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominant neurodegenerative disorder caused by the expansion of CAG trinucleotide repeats in the ATXN3 gene. This mutation induces a toxic gain-of-function of the ATXN3 protein, leading to neurodegeneration, particularly in the cerebellum and brainstem. Despite extensive research, no disease-modifying treatments are available for SCA3 patients. In this study, we developed and tested a novel therapeutic strategy using recombinant adeno-associated virus (rAAV) to deliver bicistronic artificial microRNAs designed to selectively silence the mutant ATXN3 allele. Through in vitro screening, we identified a lead construct (miATXN3-10x2) that effectively and specifically silenced the mutant allele by targeting of a single nucleotide polymorphism (SNP) associated with the repeat expansion. This construct was packaged into rAAV9 and delivered via intra-cerebellar administration into two mouse models of SCA3, resulting in robust suppression of mutant ATXN3 in the cerebellum. To assess long-term efficacy, we performed intra-cisterna magna (ICM) injections of rAAV9-miATXN3-10x2 in a severe SCA3 transgenic mouse model. Widespread distribution of viral vectors and miATXN3 copies was observed in disease-relevant brain regions. Treated animals exhibited significant and sustained improvements in motor function at 5, 8, and 11 weeks post-injection. Histological analyses showed a reduction in mutant ATXN3 aggregates and a trend toward preventing shrinkage of cerebellar molecular layer. These findings were supported by dose-dependent reductions in mutant ATXN3 mRNA levels and decreased expression of neuroinflammatory markers in the cerebellum. Additionally, a significant increase of the neuronal marker NeuN was also observed in treated animals. Finally, transcriptomic profiling of the cerebellum demonstrated that treated transgenic animals exhibited an improved transcriptomic signature, shifting toward a wild-type profile. In conclusion, our findings highlight the therapeutic potential of a single administration of rAAVs encoding bicistronic artificial microRNAs for allele-specific gene silencing in SCA3. This study provides compelling preclinical evidence supporting the translation of this approach into clinical applications for SCA3 patients.

molecular biology↗

bioRxiv2025

Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder, caused by an over-repetition of the polyglutamine-codifying region in the ATXN3 gene. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease. In order to prevent the downstream toxic pathways, our goal was to develop gene editing-based strategies to permanently inactivate the human ATXN3 gene. TALENs and CRISPR-Cas9 systems were designed to target exon 2 of this gene and functional characterization was performed in a human cell line. After the demonstration of TALENs and CRISPR-Cas9 efficiency on gene disruption, a sequence of each system was selected for further in vivo experiments. Although both TALENs and CRISPR-Cas9 systems led to a drastic reduction of ATXN3 aggregates in the striatum of a lentiviral-based mouse model of MJD/SCA3, only CRISPR-Cas9 system allowed the improvement of key neuropathological markers of the disease. Importantly, the administration of the engineered system in YAC-MJD84.2/84.2 mice mediated a delay in disease progression, when compared with non-treated littermates. These data provide the first in vivo evidence of the efficacy of a CRISPR-Cas9-based approach to permanently inactivate the ATXN3 gene in the brain of two mouse models of the disease, supporting its potential as a new therapeutic avenue in the context of MJD/SCA3.

neuroscience↗

bioRxiv2025

Circadian Rhythms are Disrupted in Patients and Preclinical Models of Machado-Joseph Disease

Machado-Joseph disease (MJD) is caused by an abnormal CAG repeat expansion in the ATXN3 gene, leading to the expression of a mutant ataxin-3 (mutATXN3) protein. MJD patients exhibit a wide range of clinical symptoms, including motor incoordination. Emerging evidence highlights circadian rhythm disruptions as early indicators and potential risk factors for the progression of neurodegenerative conditions. Circadian rhythms are regulated by internal clocks, with the suprachiasmatic nucleus (SCN) acting as the master pacemaker to synchronize timing across the bodys behavioural and physiological functions. While sleep disturbances have been observed in MJD, the role of clock regulation in its pathophysiology remains largely unexplored in spinocerebellar ataxias. This study aimed to investigate circadian rhythms, characterize associated disruptions, and uncover the mechanisms underlying clock dysregulation in patients and preclinical models of MJD. Circadian activity in MJD patients was assessed over two weeks using actigraphy, while in a YAC-MJD transgenic mouse model, circadian rhythms were examined through: (a) wheel-running experiments; (b) telemetry-based monitoring of core body temperature; (c) immunohistochemical analysis of the neuropeptides arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) in the SCN and paraventricular nucleus (PVN); and (d) RT-qPCR evaluation of clock gene expression in the cerebellum. The impact of mutATXN3 on clock mechanisms was further investigated using Bmal1/Per2-luciferase reporters. MJD patients exhibited a progressive decline in robustness of behavioural rhythms, demonstrated by negative correlations between the circadian function index, rest-activity fragmentation, and sleep efficiency with MJD clinical scales. YAC-MJD mice exhibited reduced activity levels, increased behavioural fragmentation, and required three additional days to re-entrain after a jet lag protocol, compared to controls. Disrupted core body temperature rhythms were observed, including a phase advance and elevated temperature ([~]1 {degrees}C) at the onset of the active period. Furthermore, transgenic mice showed reduced levels of VIP and AVP in the SCN and PVN, and decreased clock gene expression in the cerebellum. Lastly, we found new mechanistic evidence that WT ATXN3 activates the promoters of Bmal1 and Per2, whereas mutATXN3 loses the capacity to drive Per2 upon polyglutamine expansion. Overall, our findings indicate that central clock dysfunction in MJD is associated with impaired clock gene expression and disruptions in activity and temperature rhythms. This study provides the first robust evidence of circadian rhythm dysregulation and underlying mechanisms in MJD, paving the way for the identification of new biomarkers and the development of novel circadian-based interventions to tackle MJD and possibly other spinocerebellar ataxias. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/631212v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@25078dorg.highwire.dtl.DTLVardef@112e523org.highwire.dtl.DTLVardef@1818356org.highwire.dtl.DTLVardef@1d5a6c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Blood and cerebellar abundance of ATXN3 splice variants in spinocerebellar ataxia type 3/Machado-Joseph disease

BackgroundSpinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) is an autosomal dominant polyglutamine disease. SCA3/MJD causative gene, ATXN3, is known to undergo alternative splicing (AS) and 54 transcripts are currently annotated. Differences in the toxicity of ataxin-3 protein isoforms, harbouring on its C-terminus two or three ubiquitin interacting motifs (UIMs), were previously uncovered, raising the hypothesis that specific ATXN3 splice variants play key roles in promoting the selective toxicity displayed in SCA3/MJD. MethodsUsing RNA-seq datasets we identified and determined the abundance of annotated ATXN3 transcripts in blood (n=60) and cerebellum (n=12) of SCA3/MJD subjects and controls. ResultsGlobally, the number and the abundance of individual ATXN3 transcripts were higher in the cerebellum than in the blood. While the most abundant transcript in the cerebellum was a protein with a coding sequence not defined of unknown function (ATXN3-208), the transcript with the highest abundance in blood was the reference transcript (ATXN3-251) which translates into an ataxin-3 isoform harboring three UIMs. Noteworthy, the abundance of ATXN3-251 and ATXN3-214, two out of the four transcripts that encode full-length ataxin-3 protein isoforms but differ in the C-terminus were strongly related with tissue expression specificity: ATXN3-251 (3UIM) was expressed in blood 50-fold more than in cerebellum, whereas ATXN3-214 (2UIM) was expressed in the cerebellum 20-fold more than in blood. ConclusionsThese findings provide new insights into the elucidation of ATXN3 AS in different tissues, contributing for a better understanding of SCA3/MJD pathogenesis and providing information for the development of future effective ATXN3 mRNA-lowering therapies.

genetics↗