Mafalda Raposo

Spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease, the most common form of autosomal dominant cerebellar ataxia, is caused by a CAG repeat expansion in ATXN3 . The mutant protein is prone to aggregation and exerts toxic gain-of-function effects, whereas the wild-type protein has important neuroprotective roles as a deubiquitinase. Current treatment strategies for SCA3 lack specificity for the mutant allele. Identifying genetic variants such as single-nucleotide polymorphisms (SNPs) in linkage with the pathogenic CAG expansion offers a promising avenue to develop allele-specific therapeutic interventions, like antisense oligonucleotides (ASOs). To address this, we conducted allele-specific, targeted, multiplexed nanopore sequencing on 79 patients with SCA3 from Germany, UK, and Portugal. We identified almost 200 polymorphisms and grouped them into three haplotype blocks on the basis of linkage disequilibrium. These SNPs were strongly associated with the CAG expansion. Genotypic modeling of our subcohorts indicated that targeting only two SNPs from different blocks could enable allele-specific treatment of the majority of European patients with SCA3. We evaluated 17 candidate ASOs targeting SNPs from different haplotype blocks. Seven of these ASOs caused allele-preferential reduction of more than 65%. A single ASO application in two SCA3 iPSC-derived cortical neurons (AX1, AX2) achieved up to 90% reduction of mutant ATXN3. Toxicity assessments together with RNA sequencing analyses in AX1 did not indicate evidence of toxicity or widespread transcriptional perturbations. These findings highlight that characterizing heterozygosity rates offers valuable opportunities for selective ASO therapies. Our proof-of-principle targeting with ASOs demonstrates the feasibility of haplotype-informed allele-specific silencing in ATXN3 .

Although the CAG repeat expansion in the ATXN3 gene was identified over 30 years ago as the cause of Machado-Joseph disease (MJD), the disorder remains untreatable. Notably, MJD is the most prevalent hereditary spinocerebellar ataxia worldwide and is particularly frequent in the Azores Islands (Portugal). This results from two independent founder effects, with two major ancestral lineages – “Joseph” and “Machado” - segregating in Azorean families. Although MJD pathogenesis is mainly driven by the (CAG) _n expansion, regulatory cis- elements may modulate ATXN3 expression, thereby influencing phenotypic variation. Here, we investigated allele-specific expression (ASE) of the ATXN3 gene and examined whether distinct ATXN3 lineages modulate the differential expression of non-expanded and expanded alleles, as well as its association with age at onset. We quantified ATXN3 ASE in 38 cDNA samples from the blood of Azorean MJD expansion carriers. Notably, all 28 carriers of the Joseph lineage demonstrated higher relative expression of the expanded allele, whereas eight of the 10 Machado lineage carriers exhibited the opposite trend (Mann-Whitney U test, p < 0.0001). By incorporating genetic and clinical data from an additional 76 Azorean MJD patients we found that, based on the expanded allele size alone, Joseph carriers would be expected to develop symptoms later than Machado carriers. Both lineages, however, report similar ages at onset. suggesting a counterbalance effect of ATXN3 ASE: higher expression of the expanded allele in Joseph carriers may shift individuals toward earlier onset, while higher expression of the non-expanded allele in Machado carriers, may contribute to delayed onset. Our findings show that ATXN3 exhibits haplotype-dependent allelic imbalance. This imbalance may be tissue-specific, underscoring the need for future studies using brain samples. Furthermore, it will be important to determine whether the observed association with age at onset is driven primarily by increased levels of the expanded protein, leading to enhanced protein toxicity, or by toxic effects at the RNA level.

Background Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited adult-onset disease. We aimed to describe longitudinal changes in clinical and biological findings and to identify predictors for clinical progression. Methods We used data from participants enrolled in the ESMI cohort collected between Nov 09, 2016 and July 18, 2023. The data freeze included data from 14 sites in five European countries and the United States. We assessed ataxia with the Scale for the Assessment and Rating of Ataxia (SARA). We measured disease-specific mutant ataxin-3 protein (ATXN3) and neurofilament light chain (NfL) in plasma and performed MRIs. Data were analysed by regression modelling on a timescale defined by onset. The onset of abnormality of a marker was defined as the time at which its value, as determined by modelling, exceeded the mean ±2 SD of healthy controls. To study responsiveness of markers, we determined the sensitivity to change ratios (SCSs). Results Data from 291 SCA3 mutation carriers before and after clinical onset and 121 healthy controls were included. NfL levels became abnormal more than 20 years (-21.5 years [95% CI n.d. –9.5]) before onset. The earliest MRI abnormality was volume loss of medulla oblongata (-4.7 years [95% CI n.d. – 3.3]). The responsiveness of markers depended on the disease stage. Across all stages, pons volume had the highest responsiveness with an SCS of 1.35 [95% CI 1.11 – 1.78] exceeding that of SARA (0.99 [95% CI 0.88 – 1.11]). Lower age (p=0.0459) and lower medulla oblongata volume (pConclusion Our study provides quantitative information on the progression of biological markers in SCA3 mutation carriers before and after onset of ataxia, and allowed the identification of predictors for clinical progression. Our data could prove useful for the design of future clinical trials.

Introduction: Knowledge about the distribution and frequency of the respective haplotypes on the wildtype and mutant allele is highly relevant in the context of future gene therapy clinical studies in Spinocerebellar Ataxia Type 3, the most common autosomal dominantly inherited ataxia. Single nucleotide polymorphisms associated to the disease-causing gene, ATXN3 , have been determined. We wanted to investigate the frequency and regional distribution of two intragenic single nucleotide polymorphisms (SNPs) in a large European SCA3 cohort and their relation to the clinical phenotype. Methods: The genotypes of the two polymorphisms at base pair positions 987 and 1118 of the ATXN3 were determined for their co-localization on the normal and expanded allele, respectively, in 286 SCA3 mutation carriers and 117 healthy controls from 11 European sites. Results: The distribution of genotypes on the expanded allele differed from those of the wildtype allele of SCA3 mutation carriers and of healthy controls, and was mainly influenced by the regional origin. In our cohort, no particular clinical phenotype was associated with any specific haplotype. Conclusions: Our results confirm distinct allocations of SNPs associated to the expanded ATXN3 , and accordingly the consideration of allele-specific therapies.

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is caused by the expansion of a genetically unstable polyglutamine-encoding CAG repeat in ATXN3. Longer alleles are generally associated with earlier onset and frequent intergenerational expansions mediate the anticipation observed in this disorder. Somatic expansion of the repeat has also been implicated in disease onset and slowing the rate of somatic expansion has been proposed as a therapeutic strategy. Here, we utilised high-throughput ultra-deep MiSeq amplicon sequencing to precisely define the number and sequence of the ATXN3 repeat, the genotype of an adjacent single nucleotide variant and quantify somatic expansion in blood and buccal swab DNA of a cohort of individuals with SCA3 from the Azores islands (Portugal). We revealed systematic mis-sizing of the ATXN3 repeat and high levels of inaccuracy of the traditional fragment length analysis that have important implications for attempts to identify modifiers of clinical and molecular phenotypes. Quantification of somatic expansion in blood DNA and multivariate regression revealed the expected effects of age at sampling and CAG repeat length, although the effect of repeat length was surprisingly modest with much stronger associations with age. We also observed an association of the downstream rs12895357 single nucleotide variant with the rate of somatic expansion, and a higher level of somatic expansion in buccal swab DNA compared to blood. These data suggest that the ATXN3 locus in SCA3 patients in blood or buccal swab DNA might serve as a good biomarker for clinical trials testing suppressors of somatic expansion with peripheral exposure.

Spinocerebellar ataxia type 3 (SCA3), a currently untreatable disorder, is caused by the expansion of a genetically unstable polyglutamine-encoding complex CAG repeat in the ATXN3 gene. Longer alleles are generally associated with earlier onset and frequent intergenerational expansions mediate the anticipation observed in this disorder. Somatic expansion of the repeat has also been implicated in disease progression and slowing the rate of somatic expansion in patients has recently been proposed as a therapeutic strategy. Here, we utilised high-throughput ultra-deep MiSeq amplicon sequencing of the ATXN3 repeat to precisely define the number of repeats, the exact sequence structure, the phased genotype of an adjacent single nucleotide polymorphism and to accurately quantify somatic expansion in blood and buccal cell DNA samples of a cohort of individuals with SCA3 from the Azores islands (Portugal). We revealed systematic mis-sizing of the ATXN3 repeat and high levels of inaccuracy of the traditional fragment length analysis approach that have important implications for attempts to identify modifiers of clinical and molecular phenotypes, including genetic instability. Quantification of somatic expansion in blood DNA revealed the expected effects of age and CAG repeat length, although the effect of repeat length was surprisingly modest with much stronger associations with age at sampling. We also observed an association of the downstream rs12895357 single nucleotide polymorphism with the rate of somatic expansion, and a higher level of somatic expansion in buccal cell DNA compared to blood. Although the levels of somatic expansion are much lower per repeat unit, the average level of somatic expansion at the ATXN3 locus in SCA3 patients is much higher than is typically observed at the HTT locus in Huntington disease patients. These data suggest that the ATXN3 locus in SCA3 patients in blood or buccal cell DNA might serve as a good biomarker for clinical trials testing suppressors of somatic expansion with peripheral exposure.

Spinocerebellar ataxia type 3 (SCA3) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the ataxin-3 (ATXN3) gene. No effective treatment is available for this disorder, other than symptom-directed approaches. Bile acids have shown therapeutic efficacy in neurodegenerative disease models. Here, we pinpointed tauroursodeoxycholic acid (TUDCA) as an efficient therapeutic, improving the motor and neuropathological phenotype of SCA3 nematode and mouse models. Surprisingly, transcriptomic and functional in vivo data showed that TUDCA acts in neuronal tissue through the glucocorticoid receptor (GR), but independently of its canonical receptor, the farnesoid X receptor (FXR). TUDCA was predicted to bind to the GR, in a similar fashion to corticosteroid molecules. GR levels were decreased in disease-affected brain regions, likely due to increased protein degradation as a consequence of ATXN3 dysfunction being restored by TUDCA treatment. Analysis of a SCA3 clinical cohort showed intriguing correlations between the peripheral expression of GR and the predicted age at disease onset in presymptomatic subjects and FKBP5 expression with disease progression, suggesting this pathway as a potential source of biomarkers for future study. We have established a novel in vivo mechanism for the neuroprotective effects of TUDCA in SCA3 and propose this readily available drug for clinical trials in SCA3 patients.

Machado-Joseph disease (MJD) is a dominant neurodegenerative disease caused by an expanded CAG repeat in the ATXN3 gene encoding the ataxin-3 protein. Several cellular processes, including transcription and apoptosis, are disrupted in MJD. To gain further insights into the extent of dysregulation of mitochondrial apoptosis in MJD and to evaluate if expression alterations of specific apoptosis genes/proteins can be used as transcriptional biomarkers of disease, the expression levels of BCL2, BAX and TP53 and the BCL2/BAX ratio (an indicator of susceptibility to apoptosis) were assessed in blood and post-mortem brain samples from MJD subjects and MJD transgenic mice and controls. While patients show reduced levels of blood BCL2 transcripts, this measurement displays low accuracy to discriminate patients from matched controls. However, increased levels of blood BAX transcripts and decreased BCL2/BAX ratio are associated with earlier onset of disease, indicating a possible association with MJD pathogenesis. Post-mortem MJD brains show increased BCL2/BAX transcript ratio in the dentate cerebellar nucleus (DCN) and increased BCL2/BAX insoluble protein ratio in the DCN and pons, suggesting that in these regions, severely affected by degeneration in MJD, cells show signs of apoptosis resistance. Interestingly, a follow-up study of 18 patients further shows that blood BCL2 and TP53 transcript levels increase over time in MJD patients. Furthermore, while the similar levels of blood BCL2, BAX, and TP53 transcripts observed in preclinical subjects and controls is mimicked by pre-symptomatic MJD mice, the expression profile of these genes in patient brains is partially replicated by symptomatic MJD mice. Globally, our findings indicate that there is tissue-specific vulnerability to apoptosis in MJD subjects and that this tissue-dependent behavior is partially replicated in a MJD mouse model.

Spinocerebellar ataxia type 3/Machado–Joseph disease (SCA3) is the most common autosomal dominant ataxia. In view of the development of targeted therapies for SCA3, precise knowledge of stage-dependent fluid and MRI biomarker changes is needed. We analyzed cross-sectional data of 292 SCA3 mutation carriers including 57 pre-ataxic individuals, and 108 healthy controls from the European Spinocerebellar ataxia type 3/Machado-Joseph Disease Initiative (ESMI) cohort. Blood concentrations of mutant ATXN3 and neurofilament light (NfL) were determined, and volumes of pons, cerebellar white matter (CWM) and cerebellar grey matter (CGM) were measured on MRI. Mutant ATXN3 concentrations were high before and after ataxia onset, while NfL continuously increased and deviated from normal 11.9 years before onset. Pons and CWM volumes decreased, but the deviation from normal was only 2.0 years (pons) and 0.3 years (CWM) before ataxia onset. We propose a staging model of SCA3 that includes an initial asymptomatic carrier stage followed by the biomarker stage defined by absence of ataxia, but a significant rise of NfL. The biomarker stage leads into the ataxia stage, defined by manifest ataxia. The present analysis provides a robust framework for further studies aiming at elaboration and differentiation of the staging model of SCA3.