Recent advances in understanding dominant spinocerebellar ataxias from clinical and genetic points of view

Introduction

Spinocerebellar ataxias (SCAs) are a group of neurodegenerative diseases displaying autosomal dominant inheritance. To date, 47 SCA subtypes have been described, and 35 causal genes have been identified¹. SCAs are considered a rare group of cerebellar ataxias, with a mean prevalence of 2.7/100,000². Their most frequent forms are polyglutamine (polyQ) expansion diseases (ATXN1/SCA1, ATXN2/SCA2, ATXN3/SCA3, CACNA1A/SCA6, ATXN7/SCA7, TBP/SCA17, and ATN1/DRPLA)³. These diseases manifest above a threshold number of CAG repeats, which is different for each gene. The same mutational mechanism is found in Huntington disease⁴ and in spinal bulbar muscular atrophy⁵. Disease onset generally occurs between the ages of 20 and 40 years, and age at onset and CAG repeat expansion size are inversely correlated^6,7. Age at onset also displays variability due to interactions between polyQ genes, i.e. between the expanded allele and the alleles of normal repeat size in the other genes^8,9. The instability of expanded alleles results in genetic anticipation in successive generations³. Unsteady gait and clumsiness are usually the first clinical symptoms at onset, followed by a progressive loss of the ability to walk. Cerebellar syndrome is often associated with extracerebellar signs (pyramidal syndrome, extrapyramidal syndrome, retinal degeneration, dementia, seizures, etc.)³. There is currently no preventive or curative treatment, but different therapeutic approaches are being tested, including the use of disease-modifying compounds and therapeutic approaches, which are described below. However, in SCAs, the lack of sensitive biomarkers and the small number of patients to be included in clinical trials are a challenge for the statistical sample size estimation. Outcome measures providing information about treatment efficacy are vital, particularly for these genetic diseases that can be treated before the onset of symptoms. This review provides an overview of findings for biomarkers, recent results of clinical trials in SCA patients, and future perspectives for treatment.

Natural history of spinocerebellar ataxias (SCA1, 2, 3, and 6)

The natural history of SCA1, 2, 3, and 6 was established by the longitudinal European cohort study EUROSCA, which included 462 patients with at least one year of follow-up and a median of 49 months of observation¹⁰. This study used the Scale for the Assessment and Rating of Ataxia (SARA), an accurate measurement of cerebellar dysfunction^11,12 in SCA1, 2, 3, and 6 patients and premanifest individuals¹³. The most severe disease turned out to be SCA1, with an annual progression of 2.11 on the 40-point SARA, followed by SCA3 (1.56 points), SCA2 (1.49 points), and SCA6 (0.8 points)¹⁰. SCA1 has also been reported to have the most severe prognosis for progression in other studies, such as one in Europe¹⁴ and another conducted by the North American consortium (the Clinical Research Consortium for Spinocerebellar Ataxias)¹⁵. This may be because of motoneuron involvement, which, particularly in this form, leads to bulbar dysfunction and, thus, to respiratory and swallowing failure¹⁶. These results are consistent with 10-year survival, which is lowest for SCA1 patients, intermediate for SCA2 and SCA3, and highest for SCA6 patients¹⁷. A high SARA score has also been identified as one of the strongest risk factors for death in all subtypes. Other associated risk factors include dysphagia for SCA1, longer CAG length in pathologic allele for SCA2, and dystonia and interaction between age and CAG length in SCA3¹⁷. The Composite Cerebellar Functional Severity (CCFS) score is another quantitative score for assessing cerebellar ataxia that has been validated in adults and children^18,19. This score is calculated from scores for two tasks for the dominant hand—the nine-hole pegboard test and the click test—and is age dependent and available from an open source (https://icm-institute.org/en/tutorial-for-making-ccfs-board/). In a study on Friedreich ataxia and SCA1, 2, 3, and 7 patients, SARA and CCFS scores were higher in SCAs than in Friedreich ataxia after adjustment for disease duration, revealing a slower progression in Friedreich ataxia than in SCAs. Considering each SCA subtype separately, SCA2 and SCA1 patients had higher CCFS scores than did patients with other subtypes, despite an absence of difference in SARA scores²⁰, probably because of the more accentuate cerebellar syndrome.

Based on SARA score progression, the EUROSCA study identified, for each genotype, the sample sizes required to detect a 50% decrease in SARA score progression in a two-arm clinical trial lasting one year: 142 patients for SCA1, 172 for SCA2, 202 for SCA3, and 602 for SCA6¹⁰. These are large numbers of patients for such rare diseases, making it necessary to carry out clinical trials at an international level. Additional biomarkers are required to overcome this problem. Furthermore, these dominantly inherited cerebellar ataxias have very different progression profiles, as shown in Figure 1. Given the progressive nature of the disease and the sample sizes required, biomarkers are very important and essential.

Figure 1. Progression and severity profiles in cerebellar ataxias by underlying etiology

Multisystem atrophy (MSA) is a rapidly progressing non-monogenic alpha-synucleinopathy. It is the most severe of these diseases, followed by polyglutamine (polyQ) spinocerebellar ataxias (SCAs), such as ATXN1/SCA1, ATXN2/SCA2, ATXN3/SCA3, CACNA1A/SCA6, ATXN7/SCA7, TBP/SCA17, and ATN1/DRPLA. Very different profiles are present in the forms because of missense mutations in channel genes (CACNA1A, KCND3, KCNC3, and KCNA1).

Biomarkers

Biological biomarkers

No biological biomarkers for SCAs have yet been identified, a situation contrasting with other neurodegenerative diseases, such as Alzheimer’s disease and frontotemporal dementia. There is, therefore, a need to search for blood or cerebrospinal fluid (CSF) biomarkers of these diseases. In addition to guiding diagnostic process, biomarkers may be of use in clinical management if linked to disease progression.

In this respect, several recent studies in patients with SCA3, the most frequent SCA subtype, have yielded promising results. SIRT1 encodes sirtuin-1, an NAD⁺-dependent deacetylase involved in several cellular functions, including chromatin modulation, the cell cycle, apoptosis, and autophagy regulation in response to DNA damage. SIRT1 mRNA levels are lower in SCA3 mice than in wild-type mice and are also low in the fibroblasts of SCA3 patients⁴³. In SCA3 mice, the rescue of SIRT1 by caloric restriction or the administration of resveratrol induces motor improvement and neuropathological changes, such as a decrease in neuroinflammation and reactive gliosis with an activation of autophagy⁴³. SIRT1 overexpression has been shown to activate autophagy and thus to induce higher levels of mutant protein clearance. For these reasons, resveratrol may be a useful neuroprotective drug, as shown in some assays in Drosophila models of SCA3⁴⁴ and in the rat 3-acetylpyridine-induced cerebellar ataxia model⁴⁵. Other studies have also demonstrated an imbalance between autophagy and apoptosis in SCA3. For example, an increase has been reported in the levels of BECN1, encoding the proautophagic Beclin 1 protein⁴⁶, and BCL2/BAX ratio has been shown to be lower in pre-ataxic SCA3 carriers than in controls⁴⁷, enhancing apoptotic processes.

Oxidative stress has been implicated in several neurodegenerative disorders, and SCA3 patients have been shown to produce abnormally large amounts of reactive oxygen species⁴⁸. This phenomenon results from a decrease in antioxidant capacity: both superoxide dismutase and glutathione peroxidase (GPx) activities are lower in symptomatic than in pre-symptomatic carriers⁴⁹. Moreover, the observed correlation of the decrease in GPx levels with disease severity suggests that GPx may be a reliable biomarker⁴⁹.

Cytokines have also been investigated as possible markers of SCA3. Indeed, enhanced inflammation has been linked to stronger staining for IL1B and IL6 and higher levels of activated microglia and reactive astrocytes in the brains of SCA3 patients⁵⁰. In a study on Brazilian SCA3 patients, no difference in cytokine levels was detected between 79 carriers and 43 controls. On the contrary, higher eotaxin levels were observed in asymptomatic carriers than in symptomatic carriers. It has been suggested that the levels of eotaxin released by astrocytes are inversely correlated with disease progression⁵¹. In another cohort of SCA3 patients recruited in the Azores, lower IL6 mRNA levels, due to the presence of the IL6*C allele, were associated with an earlier age at onset than the presence of the IL6*G allele⁵². In this study, the earlier age at onset (by about 10 years on average) resulted from the presence of the APOE*e2 allele in IL6*C carriers, probably because of additional decreases in the levels of other cytokines, such as IL1B and TNF, due to the presence of the APOE*e2 allele⁵³.

In a recent study, serum neurofilament light chain (Nfl) was identified as a powerful potential serum biomarker in polyQ SCAs⁵⁴. Neurofilaments are components of the neuron cytoskeleton, and their levels in the blood reflect damage to the axons of long fiber tracts⁵⁵. In Huntington disease, which is also a polyQ disease, a correlation between serum Nfl levels and disease severity (UHDRS, cognitive decline, brain atrophy) has been reported, even after adjustment for age and CAG repeat size⁵⁶. Nfl dosage is also useful for predicting disease onset in pre-symptomatic carriers and the progression of Huntington disease⁵⁶. In a study by Wilke et al., Nfl levels were higher in SCAs patients than in controls, particularly for SCA1 and SCA3⁵⁴. The CSF has been only briefly explored in SCAs. CSF Nfl levels are a potential diagnostic and prognostic biomarker for SCAs, as in amyotrophic lateral sclerosis⁵⁷. A study in SCA1, SCA2, and SCA6 patients evaluated α-synuclein, DJ-1, and glial fibrillary acidic protein levels in CSF. The levels of all of these proteins were higher in CSF from patients than in control CSF, but this difference was significant only for tau, the levels of which were significantly higher in SCA2 patients. No correlations were found for CAG repeat size, disease severity, and disease duration⁵⁸. It will be very interesting to determine polyQ protein levels in the CSF of SCA patients, as has been done already for Huntington disease.

Genetic advances in spinocerebellar ataxias

The number of genes implicated in SCAs has steadily increased over time. The last causal gene to have been identified, in SCA47 patients, is PUM1, encoding Pumilio1, a member of the PUMILIO/FBF RNA-binding protein family. The loss of Pum1 led to an SCA1-like phenotype in mice by causing a 30–40% increase in wild-type Atxn1 protein levels in the cerebellum⁶⁵. These two proteins are functionally related: in SCA1 mice, the disease is more severe if one copy of Pum1 is removed, whereas the motor phenotype of Pum1-heterozygous mice is improved by the removal of one copy of Atxn1⁶⁵. Mutations of PUM1 were reported by Gennarino et al. in 15 patients, with different ages at disease onset (5 months to 50 years) and phenotypic presentations. A 50% loss of the protein resulted in a severe infantile disease and a developmental syndrome called Pumilio1-associated developmental disability, ataxia, and seizure (PADDAS), whereas the loss of 25% of the protein caused Pumilio1-related cerebellar ataxia (PRCA), with a later onset and incomplete penetrance⁶⁶.

Using whole-exome sequencing (WES), Nibbeling et al. identified the genes associated with SCA46 and SCA45: PLD3 and FAT2, respectively. These genes, which are strongly expressed in the cerebellum, cause pure adult-onset cerebellar ataxia. However, in some cases, SCA46 patients may present with a sensory neuropathy⁶⁷.

Genes encoding ion channels are frequently involved in dominant cerebellar ataxia: mutations of CACNA1A are the most frequent genetic cause of autosomal dominant cerebellar ataxia in patients negative for polyQ SCAs, followed by other channel-coding genes, such as KCND3, KCNC3, and KCNA1⁶⁸. These genes predispose the patient to an earlier disease onset, intellectual deficiency, and slower disease progression⁶⁸. CACNA1G mutations, underlying SCA42⁶⁹, lead to a slowly progressive pure⁷⁰ or complicated cerebellar ataxia associated with a spastic gait⁶⁹. Patients with earlier onset may also present facial dysmorphisms, microcephaly, digital abnormalities, and seizures⁷¹.

Another channel gene implicated in SCA44 is GRM1, encoding the metabotropic glutamate receptor 1 (mGluR1) responsible for two phenotypic manifestations: adult-onset cerebellar ataxia in the presence of gain-of-function mutations and early onset ataxia with intellectual deficiency when associated with loss-of-function mutations⁷².

The use of next-generation sequencing (NGS) techniques, such as WES, in everyday clinical practice has increased the diagnostic rate of complex neurological diseases^73,74. The application of NGS in ataxia patients has revealed new SCA genes and improved our knowledge of the molecular pathways involved in cerebellar ataxia. It also affects the development of new therapeutic approaches: for example, elucidation of the role of mGluR1 in the excitability of Purkinje cells led to experiments modulating mGluR1 activity in SCA1 mice with drugs such as baclofen or the negative allosteric modulator JNJ16259685, both of which improved motor function^75,76. As well, in a recent study by Bushart et al., the administration of potassium channel modulators induced improvement in SCA1 mice motor phenotype via electrophysiological changes⁷⁷; the authors also showed that chlorzoxazone and baclofen co-administration was well tolerated by SCA1 patients and should be considered as a promising approach for treating symptoms⁷⁷.

However, today, WES will not detect trinucleotide repeat disorders, mitochondrial DNA mutations, or large structural variants. Novel or de novo repeat expansions are still difficult to detect by short-read sequencing⁷⁸. Tandem repeats are usually discarded by NGS pipelines, but bioinformatics efforts will be made to detect them. Recently, a novel intronic expansion, a pentanucleotide-repeat in SAMD12, has been identified in familial myoclonic epilepsy⁷⁸. Long-read sequencing will probably allow one to identify more neurological repeat disorders.

Therapeutic approaches in spinocerebellar ataxias

Over the last few years, several disease-modifying treatments have been tested with different outcomes. The most beneficial molecules to date are riluzole and valproic acid, whereas other drugs, such as lithium carbonate, trimethoprim-sulfamethoxazole, or zinc, have no significant effect⁷⁹. A randomized placebo-controlled clinical trial in 40 SCA patients (SCA1, 2, 6, 8, and 10) and 20 Friedreich ataxia patients treated with riluzole (100 mg/day) for 12 months revealed a one-point improvement in SARA score in treated patients compared to the placebo group⁸⁰. The use of valproic acid (1,200 mg/day) in SCA3 patients resulted in a SARA score at 12 weeks lower than that obtained for patients on placebo⁸¹. However, further trials are required to identify the genotype associated with a positive response to riluzole (ClinicalTrials.gov Identifier: NCT03347344).

Based on the hypothesis that one of the main omega-3 polyunsaturated fatty acids of the cerebellum, docosahexaenoic acid (DHA), is present at low levels in SCA38 patients owing to ELOVL5 mutations, a clinical trial of DHA (600 mg/day) was performed⁸². This drug improved SARA score at 16 weeks in a small group of SCA38 patients and it also increased cerebellar metabolism, as shown by a brain 18-fluorodeoxyglucose positron emission tomography scan at 40 weeks⁸².

However, the most encouraging and innovative strategies developed to date are RNA-targeting therapies for polyQ SCAs, which have been validated in several mouse models^83,84. These approaches mostly use antisense oligonucleotides (ASOs) to downregulate levels of the pathological polyQ protein. SCA2 mice treated with intrathecal ASOs display improvements in motor abilities, a recovery of Purkinje cell firing frequency, and lower levels of mutated ATXN2 protein⁸⁵. ASOs have also been administered to SCA3 mice and were found to be well tolerated and to decrease the production of mutated protein⁸⁶. A correlation was also found with electrophysiological changes in Purkinje cells⁸⁷. These treatments may be used in patients with polyQ SCAs at early stages of the disease, even before the onset of symptoms. The long-term effects of decreasing levels of wild-type and mutated proteins and of decreasing the levels of other polyQ proteins remain unclear. The use of allele-specific ASOs may be required⁸⁸. A therapeutic approach based on ASOs is already at an advanced stage of development for Huntington disease⁸⁹, and a phase 1A clinical trial in patients has yielded promising results⁹⁰. ASO-based treatments are also being used for spinal muscular atrophy, in a different context, to retain exon 7 and produce a full-length SMN protein, with promising results^91,92.

The advent of disease-modifying treatments for SCAs, including ASOs, will require the rapid identification and validation of robust biomarkers of disease progression or processes for the assessment of treatment response.

Conclusion

This review summarizes recent clinical and genetic advances in dominant SCAs, including discoveries of novel SCAs, development of biomarkers, and therapeutic progress. The identification of biomarkers will be essential to demonstrate treatment efficacy. Future longitudinal studies based on multimodal approaches to elucidate the relationships between parameters will be required for the establishment of valid biomarkers. To date, gene suppression therapies are the most promising in polyQ SCAs, and clinical trials in the next few years are justified.

Abbreviations

ASO, antisense oligonucleotide; CCAS, cerebellar cognitive affective/Schmahmann’s syndrome; CCFS, Composite Cerebellar Functional Severity; CSF, cerebrospinal fluid; DHA, docosahexaenoic acid; fMRI, functional MRI; GPx, glutathione peroxidase; mGluR1, metabotropic glutamate receptor 1; MRS, MRI spectroscopy; Nfl, neurofilament light; NGS, next-generation sequencing; polyQ, polyglutamine; SARA, Scale for the Assessment and Rating of Ataxia; SCA, spinocerebellar ataxia; WES, whole-exome sequencing.