Novel genotype-phenotype correlations, differential cerebellar allele-specific methylation, and a common origin of the (ATTTC)n insertion in spinocerebellar ataxia type 37.

Spinocerebellar ataxia subtype 37 (SCA37) is a rare disease originally identified in ataxia patients from the Iberian Peninsula with a pure cerebellar syndrome. SCA37 patients carry a pathogenic intronic (ATTTC)n repeat insertion flanked by two polymorphic (ATTTT)n repeats in the Disabled-1 (DAB1) gene leading to cerebellar dysregulation. Herein, we determine the precise configuration of the pathogenic 5'(ATTTT)n-(ATTTC)n-3'(ATTTT)n SCA37 alleles by CRISPR-Cas9 and long-read nanopore sequencing, reveal their epigenomic signatures in SCA37 lymphocytes, fibroblasts, and cerebellar samples, and establish new molecular and clinical correlations. The 5'(ATTTT)n-(ATTTC)n-3'(ATTTT)n pathogenic allele configurations revealed repeat instability and differential methylation signatures. Disease age of onset negatively correlated with the (ATTTC)n, and positively correlated with the 3'(ATTTT)n. Geographic origin and gender significantly correlated with age of onset. Furthermore, significant predictive regression models were obtained by machine learning for age of onset and disease evolution by considering gender, the (ATTTC)n, the 3'(ATTTT)n, and seven CpG positions differentially methylated in SCA37 cerebellum. A common 964-kb genomic region spanning the (ATTTC)n insertion was identified in all SCA37 patients analysed from Portugal and Spain, evidencing a common origin of the SCA37 mutation in the Iberian Peninsula originating 859 years ago (95% CI 647-1378). In conclusion, we demonstrate an accurate determination of the size and configuration of the regulatory 5'(ATTTT)n-(ATTTC)n-3'(ATTTT)n repeat tract, avoiding PCR bias amplification using CRISPR/Cas9-enrichment and nanopore long-read sequencing, resulting relevant for accurate genetic diagnosis of SCA37. Moreover, we determine novel significant genotype-phenotype correlations in SCA37 and identify differential cerebellar allele-specific methylation signatures that may underlie DAB1 pathogenic dysregulation.

New spinocerebellar ataxia subtype caused by SAMD9L mutation triggering mitochondrial dysregulation (SCA49).

Spinocerebellar ataxias consist of a highly heterogeneous group of inherited movement disorders clinically characterized by progressive cerebellar ataxia variably associated with additional distinctive clinical signs. The genetic heterogeneity is evidenced by the myriad of associated genes and underlying genetic defects identified. In this study, we describe a new spinocerebellar ataxia subtype in nine members of a Spanish five-generation family from Menorca with affected individuals variably presenting with ataxia, nystagmus, dysarthria, polyneuropathy, pyramidal signs, cerebellar atrophy and distinctive cerebral demyelination. Affected individuals presented with horizontal and vertical gaze-evoked nystagmus and hyperreflexia as initial clinical signs, and a variable age of onset ranging from 12 to 60 years. Neurophysiological studies showed moderate axonal sensory polyneuropathy with altered sympathetic skin response predominantly in the lower limbs. We identified the c.1877C > T (p.Ser626Leu) pathogenic variant within the SAMD9L gene as the disease causative genetic defect with a significant log-odds score (Z max = 3.43; θ = 0.00; P < 3.53 × 10-5). We demonstrate the mitochondrial location of human SAMD9L protein, and its decreased levels in patients' fibroblasts in addition to mitochondrial perturbations. Furthermore, mutant SAMD9L in zebrafish impaired mobility and vestibular/sensory functions. This study describes a novel spinocerebellar ataxia subtype caused by SAMD9L mutation, SCA49, which triggers mitochondrial alterations pointing to a role of SAMD9L in neurological motor and sensory functions.

POLR3A-related spastic ataxia: new mutations and a look into the phenotype.

Adolescent-onset spastic ataxia is a proposed novel phenotype in compound heterozygous carriers of an intronic mutation (c.1909 + 22G > A) in the POLR3A gene. Here, we present ten new cases of POLR3A-related spastic ataxia and discuss the genetic, clinical and imaging findings. Patients belonged to six pedigrees with hereditary spastic paraplegia or cerebellar ataxia of unknown origin. All affected subjects presented with compound heterozygous variants, comprising c.1909 + 22G > A in combination in each pedigree with one of the following novel mutations (Thr596Met, Tyr665LeufsTer11, Glu198Ter, c.646-687_1185 + 844del). The new mutations segregated with the phenotype in all families. The phenotype combined variable cerebellar ataxia, gait and lower limb spasticity, involvement of central sensory tracts and in some cases also intention tremor. The reportedly characteristic hyperintensity along the superior cerebellar peduncle on MRI was observed in ~ 80% of the cases. Our study extends the clinical and molecular phenotype further supporting the pathogenic role of the c.1909 + 22G4A intronic mutation and identifying four novel causative mutations in POLR3A-related spastic ataxia. Certain characteristic MRI features may be useful to guide genetic diagnosis.

Clinical, genetic and neuropathological characterization of spinocerebellar ataxia type 37.

The autosomal dominant spinocerebellar ataxias (SCAs) consist of a highly heterogeneous group of rare movement disorders characterized by progressive cerebellar ataxia variably associated with ophthalmoplegia, pyramidal and extrapyramidal signs, dementia, pigmentary retinopathy, seizures, lower motor neuron signs, or peripheral neuropathy. Over 41 different SCA subtypes have been described evidencing the high clinical and genetic heterogeneity. We previously reported a novel spinocerebellar ataxia type subtype, SCA37, linked to an 11-Mb genomic region on 1p32, in a large Spanish ataxia pedigree characterized by ataxia and a pure cerebellar syndrome distinctively presenting with early-altered vertical eye movements. Here we demonstrate the segregation of an unstable intronic ATTTC pentanucleotide repeat mutation within the 1p32 5' non-coding regulatory region of the gene encoding the reelin adaptor protein DAB1, implicated in neuronal migration, as the causative genetic defect of the disease in four Spanish SCA37 families. We describe the clinical-genetic correlation and the first SCA37 neuropathological findings caused by dysregulation of cerebellar DAB1 expression. Post-mortem neuropathology of two patients with SCA37 revealed severe loss of Purkinje cells with abundant astrogliosis, empty baskets, occasional axonal spheroids, and hypertrophic fibres by phosphorylated neurofilament immunostaining in the cerebellar cortex. The remaining cerebellar Purkinje neurons showed loss of calbindin immunoreactivity, aberrant dendrite arborization, nuclear pathology including lobulation, irregularity, and hyperchromatism, and multiple ubiquitinated perisomatic granules immunostained for DAB1. A subpopulation of Purkinje cells was found ectopically mispositioned within the cerebellar cortex. No significant neuropathological alterations were identified in other brain regions in agreement with a pure cerebellar syndrome. Importantly, we found that the ATTTC repeat mutation dysregulated DAB1 expression and induced an RNA switch resulting in the upregulation of reelin-DAB1 and PI3K/AKT signalling in the SCA37 cerebellum. This study reveals the unstable ATTTC repeat mutation within the DAB1 gene as the underlying genetic cause and provides evidence of reelin-DAB1 signalling dysregulation in the spinocerebellar ataxia type 37.

Rare Neurodegenerative Diseases: Clinical and Genetic Update.

More than 600 human disorders afflict the nervous system. Of these, neurodegenerative diseases are usually characterised by onset in late adulthood, progressive clinical course, and neuronal loss with regional specificity in the central nervous system. They include Alzheimer's disease and other less frequent dementias, brain cancer, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, stroke, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease), Huntington's disease, and Prion diseases, among others. Neurodegeneration usually affects, but is not limited to, the cerebral cortex, intracranial white matter, basal ganglia, thalamus, hypothalamus, brain stem, and cerebellum. Although the majority of neurodegenerative diseases are sporadic, Mendelian inheritance is well documented. Intriguingly, the clinical presentations and neuropathological findings in inherited neurodegenerative forms are often indistinguishable from those of sporadic cases, suggesting that converging genomic signatures and pathophysiologic mechanisms underlie both hereditary and sporadic neurodegenerative diseases. Unfortunately, effective therapies for these diseases are scarce to non-existent. In this chapter, we highlight the clinical and genetic features associated with the rare inherited forms of neurodegenerative diseases, including ataxias, multiple system atrophy, spastic paraplegias, Parkinson's disease, dementias, motor neuron diseases, and rare metabolic disorders.

Ataxin-1 regulates the cerebellar bioenergetics proteome through the GSK3ß-mTOR pathway which is altered in Spinocerebellar ataxia type 1 (SCA1).

A polyglutamine expansion within the ataxin-1 protein (ATXN1) underlies spinocerebellar ataxia type-1 (SCA1), a neurological disorder mainly characterized by ataxia and cerebellar deficits. In SCA1, both loss and gain of ATXN1 biological functions contribute to cerebellar pathogenesis. However, the critical ATXN1 functions and pathways involved remain unclear. To further investigate the early signalling pathways regulated by ATXN1, we performed an unbiased proteomic study of the Atxn1-KO 5-week-old mice cerebellum. Here, we show that lack of ATXN1 expression induces early alterations in proteins involved in glycolysis [pyruvate kinase, muscle, isoform 1 protein (PKM-i1), citrate synthase (CS), glycerol-3-phosphate dehydrogenase 2 (GPD2), glucose-6-phosphate isomerase (GPI), alpha -: enolase (ENO1)], ATP synthesis [CS, Succinate dehydrogenase complex,subunit A (SDHA), ATP synthase subunit d, mitochondrial (ATP5H)] and oxidative stress [peroxiredoxin-6 (PRDX6), aldehyde dehydrogenase family 1, subfamily A1, 10-formyltetrahydrofolate dehydrogenase]. In the SCA1 mice, several of these proteins (PKM-i1, ATP5H, PRDX6, proteome subunit A6) were down-regulated and ATP levels decreased. The underlying mechanism does not involve modulation of mitochondrial biogenesis, but dysregulation of the activity of the metabolic regulators glycogen synthase kinase 3B (GSK3ß), decreased in Atxn1-KO and increased in SCA1 mice, and mechanistic target of rapamycin (serine/threonine kinase) (mTOR), unchanged in the Atxn1-KO and decreased in SCA1 mice cerebellum before the onset of ataxic symptoms. Pharmacological inhibition of GSK3ß and activation of mTOR in a SCA1 cell model ameliorated identified ATXN1-regulated metabolic proteome and ATP alterations. Taken together, these results point to an early role of ATXN1 in the regulation of bioenergetics homeostasis in the mouse cerebellum. Moreover, data suggest GSK3ß and mTOR pathways modulate this ATXN1 function in SCA1 pathogenesis that could be targeted therapeutically prior to the onset of disease symptoms in SCA1 and other pathologies involving dysregulation of ATXN1 functions.

Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia.

Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779 + 746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5' breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3' breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases.

New subtype of spinocerebellar ataxia with altered vertical eye movements mapping to chromosome 1p32.

IMPORTANCE: To provide clinical and genetic diagnoses for patients' conditions, it is important to identify and characterize the different subtypes of spinocerebellar ataxia (SCA). OBJECTIVE: To clinically and genetically characterize a Spanish kindred with pure SCA presenting with altered vertical eye movements. DESIGN Family study of ambulatory patients. Electro-oculographic and genetics studies were performed in 2 referral university centers. SETTING: Primary care institutional center in Spain. PARTICIPANTS: Thirty-six participants from a large Spanish kindred were clinically examined, and 33 family members were genetically examined. Detailed clinical data were obtained from 9 affected relatives. Two ataxic siblings and 2 asymptomatic family members were examined using an enhanced clinical protocol for a follow-up period of 7 years. MAIN OUTCOMES AND MEASURES: High-density genome-wide single-nucleotide polymorphism arrays, along with microsatellite analysis, and genetic linkage studies were performed. Whole-exome sequencing was used for 2 affected relatives. For most patients, the initial symptoms included falls, dysarthria, or clumsiness followed by a complete cerebellar syndrome. For all 9 affected relatives, we observed altered vertical eye movements, as initial ocular signs for 3 of them and for the 2 asymptomatic family members, all having inherited the risk haplotype. Neuroimaging showed isolated cerebellar atrophy. RESULTS: Initial genome-wide linkage analysis revealed suggestive linkage to chromosome 1p32. Multipoint analysis and haplotype reconstruction further traced this SCA locus to a 0.66-cM interval flanked by D1S200 and D1S2742 (z(max) = 6.539; P < .0001). The causative mutation was unidentified by exome sequencing. CONCLUSIONS AND RELEVANCE: We report a new subtype of SCA presenting in patients as slow progressing ataxia with altered vertical eye movements linked to a 11-megabase interval on 1p32. The Human Genome Nomenclature Committee has assigned this subtype of ataxia the designation SCA37.

A novel function of Ataxin-1 in the modulation of PP2A activity is dysregulated in the spinocerebellar ataxia type 1.

An expansion of glutamines within the human ataxin-1 protein underlies spinocerebellar ataxia type 1 (SCA1), a dominantly inherited neurodegenerative disorder characterized by ataxia and loss of cerebellar Purkinje neurons. Although the mechanisms linking the mutation to the disease remain unclear, evidence indicates that it involves a combination of both gain and loss of functions of ataxin-1. We previously showed that the mutant ataxin-1 interacts with Anp32a, a potent and selective PP2A inhibitor, suggesting a role of PP2A in SCA1. Herein, we found a new function of ataxin-1: the modulation of Pp2a activity and the regulation of its holoenzyme composition, with the polyglutamine mutation within Atxn1 altering this function in the SCA1 mouse cerebellum before disease onset. We show that ataxin-1 enhances Pp2a-bß expression and down-regulates Anp32a levels without affecting post-translational modifications of Pp2a catalytic subunit (Pp2a-c) known to regulate Pp2a activity. In contrast, mutant Atxn1 induces a decrease in Y307-phosphorylation in Pp2a-c, known to enhance its activity, while reducing Pp2a-b expression and inhibiting Anp32a levels. qRT-PCR and chromatin immunoprecipitation analyses show that ataxin-1-mediated regulations of the Pp2a-bß subunit, specifically bß2, and of Anp32a occur at the transcriptional level. The Pp2a pathway alterations were confirmed by identified phosphorylation changes of the known Pp2a-substrates, Erk2 and Gsk3ß. Similarly, mutant ataxin-1-expressing SH-SY5Y cells exhibit abnormal neuritic morphology, decreased levels of both PP2A-Bß and ANP32A, and PP2A pathway alterations, all of which are ameliorated by overexpressing ANP32A. Our results point to dysregulation of this newly assigned function of ataxin-1 in SCA1 uncovering new potential targets for therapy.

The spinocerebellar ataxias: clinical aspects and molecular genetics.

Spinocerebellar ataxias (SCAs) are a highly heterogeneous group of inherited neurological disorders, based on clinical characterization alone with variable degrees of cerebellar ataxia often accompanied by additional cerebellar and noncerebellar symptoms which in most cases defy differentiation. Molecular causative deficits in at least 31 genes underlie the clinical symptoms in the SCAs by triggering cerebellar and, very frequently, brain stem dysfunction. The identification of the causative molecular deficits enables the molecular diagnosis of the different SCA subtypes and facilitates genetic counselling. Recent scientific advances are shedding light into developing therapeutic strategies. The scope of this chapter is to provide updated details of the spinocerebellar ataxias with particular emphasis on those aspects aimed at facilitating the clinical and genetic diagnoses.

Cellular and molecular pathways triggering neurodegeneration in the spinocerebellar ataxias.

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of progressive neurodegenerative diseases characterised by loss of balance and motor coordination due to the primary dysfunction of the cerebellum. To date, more than 30 genes have been identified triggering the well-described clinical and pathological phenotype, but the underlying cellular and molecular events are still poorly understood. Studies of the functions of the proteins implicated in SCAs and the corresponding altered cellular pathways point to major aetiological roles for defects in transcriptional regulation, protein aggregation and clearance, alterations of calcium homeostasis, and activation of pro-apoptotic routes among others, all leading to synaptic neurotransmission deficits, spinocerebellar dysfunction, and, ultimately, neuronal demise. However, more mechanistic and detailed insights are emerging on these molecular routes. The growing understanding of how dysregulation of these pathways trigger the onset of symptoms and mediate disease progression is leading to the identification of conserved molecular targets influencing the critical pathways in pathogenesis that will serve as effective therapeutic strategies in vivo, which may prove beneficial in the treatment of SCAs. Herein, we review the latest evidence for the proposed cellular and molecular processes to the pathogenesis of dominantly inherited spinocerebellar ataxias and the ongoing therapeutic strategies.

Dissociation between neurodegeneration and caspase-11-mediated activation of caspase-1 and caspase-3 in a mouse model of amyotrophic lateral sclerosis.

Caspase-11 is a key regulator of caspase-1 and caspase-3 activation under pathological conditions. We show here that the expression of caspase-11 is upregulated in the spinal cord of superoxide dismutase 1 (SOD1) G93A transgenic mice, a mouse model of amyotrophic lateral sclerosis (ALS), before the onset of motor dysfunction and remains at the high levels throughout the course of disease. The caspase-1- and caspase-3-like activities, as well as the level of interleukin-1beta, were significantly reduced in the spinal cord of symptomatic caspase-11-/-;SOD1 G93A mice compared with that of caspase-11+/-; SOD1 G93A mice. However, neurodegeneration, inflammatory responses, and the disease onset and progression in SOD1 G93A transgenic mice were not altered by the ablation of caspase-11 gene. Thus, although caspases may contribute to certain aspects of pathology in this mouse model of ALS, their inhibition is not sufficient to prevent neurodegeneration. Our study urges caution when considering the inhibition of caspases as a direct therapeutic method for the treatment of chronic neurodegenerative diseases.

Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders.

The expansion of a CAG repeat coding for polyglutamine in otherwise unrelated gene products is central to eight neurodegenerative disorders including Huntington's disease. It has been well documented that expanded polyglutamine fragments, cleaved from their respective full-length proteins, form microscopically visible aggregates in affected individuals and in transgenic mice. The contribution of polyglutamine oligomers to neurodegeneration, however, is controversial. The azo-dye Congo red binds preferentially to beta-sheets containing amyloid fibrils and can specifically inhibit oligomerization and disrupt preformed oligomers. Here we show that inhibition of polyglutamine oligomerization by Congo red prevents ATP depletion and caspase activation, preserves normal cellular protein synthesis and degradation functions, and promotes the clearance of expanded polyglutamine repeats in vivo and in vitro. Infusion of Congo red into a transgenic mouse model of Huntington's disease, well after the onset of symptoms, promotes the clearance of expanded repeats in vivo and exerts marked protective effects on survival, weight loss and motor function. We conclude that oligomerization is a crucial determinant in the biochemical properties of expanded polyglutamine that are central to their chronic cytotoxicity.