Polyglutamine diseases comprise a cluster of genetic disorders involving neurodegeneration and movement disabilities. In polyglutamine diseases, the target proteins become aberrated due to polyglutamine repeat formation. These aberrant proteins form the root cause of associated complications. The metabolic regulation during polyglutamine diseases is not well studied and needs more attention. We have brought to light the significance of regulating glutamine metabolism during polyglutamine diseases, which could help in decreasing the neuronal damage associated with excess glutamate and nucleotide generation. Most polyglutamine diseases are accompanied by symptoms that occur due to excess glutamate and nucleotide accumulation. Along with a dysregulated glutamine metabolism, the Nicotinamide adenine dinucleotide (NAD+) levels drop down, and, under these conditions, NAD+ supplementation is the only achievable strategy. NAD+ is a major co-factor in the glutamine metabolic pathway, and it helps in maintaining neuronal homeostasis. Thus, strategies to decrease excess glutamate and nucleotide generation, as well as channelizing glutamine toward the generation of ATP and the maintenance of NAD+ homeostasis, could aid in neuronal health. Along with understanding the metabolic dysregulation that occurs during polyglutamine diseases, we have also focused on potential therapeutic strategies that could provide direct benefits or could restore metabolic homeostasis. Our review will shed light into unique metabolic causes and into ideal therapeutic strategies for treating complications associated with polyglutamine diseases.

Spinocerebellar ataxias (SCAs) are a rare autosomal dominant neurodegenerative disorder. To date, approximately 50 different subtypes of SCAs have been characterized. The prevalent types of SCAs are usually of PolyQ origin, wherein the disease pathology is a consequence of multiple glutamine residues being encoded onto the disease proteins, causing expansions. SCAs 2 and 3 are the most frequently diagnosed subtypes, wherein affected patients exhibit certain characteristic physiological manifestations, such as gait ataxia and dysarthria. Nevertheless, other clinical signs were exclusive to these subtypes. Recently, multiple molecular diagnostic methods have been developed to identify and characterize these subtypes. Despite these advancements, the molecular pathology of SCAs remains unknown. To further understand the mechanisms involved in neurodegenerative SCAs 2 and 3, patient-derived induced pluripotent stem cell (iPSC)-based modelling is a compelling avenue to pursue. We cover the present state of iPSC-based in-vitro illness modelling of SCA subtypes 2 and 3 below, along with a list of cell lines created, and the relevance of research outcomes to personalized autologous therapy.

Proteome integrity is a prerequisite for cellular functionality and organismal viability. Its compromise is considered an inherent part of the aging process and has been associated with the onset of age-related, neurodegenerative pathologies. Although the molecular underpinnings of protein homeostasis (proteostasis) have been extensively studied, several aspects of its regulation remain elusive. The nematode Caenorhabditis elegans has emerged as a versatile, heterologous model organism to study the dynamics of aggregation-prone human proteins in vivo. Here, we describe an experimental pipeline for the analysis of polyglutamine (polyQ) tract aggregation, as a measure of the state of proteostasis, during aging.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Background: Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3/MJD) are diseases due to dominant unstable expansions of CAG repeats (CAGexp). Age of onset of symptoms (AO) correlates with the CAGexp length. Repeat instability leads to increases in the expanded repeats, to important AO anticipations and to the eventual extinction of lineages. Because of that, compensatory forces are expected to act on the maintenance of expanded alleles, but they are poorly understood. Objectives: we described the CAGexp dynamics, adapting a classical equation and aiming to estimate for how many generations will the descendants of a de novo expansion last. Methods: A mathematical model was adapted to encompass anticipation, fitness, and allelic segregation; and empirical data fed the model. The arbitrated ancestral mutations included in the model had the lowest CAGexp and the highest AO described in the literature. One thousand generations were simulated until the alleles were eliminated, fixed, or 650 generations had passed. Results: All SCA2 lineages were eliminated in a median of 10 generations. In SCA3/MJD lineages, 593 were eliminated in a median of 29 generations. The other ones were eliminated due to anticipation after the 650th generation or remained indefinitely with CAG repeats transitioning between expanded and unexpanded ranges. Discussion: the model predicted outcomes compatible with empirical data - the very old ancestral SCA3/MJD haplotype, and the de novo SCA2 expansions -, which previously seemed to be contradictory. This model accommodates these data into understandable dynamics and might be useful for other CAGexp disorders.

Huntington\'s disease (HD) is an inherited neurodegenerative disorder considered a rare disease with a prevalence of 5.7 per 100,000 people. It is caused by an autosomal dominant mutation consisting of expansions of trinucleotide repeats that translate into poly-glutamine enlarged mutant huntingtin proteins (mHTT), which are particularly deleterious in brain tissues. Since there is no cure for this progressive fatal disease, searches for new therapeutic approaches are much needed. The small molecule pytren-4QMn (4QMn), a highly water-soluble mimic of the enzyme superoxide dismutase, has shown in vivo beneficial anti-inflammatory activity in mice and was able to remove mHTT deposits in a C. elegans model of HD. In this study, we assessed 4QMn therapeutic potential in zQ175 neo-deleted knock-in mice, a model of HD that closely mimics the heterozygosity, genetic injury, and progressive nature of the human disease. We provide evidence that 4QMn has good acute and chronic tolerability, and can cross the blood-brain barrier, and in male, but not female, zQ175 mice moderately ameliorate HD-altered gene expression, mHtt aggregation, and HD disease phenotype. Our data highlight the importance of considering sex-specific differences when testing new therapies using animal models and postulate 4QMn as a potential novel type of small water-soluble metal complex that could be worth further investigating for its therapeutic potential in HD, as well as in other polyglutamine diseases.

The spinocerebellar ataxias (SCAs) are a group of dominantly inherited neurodegenerative diseases, several of which are caused by CAG expansion mutations (SCAs 1, 2, 3, 6, 7 and 12) and more broadly belong to the large family of over 40 microsatellite expansion diseases. While dysregulation of alternative splicing is a well defined driver of disease pathogenesis across several microsatellite diseases, the contribution of alternative splicing in CAG expansion SCAs is poorly understood. Furthermore, despite extensive studies on differential gene expression, there remains a gap in our understanding of presymptomatic transcriptomic drivers of disease. We sought to address these knowledge gaps through a comprehensive study of 29 publicly available RNA-sequencing datasets. We identified that dysregulation of alternative splicing is widespread across CAG expansion mouse models of SCAs 1, 3 and 7. These changes were detected presymptomatically, persisted throughout disease progression, were repeat length-dependent, and were present in brain regions implicated in SCA pathogenesis including the cerebellum, pons and medulla. Across disease progression, changes in alternative splicing occurred in genes that function in pathways and processes known to be impaired in SCAs, such as ion channels, synaptic signalling, transcriptional regulation and the cytoskeleton. We validated several key alternative splicing events with known functional consequences, including Trpc3 exon 9 and Kcnma1 exon 23b, in the Atxn1154Q/2Q mouse model. Finally, we demonstrated that alternative splicing dysregulation is responsive to therapeutic intervention in CAG expansion SCAs with Atxn1 targeting antisense oligonucleotide rescuing key splicing events. Taken together, these data demonstrate that widespread presymptomatic dysregulation of alternative splicing in CAG expansion SCAs may contribute to disease onset, early neuronal dysfunction and may represent novel biomarkers across this devastating group of neurodegenerative disorders.

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare, incurable genetic disease that belongs to the group of polyglutamine (polyQ) diseases. DRPLA is the most common in the Japanese population; however, its global prevalence is also increasing due to better clinical recognition. It is characterized by cerebellar ataxia, myoclonus, epilepsy, dementia, and chorea. DRPLA is caused by dynamic mutation of CAG repeat expansion in ATN1 gene encoding the atrophin-1 protein. In the cascade of molecular disturbances, the pathological form of atrophin-1 is the initial factor, which has not been precisely characterized so far. Reports indicate that DRPLA is associated with disrupted protein-protein interactions (in which an expanded polyQ tract plays a crucial role), as well as gene expression deregulation. There is a great need to design efficient therapy that would address the underlying neurodegenerative process and thus prevent or alleviate DRPLA symptoms. An in-depth understanding of the normal atrophin-1 function and mutant atrophin-1 dysfunction is crucial for this purpose. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington\'s disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts.
To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice.
Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.