Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis.

Spinocerebellar ataxia type 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.

Emerging evidence suggests the presence of bidirectional interactions between the central nervous system and gut microbiota that may contribute to the pathogenesis of neurodegenerative diseases. However, the potential role of gut microbes in forms of spinocerebellar ataxia, such as the fatal neurodegenerative disease Machado Joseph disease (MJD), remains unexplored. Here, we examined whether gut microbiota alterations may be an early disease phenotype of MJD. We profiled the gut microbiota of male and female transgenic MJD mice (CMVMJD135) expressing human ATXN3 with expanded CAG repeats (133-143 CAG) at pre-symptomatic, symptomatic and well-established stages of the disease (7, 11 and 15 weeks of age, respectively). We compared these profiles with the gut microbiota of male and female wild-type (WT) littermate control mice at same ages. Correlation network analyses were employed to explore the relevance of microbiota changes to disease progression. The results demontrated distinct sex-dependent effects in disease development whereby male MJD mice displayed earlier motor impairments than female MJD mice. The gut microbiota community structure and composition also demonstrated sex-specific differences between MJD and WT mice. In both male and female MJD mice, the shifts in the microbiota were present by 7 weeks, before the onset of any symptoms. These pre-symptomatic microbial changes correlated with the severity of neurological impairments present at later stages of the disease. Previous efforts towards developing treatments for MJD have failed to yield meaningful outcomes. Our study reports a novel relationship between the gut microbiota and MJD development and severity. Elucidating how gut microbes are involved in MJD pathogenesis may offer new and efficacious treatment strategies for this currently untreatable disease.

Repeat diseases, such as fragile X syndrome, myotonic dystrophy, Friedreich ataxia, Huntington disease, spinocerebellar ataxias, and some forms of amyotrophic lateral sclerosis, are caused by repetitive DNA sequences that are expanded in affected individuals. The age at which an individual begins to experience symptoms, and the severity of disease, are partially determined by the size of the repeat. However, the epigenetic state of the area in and around the repeat also plays an important role in determining the age of disease onset and the rate of disease progression. Many repeat diseases share a common epigenetic pattern of increased methylation at CpG islands near the repeat region. CpG islands are CG-rich sequences that are tightly regulated by methylation and are often found at gene enhancer or insulator elements in the genome. Methylation of CpG islands can inhibit binding of the transcriptional regulator CTCF, resulting in a closed chromatin state and gene down regulation. The downregulation of these genes leads to some disease-specific symptoms. Additionally, a genetic and epigenetic interplay is suggested by an effect of methylation on repeat instability, a hallmark of large repeat expansions that leads to increasing disease severity in successive generations. In this review, we will discuss the common epigenetic patterns shared across repeat diseases, how the genetics and epigenetics interact, and how this could be involved in disease manifestation. We also discuss the currently available stem cell and mouse models, which frequently do not recapitulate epigenetic patterns observed in human disease, and propose alternative strategies to study the role of epigenetics in repeat diseases.

Since the characterization of mRNA in 1961, our understanding of the roles of RNA molecules has significantly grown. Beyond serving as a link between DNA and proteins, RNA molecules play direct effector roles by binding to various ligands, including proteins, DNA, other RNAs, and metabolites. Through these interactions, RNAs mediate cellular processes such as the regulation of gene transcription and the enhancement or inhibition of protein activity. As a result, the misregulation of RNA molecules is often associated with disease phenotypes, and RNA molecules have been increasingly recognized as potential targets for drug development efforts, which in the past had focused primarily on proteins. Although both small molecule-based and oligonucleotide-based therapies have been pursued in efforts to target RNA, small-molecule modalities are often favored owing to several advantages including greater oral bioavailability. In this review, we discuss three general frameworks (sets of premises and hypotheses) that, in our view, have so far dominated the discovery of small-molecule ligands for RNA. We highlight the unique merits of each framework as well as the pitfalls associated with exclusive focus of ligand discovery efforts within only one framework. Finally, we propose that RNA ligand discovery can benefit from using progress made within these three frameworks to move toward a paradigm that formulates RNA-targeting questions at the level of RNA structural subclasses.

Fuchs\' endothelial corneal dystrophy (FECD) is the leading indication for corneal transplantation. Seventy percent of cases are caused by an intronic CTG triplet repeat expansion in the TCF4 gene that results in accumulation of pathogenic expanded CUG repeat RNA (CUGexp) as nuclear foci in corneal endothelium. A catalytically dead Cas9 (dCas9) can serve as an effective guide to target genomic DNA or RNA transcripts. Here, we examined the utility of the clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 system to effectively target and reduce CUGexp.
We delivered dCas9 and repeat-targeting single guide RNA (sgRNA) expression plasmids to patient-derived endothelial cells using lipofection or lentiviral transduction. We used fluorescence in situ hybridization (FISH) and RNA dot-blot hybridization to quantify CUGexp foci and repeat RNA levels, respectively. TCF4 expression levels were assessed using quantitative PCR (qPCR).
Using FISH, we found that expression of both dCas9 and a (CAG)n sgRNA complementary to CUGexp are necessary to reduce foci. We observed a reduction in percentage of cells with foci from 59% to 5.6% and number of foci per 100 cells from 73.4 to 7.45 (P < 0.001) in cells stably expressing dCas9-(CAG)n sgRNA but saw no decrease in cells expressing dCas9-(CUG)n sgRNA or nontargeting control sgRNA. In cells with dCas9-(CAG)n sgRNA, we detected a reduction in CUGexp RNA by dot-blot without any reduction in TCF4 mRNA levels using qPCR.
Using CRISPR-dCas9 to target the trinucleotide repeat is a promising treatment for FECD contingent on effective in vivo delivery.
This work advances a gene therapy for a common age-related degenerative disorder.

Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG) n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG) n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?

Polyglutamine (polyQ) diseases are a group of dominantly inherited neurodegenerative disorders caused by the expansion of an unstable CAG repeat in the coding region of the affected genes. Hallmarks of polyQ diseases include the accumulation of misfolded protein aggregates, leading to neuronal degeneration and cell death. PolyQ diseases are currently incurable, highlighting the urgent need for approaches that inhibit the formation of disaggregate cytotoxic polyQ protein inclusions. Here, we screened for bisamidine-based inhibitors that can inhibit neuronal polyQ protein inclusions. We demonstrated that one inhibitor, AQAMAN, prevents polyQ protein aggregation and promotes de-aggregation of self-assembled polyQ proteins in several models of polyQ diseases. Using immunocytochemistry, we found that AQAMAN significantly reduces polyQ protein aggregation and specifically suppresses polyQ protein-induced cell death. Using a recombinant and purified polyQ protein (thioredoxin-Huntingtin-Q46), we further demonstrated that AQAMAN interferes with polyQ self-assembly, preventing polyQ aggregation, and dissociates preformed polyQ aggregates in a cell-free system. Remarkably, AQAMAN feeding of Drosophila expressing expanded polyQ disease protein suppresses polyQ-induced neurodegeneration in vivo In addition, using inhibitors and activators of the autophagy pathway, we demonstrated that AQAMAN\'s cytoprotective effect against polyQ toxicity is autophagy-dependent. In summary, we have identified AQAMAN as a potential therapeutic for combating polyQ protein toxicity in polyQ diseases. Our findings further highlight the importance of the autophagy pathway in clearing harmful polyQ proteins.

Microsatellite expansions cause more than 40 neurological disorders, including Huntington\'s disease, myotonic dystrophy, and C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). These repeat expansion mutations can produce repeat-associated non-ATG (RAN) proteins in all three reading frames, which accumulate in disease-relevant tissues. There has been considerable interest in RAN protein products and their downstream consequences, particularly for the dipeptide proteins found in C9ORF72 ALS/FTD. Understanding how RAN translation occurs, what cellular factors contribute to RAN protein accumulation, and how these proteins contribute to disease should lead to a better understanding of the basic mechanisms of gene expression and human disease.