Background: RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting. Results: Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge. Conclusions: Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.

A common mechanism in inherited ataxia is a vulnerability of DNA damage. Spinocerebellar ataxia type 7 (SCA7) is a CAG-polyglutamine-repeat disorder characterized by cerebellar and retinal degeneration. Polyglutamine-expanded ataxin-7 protein incorporates into STAGA co-activator complex and interferes with transcription by altering histone acetylation. We performed chromatic immunoprecipitation sequencing ChIP-seq on cerebellum from SCA7 mice and observed increased H3K9-promoter acetylation in DNA repair genes, resulting in increased expression. After detecting increased DNA damage in SCA7 cells, mouse primary cerebellar neurons, and patient stem-cell-derived neurons, we documented reduced homology-directed repair (HDR) and single-strand annealing (SSA). To evaluate repair at endogenous DNA in native chromosome context, we modified linear amplification-mediated high-throughput genome-wide translocation sequencing and found that DNA translocations are less frequent in SCA7 models, consistent with decreased HDR and SSA. Altered DNA repair function in SCA7 may predispose the subject to excessive DNA damage, leading to neuron demise and highlights DNA repair as a therapy target.

Spinocerebellar ataxia type 7 (SCA7) is a genetic neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion. Purkinje cells (PCs) are central to the pathology of ataxias, but their low abundance in the cerebellum underrepresents their transcriptomes in sequencing assays. To address this issue, we developed a PC enrichment protocol and sequenced individual nuclei from mice and patients with SCA7. Single-nucleus RNA sequencing in SCA7-266Q mice revealed dysregulation of cell identity genes affecting glia and PCs. Specifically, genes marking zebrin-II PC subtypes accounted for the highest proportion of DEGs in symptomatic SCA7-266Q mice. These transcriptomic changes in SCA7-266Q mice were associated with increased numbers of inhibitory synapses as quantified by immunohistochemistry and reduced spiking of PCs in acute brain slices. Dysregulation of zebrin-II cell subtypes was the predominant signal in PCs of SCA7-266Q mice and was associated with the loss of zebrin-II striping in the cerebellum at motor symptom onset. We furthermore demonstrated zebrin-II stripe degradation in additional mouse models of polyglutamine ataxia and observed decreased zebrin-II expression in the cerebella of patients with SCA7. Our results suggest that a breakdown of zebrin subtype regulation is a shared pathological feature of polyglutamine ataxias.

The polyglutamine (polyQ) family of disorders comprises 9 genetic diseases, including several types of ataxia and Huntington disease. Approximately two decades of investigation and the creation of more than 130 mouse models of polyQ disorders have revealed many similarities between these diseases. The disorders share common mutation types, neurological characteristics and certain aspects of pathogenesis, including morphological and physiological neuronal alterations. All of the diseases still remain incurable. The large volume of information collected as a result of the investigation of polyQ models currently represents a great potential for searching, comparing and translating pathogenesis and therapeutic information between diseases. Therefore, we generated a public database comprising the polyQ mouse models, phenotypes and therapeutic interventions tested in vivo. The database is available at http://conyza.man.poznan.pl/ . The use of the database in the field of polyQ diseases may accelerate research on these and other neurodegenerative diseases and provide new perspectives for future investigation.

Huntington's disease (HD) is a neurodegenerative polyglutamine (polyQ) disease resulting from the expansion of CAG repeats located in the ORF of the huntingtin gene ( HTT ). The extent to which mutant mRNA‐driven disruptions contribute to HD pathogenesis, particularly in comparison to the dominant mechanisms related to the gain‐of‐function effects of the mutant polyQ protein, is still debatable. To evaluate this contribution in vivo, we generated two mouse models through a knock‐in strategy at the Rosa26 locus. These models expressed distinct variants of human mutant HTT cDNA fragment: a translated variant (HD/100Q model, serving as a reference) and a nontranslated variant (HD/100CAG model). The cohorts of animals were subjected to a broad spectrum of molecular, behavioral, and cognitive analysis for 21 months. Behavioral testing revealed alterations in both models, with the HD/100Q model exhibiting late disease phenotype. The rotarod, static rod, and open‐field tests showed some motor deficits in HD/100CAG and HD/100Q model mice during the light phase, while ActiMot indicated hyperkinesis during the dark phase. Both models also exhibited certain gene deregulations in the striatum that are related to disrupted pathways and phenotype alterations observed in HD. In conclusion, we provide in vivo evidence for a minor contributory role of mutant RNA in HD pathogenesis. The separated effects resulting from the presence of mutant RNA in the HD/100CAG model led to less severe but, to some extent, similar types of impairments as in the HD/100Q model. Increased anxiety was one of the most substantial effects caused by mutant HTT RNA.

We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.