Dose-dependent reduction of somatic expansions but not Htt aggregates by di-valent siRNA-mediated silencing of MSH3 in HdhQ111 mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the HTT gene. In addition to germline CAG expansions, somatic repeat expansions in neurons also contribute to HD pathogenesis. The DNA mismatch repair gene, MSH3, identified as a genetic modifier of HD onset and progression, promotes somatic CAG expansions, and thus presents a potential therapeutic target. However, what extent of MSH3 protein reduction is needed to attenuate somatic CAG expansions and elicit therapeutic benefits in HD disease models is less clear. In our study, we employed potent di-siRNAs to silence mouse Msh3 mRNA expression in a dose-dependent manner in HdhQ111/+ mice and correlated somatic Htt CAG instability with MSH3 protein levels from simultaneously isolated DNA and protein after siRNA treatment. Our results reveal a linear correlation with a proportionality constant of ~ 1 between the prevention of somatic Htt CAG expansions and MSH3 protein expression in vivo, supporting MSH3 as a rate-limiting step in somatic expansions. Intriguingly, despite a 75% reduction in MSH3 protein levels, striatal nuclear HTT aggregates remained unchanged. We also note that evidence for nuclear Msh3 mRNA that is inaccessible to RNA interference was found, and that MSH6 protein in the striatum was upregulated following MSH3 knockdown in HdhQ111/+ mice. These results provide important clues to address critical questions for the development of therapeutic molecules targeting MSH3 as a potential therapeutic target for HD.

Optimization of small-scale sample preparation for high-throughput OpenArray analysis.

OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.

The Parkinson's disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Altered muscle electrical tissue properties in a mouse model of premature aging.

INTRODUCTION: Improved methods are needed to detect and quantify age-related muscle change. In this study we assessed the electrical properties of muscle impacted by acquired mitochondrial DNA mutations via the PolG mouse, which exhibits typical age-associated features, and the impact of a potential therapy, nicotinamide mononucleotide (NMN). METHODS: The gastrocnemii of 24 PolG and 30 wild-type (WT) mice (8 PolG and 17 WT treated with NMN) were studied in an electrical impedance-measuring cell. Conductivity and relative permittivity were determined from the impedance data. Myofiber cross-sectional area (CSA) was quantified histologically. RESULTS: Untreated PolG mice demonstrated alterations in several impedance features, including 50-kHz relative permittivity and center frequency. A potential effect of NMN was also observed in these parameters in PolG but not WT animals. Impedance values correlated with myofiber CSA. DISCUSSION: Electrical impedance is sensitive to myofiber features considered characteristic of aging and to the impact of a potential therapy.