Biology subjects

Kegel-Gleason, K.

Publications and source records attributed to Kegel-Gleason, K..

3 recordsLinked to original sources

bioRxiv2025

Early reduction and impaired targeting of myelin-associated glycoprotein to myelin membranes in Huntington disease

BackgroundHuntingtons disease (HD) is a hereditary life-threatening disease marked by progressive neuronal loss and atrophy of grey matter structures, particularly the caudate putamen. Brain imaging studies have revealed that the degradation of the white matter occurs many years prior to symptomatic onset and neuronal loss, suggesting that the decay of brain white matter is an active contributor to the disease progression. However, the mechanisms by which the HD mutation triggers white matter loss is not well understood. MethodsWestern blot, immunohistochemistry, and electron microscopy were conducted to assess white matter pathology and explore the relevant mechanisms in CAG140 knock-in mice, which express the HD protein in the same way as patients suffering from HD and thus biologically replicate HD in human. ResultsWestern blot analysis of proteins localized at different layers of the myelin coat revealed that the myelin-associated glycoprotein (MAG), which is localized at the innermost layer of the myelin coat and essential for maintaining the periaxonal space and the integrity of the myelin sheath, manifested as an early and progressive decline in HD mouse caudate putamen. The loss of MAG was detected at myelinated axons and in fiber bundles in HD mouse brains at an age when the abundance of myelinated axons was normal. Fluorescence immunohistochemical studies found that MAG labeling was concentrated in the soma of a subset of oligodendrocytes, which expressed breast carcinoma amplified sequence 1, a marker for new oligodendrocytes. While their abundance was normal, new oligodendrocytes in HD mouse caudate appeared to be impeded in acquiring the expression of MAG and in targeting MAG away from perinuclear punctate structures to processes, signs of impaired maturation. Compared with those in wildtype mouse brains, oligodendrocytes in HD mouse brains had a reduced abundance of small vesicles whereas an increased abundance of large punctate structures in the perinuclear region, implying defective generation of small vesicles transporting MAG from large punctate structures in the soma to processes. The MAG-containing perinuclear punctate structures were negative for proteins specifying trans-Golgi networks, early endosomes, or exosomes but had a minor portion labeled with lysosome-associated membrane protein 1, indicating that the structures where MAG accumulates in the soma are derived from the late endosomal lysosomal compartment. ConclusionsOur study suggests that the decay of the brain white matter in Huntingtons disease involves a deficit in trafficking of myelin-associated glycoprotein, preventing its proper delivery from the soma of oligodendrocytes to myelin-forming processes.

neuroscience↗

bioRxiv2025

Detection of HTTex1p by western blot and immunostaining of HD human and mouse brain using neo-epitope antibody P90 highlights impact of CAG repeat expansion on its size, solubility, and response to MSH3 silencing

HTT1a was identified in human and mouse Huntingtons disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction.1 Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with fluorescence-based reporter assays (Meso Scale Discovery, Homogeneous Time Resolved Fluorescence) and immunoprecipitation assays in Huntingtons disease knock-in mice but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntingtons disease brain were separated by gel electrophoresis and probed by western blot with neo-epitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6 month old Huntingtons disease knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTT1a migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in WT mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear indicating that the effects of curbing CAG repeat expansion were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntingtons disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinsons disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate HTT proteins from either Huntingtons disease mouse or human brain lysates using conditions that pulled down full length HTT with anti-HTT antibody 2B7. Altogether these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility, and subcellular localization in Huntingtons disease mouse brain. Abbreviated SummarySapp et al., report that pathogenic exon 1 protein HTT1a is detected in brain of mouse models of Huntingtons disease by direct western blot assay using monoclonal antibodies 11G2 and 1B12. Lowering MSH3 mRNA in the caudate putamen to prevent CAG repeat expansion reduced levels of HTT1a.

neuroscience↗

bioRxiv2022

Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in vivo in CNS

Small interfering RNAs (siRNAs) are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS. Many genetically-defined neurodegenerative disorders are autosomal dominant favoring selective silencing of the mutant allele. In some cases, successful targeting of the mutant allele requires targeting of a single nucleotide polymorphism (SNP) heterozygosity. Using Huntingtons disease as a model, we demonstrate allele-specific RNAi-based silencing of gene expression in vivo and in neurons differentiated from HD patient-derived iPSCs. A series of in vitro screens, with chemical and thermodynamic optimization, identified compounds with >50-fold selectivity for the mutant HD-causing allele, based on a single nucleotide difference. The optimized compound exhibits selective silencing of mutant huntingtin (HTT) protein in patient derived cells and throughout the HD mouse brain, providing a demonstration of SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. The ability to target a disease-causing allele using RNAi-based therapies could be applied to a wide range of dominant CNS disorders, where maintenance of wild-type expression is essential.

neuroscience↗