Surveying Glial Disparities in Two Rare and Neglected Diseases: Non-Ketotic Hyperglycinemia and Kabuki Syndrome

Studying rare neurodegenerative diseases presents significant challenges due to limited epidemiological data, incomplete understanding of pathophysiological mechanisms, and low research investment. In the United States, a disease is classified as rare if it affects fewer than 200,000 individuals – approximately 1 in 2,000 people in a population of 330 million. In this thesis research, I examined two rare disorders: Non-Ketotic Hyperglycinemia (NKH) and Kabuki Syndrome (KS). Both conditions are associated with abnormalities in glial cell populations and result in impaired neurodevelopment. My findings indicate that astrocyte function in NKH and microglial activity in KS are significantly suppressed in their respective mouse models. Both cell types exhibit functional recovery following treatment. These results potentially reveal novel pathophysiological mechanisms in both NKH and KS that have not been previously described. NKH is an autosomal recessive, mitochondrial disorder caused by defects in the glycine cleavage system (GCS) that results in the elevation of glycine both systemically and in the central nervous system. Glycine decarboxylase (GLDC, also called P protein) is a major component of the GCS. Mutations in Gldc cause over 80% of NKH cases. The current estimated prevalence is ~1:76,000 births, with some regional variations; for instance, within the Amish community, the estimate is at ~1:2,000. Clinical presentation is highly heterogeneous with broad, neurometabolic range and is often severe. Adjunctive treatment of glycine reduction frequently fails to confer neurological benefit. In our study, we used an attenuated CRISPR Cas9-edited humanized mouse expressing a prevalent clinical mutation, administered with a single intraperitoneal dose of a novel recombinant of adeno-associated viral vector 9 and GLDC (rAAV9-GLDC). Mice were assessed over five and ten months and validated by assessing the brain and liver. Our control was a single intraperitoneal dose of rAAV9 containing green fluorescent protein (GFP). Our results present a significant increase in astrocytes with minimal inflammatory distress. Additionally, whole brain protein analysis revealed increased levels of neurons and neuronal precursors. However, there were no changes in oligodendrocytes and microglia. Overall, our novel single dose gene therapy increased astrocyte proliferation and eliminated long term neurological defects and death. KS is a rare autosomal heterozygous intellectual disability disorder caused by defects in lysine-specific methyltransferase 2D protein (KMT2D), which results in a decrease in open chromatin mark H3K4me3. Mutations in Kmt2D cause over 55-80% of KS cases. The current estimated prevalence of KS is ~1:86,000 births, with some regional variations; for example, in Japan, the estimate is ~1:32,000. A decrease in open chromatin and trimethylation of H3K4me3 leads to reduced hippocampal functions, low proliferation levels of microglia, and overall cerebral function. To alleviate symptoms, we introduced a triple combination formulation (TCF) that delivers the HDACi vorinostat (Vo) to the brain, bypassing the blood brain barrier (BBB). TCF administration increased the acetylation at H3K4me3, which resulted in promoting microglial arborization, proliferation, and surveillance phenotype. Our results point to an unprecedented defect in KS and provide an effective method of microglial differentiation without a major inflammatory response. My research highlights previously unrecognized glial cell dysfunctions in NKH and KS, offering new insights into the underlying pathophysiology of these rare neurodevelopmental disorders. By demonstrating that targeted therapeutic interventions can restore astrocyte and microglial function without provoking significant inflammatory responses, these findings open new avenues for the development of effective treatments. Overall, this work emphasizes the importance of glial cells in rare neurological diseases and supports further exploration of gene therapy and epigenetic modulation as feasible strategies for addressing unmet clinical needs in these patient groups.<p></p>