Studies on RNA polymerase III-related leukodystrophy

Project Summary RNA polymerase (Pol) III synthesizes abundant non-coding RNAs such as tRNA, 5S rRNA, U6 snRNA and 7SL RNA that are involved in essential cellular processes including protein synthesis, RNA processing and protein secretion. Despite the essential roles of these RNAs in all eukaryotic cells, mutations in Pol III can have remarkably selective effects on the central nervous system (CNS) as demonstrated by patients with Pol III- related leukodystrophy. Mutations causing this autosomal recessive neurodegenerative disease have been identified in five different Pol III subunits with disease onset occurring most often in early childhood. Pol III- related leukodystrophy is typically a hypomyelinating disorder although patients present with a range of both neurological and non-neurological deficits. Neurological deficits typically include developmental delay with a progressive decline in cognitive and motor function, ataxia and cerebellar atrophy. Non-neurological deficits may include hypodontia, myopia and endocrine abnormalities. An understanding of the disease mechanisms underlying these clinical characteristics is currently lacking and treatments are limited to palliative care. A significant barrier to progress on both these fronts has been the absence of an animal model of the disease. However, recent work has resulted in a mouse model of Pol III-related leukodystrophy that recapitulates a subset of clinical features. Conditional knock-in of pathogenic mutations in Polr3a, which encodes the largest Pol III subunit, specifically in Olig2-expressing cells in the CNS, leads to reduce growth, neurobehavioral deficits and hypomyelination in multiple regions of the brain and spinal cord. Moreover, electron microscopy along with an analysis of oligodendrocyte cell populations and specific protein markers has identified two distinct mechanisms contributing to disease pathogenesis: Defective differentiation of oligodendrocyte precursor cells (OPCs) and defective function of mature oligodendrocytes (OLs). Nevertheless, the molecular mechanisms of pathogenesis remain poorly characterized. To advance understanding in this regard, the long- term goals of this work are to determine how the Pol III transcriptome has been altered in OPCs and OLs in the mutant mice and to examine the consequences of these changes for protein synthesis and for the proteome of OPCs and OLs and the lipidome of myelin. Additionally, we will test a model for genetic suppression of Pol III- related leukodystrophy phenotypes in the mice. Together, these studies will inform how decreasing Pol III transcription interferes with oligodendrogenesis and myelination, yielding important mechanistic insights, and a potential therapeutic approach that may benefit patients with Pol III-related leukodystrophy.