Pdgfrα-dependent Polr3b Exon Loss Recapitulates POLR3-related Hypomyelinating Leukodystrophy Phenotypes in vivo (S2.004)

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Objective: To develop the first model of RNA Polymerase III (Pol III)-Related Hypomyelinating Leukodystrophy (POLR3-HLD) based on a POLR3B mutation. Background: POLR3-HLD is a devastating neurological disease characterized by severe diffuse hypomyelination and progressive functional decline leading to early death. It is caused by biallelic pathogenic variants in genes encoding Pol III subunits. Patients are most commonly affected by mutations in POLR3A or POLR3B, but there are currently no disease models based on POLR3B. Furthermore, existing models of the disease based on POLR3A mutation fail to replicate the hypomyelination severity commonly observed in patients. We hypothesize that mutations in Pol III subunits such as POLR3B reduce enzyme function during a critical developmental period causing defective myelinogenesis and hypomyelination. Design/Methods: We characterized the impact of a POLR3B mutant lacking exon 10 (POLR3BΔ10) on Pol III complex assembly, nuclear import, and protein expression in human cells. We developed an inducible/conditional animal model using the cre/lox system to express the orthologous mutation in a Pdgfrα-dependent manner during postnatal development in mice. The animal model was characterized using a variety of techniques including tissue biochemistry, histology, and advanced imaging (microCT, ex vivo MRI). Results: POLR3BΔ10 expression was shown to cause a severe Pol III assembly defect accompanied by reduced expression and nuclear import of the mutant protein in human cells. Inducing Pdgfrα-dependent expression of orthologous Polr3bΔ10 during postnatal development in mice causes severe hypomyelination, craniofacial defects, and hypodontia. The hypomyelination phenotype was caused by proliferation and maturation defects in oligodendrocyte-lineage cells carrying homozygous Polr3bΔ10, which preceded hypomyelination and led to non-apoptotic cell loss from the brain parenchyma. Conclusions: We describe the first severe model of POLR3-HLD and the first working disease model based on mutation of Polr3b. This work advances our understanding of POLR3-HLD and implicates defective proliferation and differentiation of oligodendrocyte-lineage cells as key features of POLR3-HLD pathogenesis. Disclosure: Mackenzie A. Michell-Robinson has nothing to disclose. Dr. Watt has received research support from National Institute of Dental and Craniofacial Research. Mr. Grouza has nothing to disclose. Ms. Macintosh has nothing to disclose. Dr. Pinard has nothing to disclose. Mr. Tuznik has nothing to disclose. Dr. Chen has nothing to disclose. Lama Darbelli has nothing to disclose. Chia-Lun Wu has nothing to disclose. Dr. Perrier has received research support from the Fonds de Recherche du Quebec-Santé (FRQS) Doctoral Scholarship, the Fondation du Grand défi Pierre Lavoie Doctoral Scholarship, the McGill Faculty of Medicine F.S.B. Miller Fellowship, and the Research Institute of the McGill University Health Centre Desjardins Studentship in Child Health Research. Mr. Chitsaz has received research support from Multiple Sclerosis Society of Canada, Fonds de Recherche du Quebec. Dr. Uccelli has received research support from Hoffmann-La Roche Limited. Dr. Liu has nothing to disclose. The institution of Prof. Cox has received research support from NIH. Dr. Mueller has nothing to disclose. Timothy Kennedy has received research support from Canadian Institutes of Health Research. Benoit Coulombe has nothing to disclose. David Rudko has nothing to disclose. Dr. Trainor has received personal compensation for serving as an employee of Stowers Institute for Medical Researh. Dr. Trainor has received personal compensation in the range of $10,000-$49,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Developmental Dynamics. The institution of Dr. Trainor has received research support from Stowers Institute for Medical Research. Geneviève Bernard has nothing to disclose.

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