Role of Microglia in Vanishing White Matter Disease Leukodystrophy

Leukoencephalopathy with Vanishing White Matter (VWM) is a rare neurodegenerative leukodystrophy that presents with ataxia, seizures, and progression to death. There are no clinicaltreatments for VWM, and our ultimate goal is the development of a therapy for VWM. VWM is caused by autosomal recessive mutations in the five subunit genes of the Eukaryotic Initiation Factor 2B (EIF2B) complex, which is necessary for regulation of the integrated stress response (ISR). VWM has abnormal, persistent activation of the ISR. Current dogma in the field has settled on astrocytes as responsible for VWM pathophysiology. We developed adeno-associated virus (AAV)- mediated EIF2B5 gene (the most commonly mutated subunit) replacement therapy for VWM with targeted astrocyte expression. In both the classic R191H mouse model and a more severe I98M mouse model, we showed rescue of VWM-disease related phenotypes (Herstine et al., 2024). However, we observed waning efficacy in treated VWM mice. Interestingly, our preliminary data showed recrudescent ISR activation that mirrored presence of activated microglia. Further, we found that untreated mice have extensive infiltration of Iba1+/CD68+ microglia into the CNS. Activated microglia secrete cytokines which induce reactive astrocytes in vitro and in vivo. Our hypothesis is that activated microglia contribute to VWM pathophysiology, and that transplant of wild-type microglia is necessary for full gene therapy rescue of VWM by interrupting the cycle of microglia-driven reactive astrocyte activation.To test our hypothesis, we propose first, to Characterize microglial phenotypes in wild-type, mutant, and AAV9-treated mutant VWM mice. We will characterize the timing and extent of activated microglia involvement in the CNS, in R191H and I98M mice, at defined ages; in comparison to wild-type and AAV9-treated VWM affected mice. Histological, molecular, and biochemical targets include CD68 and Iba1 (for activated and stable microglia); and CNS tissue markers (GFAP, astrocytes; NeuN and neurofilament light, neurons; and Olig2, fluoromyelin, oligodendrocytes and myelin). Second, we will Perform microglia replacement using circulation-derived myeloid cells (CDMCs) in VWM mice in conjunction with or without AAV9 gene therapy. Since dysregulated microglia may interfere with gene therapy rescue, we will test a combination approach of microglia transplantation and gene therapy. We will use a published protocol, which we have demonstrated in preliminary data is effective for VWM mice at our lab (busulfan ablation of endogenous hematopoietic cells, followed by bone marrow transplant with donor circulation derived myeloid cells (CDMC)). With use of PLX5622 (a CSF1R inhibitor required for native microglia), the CDMC cross the blood-brain barrier and replace endogenous microglia. Our experiments will include 3 mouse groups: VWM mice; VWM mice receiving CDMC replacement and AAV9-EIF2B5 gene therapy; and VWM mice with CDMC only (no gene therapy). Project Number: 1R21HD119649-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Joshua Bonkowsky (+1 co-PI) | Institution: UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH, SALT LAKE CITY, UT | Award Amount: $424,655 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 BN-S (90)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21HD11964901