Abstract: The myelin sheaths that wrap around axons in the Central Nervous System (CNS) are damaged or formed incorrectly in many developmental brain disorders. We thus sought to better understand the control of myelination and how it may be impaired in such conditions. Essentially all of the major myelin proteins are covalently modified with the lipid palmitate, a process that is often critical for correct protein subcellular localization and function, but how myelin protein palmitoylation is controlled is unknown. We were therefore struck by findings that the X-linked Intellectual Disability (XLID) gene ZDHHC9 codes for a specific palmitoyl acyltransferase enzyme that is highly expressed in oligodendrocytes (OLs), the myelinating cells of the CNS. Importantly, human patients with ZDHHC9 loss-of-function mutations and Zdhhc9 knockout (KO) mice display cognitive deficits and have markedly reduced forebrain white matter volume, suggesting a key role for ZDHHC9 in OL formation and/or function. Our own studies revealed that Zdhhc9 KO mice have impaired corpus callosal myelination without loss of neuronal axons and that Zdhhc9 knockdown cell-autonomously impairs OL maturation after differentiation from Oligodendrocyte Precursor Cells (OPCs) in culture. These striking phenotypes may account for the intellectual disability and/or epileptic seizures seen in human patients with ZDHHC9 mutations. In this project we will more precisely define links between Zdhhc9 mutation and myelination impairments. In Aim 1, we will combine well-established Electron Microscopic (EM) and immunostaining analyses with a genetic fate-mapping strategy to comprehensively define how Zdhhc9 loss impacts OL distribution and axonal myelination in vivo. In Aim 2 we will more precisely determine how cell- autonomous Zdhhc9 loss affects OL differentiation and morphological elaboration. In each Aim we will compare the ability of wild type (wt) and XLID mutant forms of ZDHHC9 to rescue the observed phenotypes. These studies will provide new insights regarding not just the impact of Zdhhc9 loss, but also palmitoylation- dependent control of myelination, a process first reported decades ago but about which almost nothing is known. Insights from this work could thus be invaluable to those studying a range of brain disorders marked by White Matter Impairments.

Project Narrative: The proposed study focuses on how mutations in the X-linked Intellectual Disability (XLID) gene ZDHHC9 cause impairments in the maturation and morphological elaboration of oligodendrocytes (OLs), the myelin- forming cells of the Central Nervous System (CNS). We will define how ZDHHC9 loss affects myelin formation in vivo and, in complementary studies, will determine which aspects of development of OLs in culture are ZDHHC9-dependent. This work will provide insights not just into ZDHHC9-dependent XLID, but also into other forms of ID and conditions such as cerebral palsy, which are marked by impairments to CNS myelin.