14800-DvRappard

91 Donor macrophages and remyelination in metachromatic leukodystrophy 6 INTRODUCTION Metachromatic leukodystrophy (MLD, MIM 250100) is a devastating inherited white matter (WM) disorder caused by biallelic mutations in ARSA , leading to deficient activity of arylsulfatase A (ASA), a lysosomal enzyme digesting sulfatides (figure 1a). 1 Sulfatides are enriched in myelin sheaths; their accumulation in MLD causes demyelination and inhibits oligodendrocyte differentiation from precursor cells. 6 Depending on the age at onset, MLD is divided into late-infantile (onset before 30 months of age), juvenile (onset between 30 months and 16 years of age) and adult (onset after 16 years of age) forms. If MLD is diagnosed early, its later-onset forms (juvenile and adult) can be treated by allogenic hematopoietic cell transplantation (HCT). 2,3 Ex vivo gene therapy with ASA overexpression has been shown to treat the late-infantile form when done early, before symptoms develop. 7 HCT halts further demyelination, and in some patients brain white matter abnormalities even improve on MRI (figure 1b, c). 4,5 Mechanisms of HCT action in MLD, however, are not yet fully understood. HCT is supposed to provide cross-correction of deficient enzyme levels by secreting ASA from donor cells migrated into the brain, thus restoring sulfatide degradation, but its exact mechanism remains elusive. 8 Until HCT becomes effective, there is an estimated six to twelve month delay possibly causing treatment failure in the rapidly progressive late-infantile form and in juvenile cases presenting with significant disability and MRI abnormalities. Using tissue of children with MLD, two of whom HCT-treated 2 and six untreated, we ascertained the presence of metabolically competent cells in the brain following HCT and compared inflammatory response and oligodendrocyte numbers in the two groups. METHODS The study was approved by the IRB of the VU University Medical Center, with informed parental consent. Brain autopsies of patients 1, 2 and 3 were performed within six hours post-mortem in our center; brain tissue of patients 4 to 8 was obtained through the NIH NeuroBioBank ( https://neurobiobank.nih.gov ) . Tissue staining Six-μm-thick formalin-fixed paraffin-embedded tissue sections were routinely stained with Haematoxylin&Eosin and Toluidine blue. Immunohistochemistry was done as described 9 usingantibodies targeting themicroglia/macrophagesmarkers CD68 (1:1600 Dako, M0814) and CD45 (1:100 Dako, 0701), the myelin marker proteolipid protein (PLP, 1:3000, Biorad, MCA839G), and the M2 marker CD163 (1:300, Cell Marque, MRQ-26). 10