The Infantile Leukoencephalopathy-Associated Mutation of C11ORF73/HIKESHI Proteins Generates De Novo Interactive Activity with Filamin A, Inhibiting Oligodendroglial Cell Morphological Differentiation

Abstract

Background: Genetic hypomyelinating diseases are a heterogeneous group of disorders involving the white matter. One infantile hypomyelinating leukoencephalopathy is associated with the homozygous variant (Cys4-to-Ser (C4S)) of the c11orf73 gene. Methods: We observed that in mouse oligodendroglial FBD-102b cells, the C4S mutant proteins but not the wild type ones of C11orf73 are microscopically localized in the lysosome. And, they downregulate lysosome-related signaling in an immunoblotting technique. Results: The C4S mutant proteins specifically interact with Filamin A, which is known to anchor transmembrane proteins to the actin cytoskeleton; the C4S mutant proteins and Filamin A are also observed in the lysosome fraction. While parental FBD-102b cells and cells harboring the wild type constructs exhibit morphological differentiation, cells harboring C4S mutant constructs do not. It may be that morphological differentiation is inhibited because expression of these C4S mutant proteins leads to defects in the actin cytoskeletal network involving Filamin A. Conclusions: The findings that leukoencephalopathy-associated C11ORF73 mutant proteins specifically interact with Filamin A, are localized in the lysosome, and inhibit morphological differentiation shed light on the molecular and cellular pathological mechanisms that underlie infantile hypomyelinating leukoencephalopathy.

Hypomyelinating Leukodystrophy 15 (HLD15)-Associated Mutation of EPRS1 Leads to Its Polymeric Aggregation in Rab7-Positive Vesicle Structures, Inhibiting Oligodendroglial Cell Morphological Differentiation

“…African green monkey kidney epithelial cell-like COS-7 cells (Human Health Science Research Resource Bank, Osaka, Japan) were cultured on cell culture dishes (Greiner, Oberösterreich, Germany) in a culture medium consisting of Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) containing 10% heat-inactivated FBS and PenStrep reagent (Thermo Fisher Scientific) in 5% CO 2 at 37 • C [12][13][14][15].…”

“…Cells from the oligodendroglial FBD-102b cell line (a mouse brain neuronal stem cell line) were kindly provided by Dr. Y. Tomo-oka (Tokyo University of Science, Chiba, Japan). These FBD-102b cells were cultured on cell culture dishes in a culture medium consisting of DMEM/Nutrient Mixture F-12 containing 10% heat-inactivated FBS and PenStrep reagent in 5% CO 2 at 37 • C [13][14][15]. To induce differentiation, FBD-102b cells were cultured for several days in the same culture medium without FBS on cell culture dishes (Greiner) with advanced TC polymer modification in 5% CO 2 at 37 • C [13][14][15].…”

“…These FBD-102b cells were cultured on cell culture dishes in a culture medium consisting of DMEM/Nutrient Mixture F-12 containing 10% heat-inactivated FBS and PenStrep reagent in 5% CO 2 at 37 • C [13][14][15]. To induce differentiation, FBD-102b cells were cultured for several days in the same culture medium without FBS on cell culture dishes (Greiner) with advanced TC polymer modification in 5% CO 2 at 37 • C [13][14][15]. Cells with myelin-like wide membranes bearing multiple processes from the cell bodies, i.e., with cellular surface areas of 50-square-micrometers, were identified as differentiated [13][14][15].…”

“…To induce differentiation, FBD-102b cells were cultured for several days in the same culture medium without FBS on cell culture dishes (Greiner) with advanced TC polymer modification in 5% CO 2 at 37 • C [13][14][15]. Cells with myelin-like wide membranes bearing multiple processes from the cell bodies, i.e., with cellular surface areas of 50-square-micrometers, were identified as differentiated [13][14][15].…”

“…Transfected cells were generally used for experiments 48 h after transfection. The transfection efficiencies of COS-7 and FBD-102b cells were approximately 75% and 25%, respectively [15]. Since COS-7 cells had a higher transfection efficiency, they were used for the subsequent biochemical experiments.…”