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

Abstract

Pelizaeus–Merzbacher disease (PMD), also known as hypomyelinating leukodystrophy 1 (HLD1), is an X-linked recessive disease affecting in the central nervous system (CNS). The gene responsible for HLD1 encodes proteolipid protein 1 (plp1), which is the major myelin structural protein produced by oligodendroglial cells (oligodendrocytes). HLD15 is an autosomal recessive disease affecting the glutamyl-prolyl-aminoacyl-tRNA synthetase 1 (eprs1) gene, whose product, the EPRS1 protein, is a bifunctional aminoacyl-tRNA synthetase that is localized throughout cell bodies and that catalyzes the aminoacylation of glutamic acid and proline tRNA species. Here, we show that the HLD15-associated nonsense mutation of Arg339-to-Ter (R339X) localizes EPRS1 proteins as polymeric aggregates into Rab7-positive vesicle structures in mouse oligodendroglial FBD-102b cells. Wild-type proteins, in contrast, are distributed throughout the cell bodies. Expression of the R339X mutant proteins, but not the wild-type proteins, in cells induces strong signals regulating Rab7. Whereas cells expressing the wild-type proteins exhibited phenotypes with myelin web-like structures bearing processes following the induction of differentiation, cells expressing the R339X mutant proteins did not. These results indicate that HLD15-associated EPRS1 mutant proteins are localized in Rab7-positive vesicle structures where they modulate Rab7 regulatory signaling, inhibiting cell morphological differentiation. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD15.

Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

“…To determine whether, similar to other HLD-related cellular phenotypes [ 18 , 19 , 20 , 21 , 22 ], the R140X mutant proteins of POLR3A form protein aggregates in cells, we transfected the plasmid encoding POLR3A harboring the R140X mutation or the wild type into FBD−102b cells. The transfected cell images showed that the wild type POLR3A proteins were distributed throughout the cytoplasm and slightly in nuclei ( Figure 1 A).…”

“…Cells from the oligodendroglial FBD−102b cell line (a mouse brain oligodendroglial precursor cell line) were kindly provided by Dr. Y. Tomo-oka (Tokyo University of Science, Chiba, Japan; and Riken, Saitama, Japan). FBD−102b cells [ 18 , 19 , 20 , 21 , 22 ] were cultured on standard cell culture dishes (Greiner, Oberösterreich, Germany) in a culture medium consisting of DMEM/Nutrient Mixture F-12 (Nacalai Tesque, Kyoto, Japan) containing 10% heat-inactivated FBS (Thermo Fisher Scientific, Waltham, MA, USA) and PenStrep (Thermo Fisher Scientific) in 5% CO 2 at 37 °C. To induce differentiation, FBD−102b cells were cultured for 5 days in the same culture medium without FBS on cell culture dishes with advanced TC polymer surface modification (Greiner) in 5% CO 2 at 37 °C [ 18 , 19 , 20 , 21 , 22 ].…”

“…FBD−102b cells [ 18 , 19 , 20 , 21 , 22 ] were cultured on standard cell culture dishes (Greiner, Oberösterreich, Germany) in a culture medium consisting of DMEM/Nutrient Mixture F-12 (Nacalai Tesque, Kyoto, Japan) containing 10% heat-inactivated FBS (Thermo Fisher Scientific, Waltham, MA, USA) and PenStrep (Thermo Fisher Scientific) in 5% CO 2 at 37 °C. To induce differentiation, FBD−102b cells were cultured for 5 days in the same culture medium without FBS on cell culture dishes with advanced TC polymer surface modification (Greiner) in 5% CO 2 at 37 °C [ 18 , 19 , 20 , 21 , 22 ]. We incubated cells with or without 300 μM ibuprofen [ 14 , 15 , 16 , 17 ] and/or 500 nM AH6809 (IC 50 = 6.8 nM for prostaglandin EP1 receptor in a cell-free system; see the Tocris Biological Activity website, accessed on 1 April 2020) for 2 days.…”

“…In addition, we changed the culture medium into a newly-prepared medium containing ibuprofen and/or AH6809, and further incubated cells with each or both of them for 3 days. Cells with widespread membranes (more than 75 μm square fields; Image J software [Bethesda, MD, USA]) were identified as differentiated [ 18 , 19 , 20 , 21 , 22 ]. We confirmed that FBD−102b cells were viable under each experimental condition by verifying that the attached trypan-blue (Nacalai Tesque)-incorporating cells made up less than 5% of all the cells in each culture.…”

“…Along with these observations, the expression levels of myelin marker proteins were confirmed by immunoblotting techniques. FBD−102b cells form widespread membranes and express myelin marker proteins [ 18 , 19 , 20 , 21 , 22 ]. However, it is unlikely that FBD−102b cells are able to achieve the terminal differentiated phenotype, since they fail to exhibit the myelin web-like membrane structures needed to wrap neuronal axons with their membranes.…”