Hypomyelinating Leukodystrophy 10 (HLD10)-Associated Mutations of PYCR2 Form Large Size Mitochondria, Inhibiting Oligodendroglial Cell Morphological Differentiation.

Hypomyelinating leukodystrophy 10 (HLD10) is an autosomal recessive disease related to myelin sheaths in the central nervous system (CNS). In the CNS, myelin sheaths are derived from differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve neuronal functions. Nucleotide mutations of the pyrroline-5-carboxylate reductase 2 (PYCR2) gene are associated with HLD10, likely due to PYCR2's loss-of-function. PYCR2 is a mitochondrial residential protein and catalyzes pyrroline-5-carboxylate to an amino acid proline. Here, we describe how each of the HLD10-associated missense mutations, Arg119-to-Cys [R119C] and Arg251-to-Cys [R251C], lead to forming large size mitochondria in the FBD-102b cell line, which is used as an oligodendroglial cell differentiation model. In contrast, the wild type proteins did not participate in the formation of large size mitochondria. Expression of each of the mutated R119C and R251C proteins in cells increased the fusion abilities in mitochondria and decreased their fission abilities relatively. The respective mutant proteins, but not wild type proteins also decreased the activities of mitochondria. While cells expressing the wild type proteins exhibited differentiated phenotypes with widespread membranes and increased expression levels of differentiation marker proteins following the induction of differentiation, cells harboring each of the mutant proteins did not. Taken together, these results indicate that an HLD10-associated PYCR2 mutation leads to the formation of large mitochondria with decreased activities, inhibiting oligodendroglial cell morphological differentiation. These results may reveal some of the pathological mechanisms in oligodendroglial cells underlying HLD10 at the molecular and cellular levels.

The Antiepileptic Valproic Acid Ameliorates Charcot-Marie-Tooth 2W (CMT2W) Disease-Associated HARS1 Mutation-Induced Inhibition of Neuronal Cell Morphological Differentiation Through c-Jun N-terminal Kinase.

Hereditary peripheral neuropathies called Charcot-Marie-Tooth (CMT) disease affect the sensory nerves as well as motor neurons. CMT diseases are composed of a heterogeneous group of diseases. They are characterized by symptoms such as muscle weakness and wasting. Type 2 CMT (CMT2) disease is a neuropathy with blunted or disrupted neuronal morphological differentiation phenotypes including process formation of peripheral neuronal axons. In the early stages of CMT2, demyelination that occurs in Schwann cells (glial cells) is rarely observed. CMT2W is an autosomal-dominant disease and is responsible for the gene encoding histidyl-tRNA synthetase 1 (HARS1), which is a family molecule of cytoplasmic aminoacyl-tRNA synthetases and functions by ligating histidine to its cognate tRNA. Despite increasing knowledge of the relationship of mutations on responsible genes with diseases, it still remains unclear how each mutation affects neuronal differentiation. Here we show that in neuronal N1E-115 cells, a severe Asp364-to-Tyr (D364Y) mutation of HARS1 leads to formation of small aggregates of HARS1 proteins; in contrast, wild type proteins are distributed throughout cell bodies. Expression of D364Y mutant proteins inhibited process formation whereas expression of wild type proteins possessed the normal differentiation ability to grow processes. Pretreatment with the antiepileptic valproic acid recovered inhibition of process formation by D364Y mutant proteins through the c-Jun N-terminal kinase signaling pathway. Taken together, these results indicate that the D364Y mutation of HARS1 causes HARS1 proteins to form small aggregates, inhibiting process growth, and that these effects are recovered by valproic acid. This could be a potential therapeutic drug for CMT2W at the cellular levels.

Hypomyelinating Leukodystrophy 8 (HLD8)-Associated Mutation of POLR3B Leads to Defective Oligodendroglial Morphological Differentiation Whose Effect Is Reversed by Ibuprofen.

POLR3B and POLR3A are the major subunits of RNA polymerase III, which synthesizes non-coding RNAs such as tRNAs and rRNAs. Nucleotide mutations of the RNA polymerase 3 subunit b (polr3b) gene are responsible for hypomyelinating leukodystrophy 8 (HLD8), which is an autosomal recessive oligodendroglial cell disease. Despite the important association between POLR3B mutation and HLD8, it remains unclear how mutated POLR3B proteins cause oligodendroglial cell abnormalities. Herein, we show that a severe HLD8-associated nonsense mutation (Arg550-to-Ter (R550X)) primarily localizes POLR3B proteins as protein aggregates into lysosomes in the FBD-102b cell line as an oligodendroglial precursor cell model. Conversely, wild type POLR3B proteins were not localized in lysosomes. Additionally, the expression of proteins with the R550X mutation in cells decreased lysosome-related signaling through the mechanistic target of rapamycin (mTOR). Cells harboring the mutant constructs did not exhibit oligodendroglial cell differentiated phenotypes, which have widespread membranes that extend from their cell body. However, cells harboring the wild type constructs exhibited differentiated phenotypes. Ibuprofen, which is a non-steroidal anti-inflammatory drug (NSAID), improved the defects in their differentiation phenotypes and signaling through mTOR. These results indicate that the HLD8-associated POLR3B proteins with the R550X mutation are localized in lysosomes, decrease mTOR signaling, and inhibit oligodendroglial cell morphological differentiation, and ibuprofen improves these cellular pathological effects. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD8 and their amelioration.

Knockdown of Golgi Stress-Responsive Caspase-2 Ameliorates HLD17-Associated AIMP2 Mutant-Mediated Inhibition of Oligodendroglial Cell Morphological Differentiation.

Hypomyelinating leukodystrophy 17 is an autosomal recessive disease affecting myelin-forming oligodendroglial cells in the central nervous system. The gene responsible for HLD17 encodes aminoacyl-tRNA synthase complex-interacting multifunctional protein 2, whose product proteins form a scaffold that supports aminoacyl-tRNA synthetases throughout the cell body. Here we show that the HLD17-associated nonsense mutation (Tyr35-to-Ter [Y35X]) of AIMP2 localizes AIMP2 proteins as aggregates into the Golgi bodies in mouse oligodendroglial FBD-102b cells. Wild type AIMP2 proteins, in contrast, are distributed throughout the cell body. Expression of the Y35X mutant proteins, but not the wild type proteins, in cells upregulates Golgi stress signaling involving caspase-2 activation. Cells expressing the wild type proteins exhibit differentiated phenotypes with web-like structures bearing many processes following the induction of differentiation, whereas cells expressing the Y35X mutant proteins fail to differentiate. Furthermore, CASP2 knockdown but not control knockdown reverses the phenotypes of cells expressing the mutant proteins. These results suggest that HLD17-associated AIMP2 mutant proteins are localized in the Golgi bodies where their proteins stimulate Golgi stress-responsive CASP2 to inhibit differentiation; this effect is ameliorated by knockdown of CASP2. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD17 and possible approaches to ameliorating the disease's effects.

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

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.

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

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

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD-102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.