Abstract
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.
Highlights
POLR3A and POLR3B are important subunits of RNA polymerase III, which is a synthesizing non-coding RNA that includes a variety of tRNAs and rRNAs
While the wild type POLR3B proteins were distributed throughout the cell body (Figure 1A), mutated POLR3B proteins were localized in punctate structures in the cytoplasmic regions in over 90% of the transfected cells (Figure 1B,C)
Because wild type POLR3B proteins were localized throughout cell bodies, wild type POLR3B proteins may be at least partially co-localized with major organelle antigens
Summary
POLR3A and POLR3B are important subunits of RNA polymerase III, which is a synthesizing non-coding RNA that includes a variety of tRNAs and rRNAs. HLD8 is a typical oligodendroglial cell genetic disease [1–4]. Despite these detailed studies on the relationship between genes encoding POLR3A or POLR3B and disease pathologic features, therapeutic drugs and strategies for HLD8 have not been established. Hypomyelinating leukodystrophies (HLDs) are a recently classified group of hereditary neuropathies [5–8]. These diseases are rare, affecting one out of every 250,000 to 500,000 people [5–8]. Oligodendrocytes ( called oligodendroglial cells) play a role in generating the myelin sheath [9–12]. They functionally contribute to the propagation of saltatory conduction, and they protect neuronal axons from a variety of stresses such as physical and physiological stresses [9–12]
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