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.

Because the HLD8-associated mutation of POLR3B decreases the POLR3B protein expression level, it is thought to be a loss-of-function mutation. However, some residual proteins with the HLD8-associated mutation seem likely to remain in pathological cells and tissues [2][3][4]. Therefore, these residual proteins may cause molecular and cellular pathological effects of HLD8. Herein, we report that a severe HLD8-associated nonsense mutation of Arg550-to-Ter (R550X) localizes POLR3B proteins as aggregates, which is similar to other HLD-associated mutated proteins [13][14][15][16][17][18] in FBD-102b cell lysosomes, and it is used as an oligodendroglial precursor cell model. However, the wild type proteins were not localized in lysosomes. Additionally, in cells expressing proteins with the R550X mutation, lysosomerelated signaling of the mechanistic target of rapamycin (mTOR) such as phosphorylation of ribosomal S6 and 4E-BP1 proteins [19][20][21][22][23][24] was greatly decreased. Additionally, while cells harboring the R550X mutant constructs exhibited decreased morphological differentiation, cells harboring wild type constructs showed differentiated phenotypes. Furthermore, molecular and cellular phenotypes harboring the mutant constructs were improved by ibuprofen, which is a non-steroidal anti-inflammatory drug (NSAID) that has been reported to have protective effects preferentially in neuronal cells but not in glial cells [25,26].
Thus, the HLD8-associated mutation of POLR3B proteins affects oligodendroglial cell morphological differentiation possibly through decreased lysosome-related mTOR signaling, and treatment with ibuprofen can cause its amelioration in glial cells such as oligodendroglial cells. This suggests some underlying molecular and cellular pathological mechanisms for HLD8.

Plasmid Constructions and Primary and Secondary Antibodies
The full-length human polr3b (Gene ID 55703) gene, which is inserted into a vector expressing GFP, was purchased from Sino Biological, Inc. (Beijing, China). The polr3b cDNA harboring the Arg550-to-Ter (R550X) mutation (OMIM ID 614366) was generated with a Gflex DNA polymerase (Takara Bio, Shiga, Japan)-based method using human polr3b gene as template. DNA sequences were confirmed by Fasmac sequencing service (Kanagawa, Japan). Antibodies that were used in these experiments are listed in Table 1.

Cell Differentiation
FBD-102b cells were cultured in medium without FBS on the cell culture dishes using advanced TC polymer modification (Greiner) in 5% CO 2 at 37 • C to induce differentiation. After a few days, cells grow to have a widespread membrane that is similar to the myelin membrane. Their cellular morphological phenotypes were identified as differentiated cells [27]. We confirmed that FBD-102b cells were viable under each differentiation experimental condition by verifying that attached trypan-blue (Nacalai Tesque)-incorporating cells comprised less than 5% of all cells in each culture.

Cell Transfection
Cells were transfected with the respective plasmids using a Screen Fect A or Screen Fect A Plus transfection kit (Fujifilm, Tokyo, Japan), in accordance with the manufacturer's instructions. The medium was replaced 4 h after transfection. Transfected cells were generally used for experiments 48 h after transfection. Otherwise, G418 (2500 microgram/mL)resistant clones were collected as stable clones.
We confirmed that FBD-102b cells were viable under each transfection experimental condition by verifying that attached trypan-blue-incorporating cells made up less than 5% of all cells in each culture.

Confocal Microscopic Images
Cells on glass coverslips were fixed with 4% paraformaldehyde or 100% cold methanol and blocked with a Blocking One reagent (Nacalai Tesque). Then, cells were incubated with primary antibodies and secondary antibodies that were conjugated with Alexa Fluor dyes, in accordance with the manufacturer's instructions. Each glass coverslip was placed on each slide glass and mounted with a Vectashield reagent (Vector Laboratories, Burlingame, CA, USA).
TIFF images of the cells were collected using a microscope equipped with a laserscanning Fluoview apparatus (FV1000D or FV1200, Olympus, Tokyo, Japan) and processed using Fluoview software (ver. 2016, Olympus). The resulting color images were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Each image in each figure is representative of three independent experimental results.
Images were captured as TIFF files using LiDE scanners (Canon, Tokyo, Japan) and processed using LiDE driver software (ver. 2017, Canon). The band pixels were measured using Image J software (ver. 1.53). Each image in each figure is representative of three independent experimental results ( Figure S1).

Statistical Analysis
Values are presented as the average ± standard deviation (SD) for separate experiments. Intergroup comparisons were made using the unpaired Student's t-test and Excel software (ver. 2019, Microsoft, Redmond, WA, USA). A one-way analysis of variance (ANOVA) was followed by a Fisher's protected least significant difference (PLSD) test as a post hoc comparison using StatPlus (add-in Exel Ver. 2019, AnalystSoft, Walnut, CA, USA). Differences were considered significant when p < 0.05.

Ethics Statement
Gene recombination techniques were performed in accordance with a protocol that was approved by the Tokyo University of Pharmacy and Life Sciences Gene and Animal Care Committee (Approved Nos. LS28-20 and LSR3-011).

The R550X Mutant Proteins of POLR3B Are Aggregated in Lysosomes in FBD-102b Cells
Because R550X mutant proteins in POLR3B may be localized in punctate structures [13][14][15][16][17][18], we transfected the plasmid encoding the wild type or mutated POLR3B into FBD-102b cells. 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).
We then investigated which organelle corresponds to punctate structures. Wild type POLR3B proteins were not significantly co-localized with the endoplasmic reticulum (ER) antigen Lys-Asp-Asn-Leu (KDEL) ( Figure 2). Additionally, mutated POLR3B proteins were not significantly co-localized with the KDEL antigen ( Figure 3). However, 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. Such localization for wild type POLR3B proteins was obtained for the Golgi body antigen Golgi matrix protein of 130 kDa (GM130) (Figure 4). Mutated POLR3B proteins were not co-localized with the GM130 antigen ( Figure 5). Moreover, wild type POLR3B proteins throughout cell bodies exhibit a co-localization with the lysosome antigen lysosomal-associated membrane protein 1 (LAMP1) ( Figure 6). Conversely, mutated POLR3B proteins were mostly co-localized with LAMP1, indicating that HLD8-associated mutation causes POLR3B proteins to localize in lysosomes ( Figure 7). Additionally, although the wild type POLR3B proteins appeared to overlap with LAMP1 antigens, LAMP1 antigens in normal cellular states can be present throughout the cell body.    We, thus, investigated whether mutated POLR3B proteins are aggregated and localized in lysosomes. The lysates of mock-transfected cells or cells expressing the wild type or mutated proteins were subjected to non-denaturing or denaturing polyacrylamide gel electrophoresis. In non-denaturing or denaturing polyacrylamide gel electrophoresis, the wild type proteins corresponded to the predicted molecular weight (~200 kDa), whereas mutated proteins displayed very high molecular weights ( Figure 8A). In denaturing polyacrylamide gel electrophoresis, the mutated proteins corresponded to the predicted small molecular weight, indicating that HLD8-associated mutation leads to protein aggregation ( Figure 8B). dotted lines in the direction of the arrows can be seen in the bottom panels. (C) Percentages of cells with punctate structures were statistically assessed (** p < 0.01; n = 3 fields).

Cells Expressing the R550X Mutant Proteins of POLR3B Fail to Exhibit Differentiation Phenotypes
Because POLR3B mutated proteins have some molecular pathological properties, we further asked whether mutated proteins have effects on cell differentiation in FBD-102b cells. While cells harboring the wild type constructs exhibited differentiated phenotypes with widespread membranes following the induction of differentiation, cells harboring the R550X mutant constructs failed to show these differentiated phenotypes ( Figure 9A,B). These results were supported by decreased expression levels of the differentiation and myelin marker proteins PLP1 and CNPase ( Figure 9C,D). The oligodendrocyte-lineage marker Sox10 and the internal control protein actin were comparable in both cells, revealing that HLD8-associated mutation is related to an inhibition of morphological differentiation.
Because mutated proteins are localized as aggregates in lysosomes, we examined whether cells expressing the mutated proteins decrease signaling through mTOR. Signaling through mTOR is present around lysosomes that have activities that are strictly correlated with their signaling [19,20]. Additionally, mTOR signaling, including ribosomal S6 and 4E-BP1 protein phosphorylation, is required for oligodendroglial cell differentiation and subsequent myelination [21][22][23][24]. As expected, cells expressing the mutated, but not the wild type, proteins showed decreased ribosomal S6 protein phosphorylation ( Figure  10A,B shows immunofluorescence; Figure 10C,D shows immunoblotting). Similar results were obtained for 4E-BP1 protein phosphorylation.

Cells Expressing the R550X Mutant Proteins of POLR3B Fail to Exhibit Differentiation Phenotypes
Because POLR3B mutated proteins have some molecular pathological properties, we further asked whether mutated proteins have effects on cell differentiation in FBD-102b cells. While cells harboring the wild type constructs exhibited differentiated phenotypes with widespread membranes following the induction of differentiation, cells harboring the R550X mutant constructs failed to show these differentiated phenotypes ( Figure 9A,B). These results were supported by decreased expression levels of the differentiation and myelin marker proteins PLP1 and CNPase ( Figure 9C,D). The oligodendrocyte-lineage marker Sox10 and the internal control protein actin were comparable in both cells, revealing that HLD8-associated mutation is related to an inhibition of morphological differentiation.
Because mutated proteins are localized as aggregates in lysosomes, we examined whether cells expressing the mutated proteins decrease signaling through mTOR. Signaling through mTOR is present around lysosomes that have activities that are strictly correlated with their signaling [19,20]. Additionally, mTOR signaling, including ribosomal S6 and 4E-BP1 protein phosphorylation, is required for oligodendroglial cell differentiation and subsequent myelination [21][22][23][24]. As expected, cells expressing the mutated, but not the wild type, proteins showed decreased ribosomal S6 protein phosphorylation ( Figure 10A,B shows immunofluorescence; Figure 10C,D shows immunoblotting). Similar results were obtained for 4E-BP1 protein phosphorylation.

Ibuprofen Specifically Improves the Phenotypes of Cells Expressing the R550X Mutant Proteins of POLR3B
Ibuprofen is an activator of mTOR signaling [31][32][33][34], and we investigated whether ibuprofen improves the phenotypes of FBD-102b cells harboring the R550X mutant constructs of POLR3B. Treatment with ibuprofen recovered an inhibition of morphological differentiation in cells harboring the mutated constructs ( Figure 11A,B), which is consistent with the increasing expression levels of differentiation and myelin marker proteins ( Figure 11C,D). Additionally, ibuprofen recovered the decreased ribosomal S6 and 4E-BP1 protein phosphorylation levels ( Figure 12A,B). We also explored the effects of ibuprofen on POLR3B mutated protein aggregation. Treatment with ibuprofen recovered the mutated protein aggregation ( Figure 12C,D).
However, when cells expressing wild type proteins were treated with ibuprofen, ibuprofen had no significant effects on cell morphological differentiation ( Figure 13A,B). These effects were supported by the comparable differentiation and myelin marker protein expression levels ( Figure 13C,D). Additionally, ibuprofen neither affected phosphorylation levels of ribosomal S6 and 4E-BP1 proteins ( Figure 14A,B) nor caused aggregation ( Figure 14C,D).
Taken together with the results in cells expressing the wild type proteins, ibuprofen specifically recovers protein aggregation and inhibitory morphological differentiation as well as downregulation of the related signaling in cells expressing the R550X mutant proteins of POLR3B.

Discussion
Here, we showed that HLD8-associated POLR3B nonsense mutant proteins are localized as aggregates in lysosomes. However, the wild type proteins are not localized in lysosomes. These findings are supported by the results that the mutated but not the wild type proteins were co-localized with a LAMP1 antigen but not KDEL and GM130 antigens. Additionally, in non-denaturing polyacrylamide gel electrophoresis, mutated proteins exhibit a high molecular weight, whereas wild type proteins correspond to the predicted molecular weight. In denaturing polyacrylamide gel electrophoresis, the mutated and wild type proteins correspond to the predicted molecular weight. We also found that cells expressing the mutated proteins show a decreased ability to differentiate morphologically and decreased phosphorylation/activation levels of molecules associated with mTOR signaling; conversely, cells expressing wild type proteins retain their normal abilities. These phenotypes were consistent with changes in differentiation marker protein expression levels. Inhibition of differentiation by the mutated proteins might be consistent with the fact that HLD8 patients show hypomyelination in various brain regions [2][3][4]. Finally, we have identified ibuprofen as a drug that recovers the decreased differentiating ability in cells expressing the mutated proteins. Ibuprofen likely has glial cell-protective effects and the ability to promote cell morphological differentiation. Despite increasing identification of HLD-responsible genes, the molecular mechanisms underlying HLDs have remained unclear. Additionally, the therapeutic procedure has not been established. This is the first report that shows how HLD8-associated POLR3B nonsense mutant proteins inhibit oligodendroglial cell morphological differentiation and how ibuprofen recovers HLD8-associated defective morphological differentiation.
R550X mutation results in a premature stop of POLR3B protein synthesis, which generates proteins with deficient functional domains, and this mutation is thought be a loss-of-function mutation. In this study, the R550X mutation was suggested to cause protein aggregation and its accumulation in lysosomes. This accumulation probably affects signals around lysosomes and morphological differentiation, and on the basis of these results, the R550X mutation can correspond to a gain of function. Although HLD8 includes some other POLR3B mutations, the respective mutations may increase protein aggregation and/or may display a functional deficiency in POLR3B. Thus, possible molecular and cellular pathological effects of mutations in POLR3B may be similarly observed for HLD3associated mutation of aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (AIMP1) [26], HLD15-associated mutation of glutamyl-prolyl-tRNA synthetase 1 (EPRS1) [17], and HLD17-associated mutation of AIMP2 [18].
It remains unclear which ibuprofen target molecule is involved in promoting morphological differentiation. In contrast to an unanticipated involvement of ibuprofen in oligodendroglial cells in this study, ibuprofen is known to have multiple effects on neuronal cells and tissues. Whether direct or indirect, many ibuprofen target molecules and pathways with protective effects have been described besides cyclooxygenases, such as Cox1/2 [25], which show antipyretic effects ( Table 2). Among them, one target is amyloid precursor protein (APP) or amyloid oligomers such as an APP pathological product peptide or other protein oligomers [35][36][37][38]. Ibuprofen inhibits APP secretion and binds pathological amyloid to decrease its toxicity. These reactions are direct effects of ibuprofen. It tightly binds to protein structures such as pathological amyloid proteins to decrease their structural toxicity. Because treating cells with ibuprofen decreases mutated POLR3B protein aggregation, it is possible that ibuprofen can eliminate protein aggregation by binding to the mutated proteins. However, it remains unclear whether the mutated POLR3B proteins display a beta-amyloid-like protein structure.

Molecular Target and/or Mechanism
Another ibuprofen target in neuronal cells is the inhibition of molecule(s) that may be associated with Rho signaling [39][40][41][42][43]. RhoA and related small GTPases as well as Rho-kinases of their effectors are negative regulators of axonal process elongation, whereas Rac1 and Cdc42, which are Rho-family small GTPase members, act as positive regulators of neuronal process elongation. The inhibition of Rho signaling in vitro or in vivo leads to a reverse of neuronal damage following pathological situations such as spinal cord injury. Because oligodendroglial cells also grow multiple processes during their morphological differentiation [9][10][11][12], it is conceivable that ibuprofen participates in oligodendroglial process elongation. Considering all the findings concerning potential ibuprofen targets, ibuprofen may act on all intracellular target molecules at the same or at different times, probably to protect cells and to promote cell differentiation with or without decreasing protein aggregation. No matter which molecular mechanisms underlie treatment with ibuprofen, the results of this study may lead to new disease applications for ibuprofen such as hypomyelinating or demyelinating disease. Ibuprofen may be added to the list of potential HLD8 or other HLD therapeutic drugs.
POLR3B is a key subunit that constitutes RNA polymerase III [1][2][3][4]. Because RNA polymerase III plays a key role in synthesizing essential non-coding RNAs, such as tRNAs and rRNAs, functional defects in POLR3B are thought to have serious effects on normal tissue and organ development. The defects are typically associated with hypodontia and hypogonadotropic hypogonadism, and specially with brain hypomyelination. However, it is unclear why POLR3B abnormalities have particularly prominent and serious effects on oligodendroglial cells. It might be because during development, oligodendroglial cells need a large amount of tRNAs and rRNAs to undergo dynamic morphological changes. Alternatively, POLR3B may have an unidentified oligodendroglial cell-specific particular role. For example, POLR3B may participate in producing RNAs, such as tRNAs and rRNAs, that have high levels of or that are preferentially specific for oligodendroglial cells [44][45][46][47]. Furthermore, POLR3B may have a dual function, similar to AIMP1, which functions as an adaptor protein of amino acid-tRNA ligases and has a role as a precursor for a cytokine called endothelial monocyte-activating polypeptide II (EMAPII) [48]. If this is the case, one of POLR3B's identified or unidentified roles may be specific for oligodendroglial cells.
Here, we have demonstrated that the HLD8-associated mutation of POLR3B causes POLR3B proteins to aggregate in lysosomes, which decreases the related signaling. The decreased signaling is probably related to the inhibition of oligodendroglial cell morphological differentiation. Ibuprofen recovers the POLR3B mutated protein-mediated inhibition of morphological differentiation and protein aggregation. Similar observations were obtained in studies with HLD7 [49]. Further studies will allow us to understand the detailed molecular mechanisms of how the HLD8-associated mutation of POLR3B inhibits morphological differentiation and also how ibuprofen recovers these possible molecular and cellular pathological effects. Additionally, it can be an important issue to determine whether ibuprofen has effects on HLD8 model mice as well as whether molecular and cellular mechanistic phenotypes seen in cells are preserved in HLD8 model mice. Such studies may lead to the development of the target-specific medications for HLD8 and possibly other HLDs.

Limitation of This Study and Perspective
The molecular mechanisms and amelioration underlying HLD8 have been clarified at molecular and cellular levels in this study. However, defective morphological differentiation remains unclear, specifically the relationship between no widespread membrane phenotypes and hypomyelinating phenotypes. Future research directions may include clarifying these relationships using disease-specific human induced pluripotent stem cells (iPSCs) or human induced oligodendroglial cells (iOCs). Studies will also lead to experiments in human or mouse oligodendroglial cell-neuronal cocultures and/or crossbreeding of disease model mice with genetically modified animals. In the future, ibuprofen might be used with caution in HLD8 patients because it has many applications in humans. Further studies will clarify if known drug(s) can be used for new applications using a model in this study or the improved models in mice and humans.