Knockdown of Rab7B, But Not of Rab7A, Which Antagonistically Regulates Oligodendroglial Cell Morphological Differentiation, Recovers Tunicamycin-Induced Defective Differentiation in FBD-102b Cells

In the central nervous system (CNS), insulative myelin sheaths are generated from the differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve saltatory conduction. Despite the functional involvement of myelin sheaths in the CNS, the molecular mechanism by which oligodendroglial cells themselves undergo differentiation of plasma membranes remains unclear. It also remains to be explored whether their signaling mechanisms can be applied to treating diseases of the oligodendroglial cells. Here, we describe that Rab7B of Rab7 subfamily small GTPases negatively regulates oligodendroglial cell morphological differentiation using FBD-102b cells, which are model cells undergoing differentiation of oligodendroglial precursors. Knockdown of Rab7B or Rab7A by the respective specific siRNAs in cells positively or negatively regulated morphological differentiation, respectively. Consistently, these changes were supported by changes on differentiation- and myelination-related structural protein and protein kinase markers. We also found that knockdown of Rab7B has the ability to recover inhibition of morphological differentiation following tunicamycin-induced endoplasmic reticulum (ER) stress, which mimics one of the major molecular pathological causes of hereditary hypomyelinating disorders in oligodendroglial cells, such as Pelizaeus-Merzbacher disease (PMD). These results suggest that the respective molecules among very close Rab7 homologues exhibit differential roles in morphological differentiation and that knocking down Rab7B can recover defective differentiating phenotypes under ER stress, thereby adding Rab7B to the list of molecular therapeutic cues taking advantage of signaling mechanisms for oligodendroglial diseases like PMD.


Introduction
Rab family proteins belong to a small GTPase molecular branch (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995). Rab proteins are the master regulator molecules that control basic cellular functions to mediate intracellular vesicle trafficking to fuse with their cognate target membrane or vesicle (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995). Different or similar Rab proteins are localized on the respective organelles such as the endoplasmic reticulum (ER), Golgi body, and intermediate components between organelles including ones between the ER and the Golgi body as well as the early, recycling, and late endosomes. Thus, to distinguish these intracellular vesicles or components, it is believed that there are over 40 species of Rab proteins (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995;Snider 2003). And, some Rab proteins are redundant in organelles or transporting systems whereas others are unique and may be essential for them (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995;Snider 2003).
Among Rab proteins, Rab7A and Rab42 (also called Rab7B) subfamily molecules are unique, since only Rab7 subfamily of the Rab proteins specifically participates in membrane trafficking around lysosomes and late endosomes (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995;Snider 2003). Rab7A is well studied compared to Rab7B (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995;Snider 2003). It is known that Rab7A and to a lesser extent Rab7B play roles not only in lysosomal biosynthesis, positioning, and function but also in the transport and degradation of cell surface receptors and adhesion molecules (Borg Distefano et al. 2015;Borg et al. 2014;Marubashi and Fukuda 2020;Progida et al. 2010;Snider 2003;Vestre et al. 2021). It is likely that Rab7A and Rab7B actually display differential localization; Rab7A exhibits probable specific localization in late endosomes and lysosomes whereas Rab7B is localized in the trans-Golgi networks, late endosomes, and lysosomes (Borg et al. 2014;Borg Distefano et al. 2015;Marubashi and Fukuda 2020;Snider 2003;Progida et al. 2010;Tian et al. 2021;Vestre et al. 2021).
During development, myelin sheaths are formed from differentiated plasma membranes of oligodendrocytes (also called oligodendroglial cells) in the central nervous system and Schwann cells in the peripheral nervous system. The membranes wrap around neuronal axons where they become multiple layers of myelin. The myelin sheaths often grow to become much larger than the collective surface areas of the plasma membranes prior to myelination. Mature myelin not only aids in conduction propagation but also protects axons from some stresses (Adams et al. 2021;Barateiro et al. 2016;Elbaz and Popko 2019;Simons and Nave 2015). It also contributes to supplying some nutrients to neuronal axons (Adams et al. 2021;Barateiro et al. 2016;Bercury et al. 2014;Elbaz and Popko 2019;Figlia et al. 2018;Simons and Nave 2015).
The question of whether Rab proteins that hold basic and key cell biological roles are actually involved in the regulation of morphological differentiation in oligodendroglial cells remains unanswered. It also remains unclear whether the unique Rab7 protein contributes to differentiation. If so, how Rab7A and Rab7B regulate differentiation needs to be determined. Herein, we describe that Rab7A and Rab7B differentially regulate morphological differentiation in FBD-102b cells, which are models of differentiation of oligodendroglial precursor cells Miyamoto et al. 2007;Nishino et al. 2022;Urai et al. 2018). It is of note that knockdown of Rab7B greatly promoted differentiation. This finding reminded us of the possibility that knockdown of Rab7B recovers disease-associated undifferentiated states. As expected, Rab7B knockdown recovered undifferentiated morphologies induced by treatment with tunicamycin, which mimics endoplasmic reticulum (ER) stress (Morimura et al. 2014;Wilding et al. 2018). It is thought that ER stress is one of the major pathological causes observed in Pelizaeus-Merzbacher disease (PMD, also called hypomyelinating leukodystrophy 1 [HLD1]) and some other neuropathies (Dhaunchak et al. 2011;Simons and Nave 2015;Wrabetz et al. 2006). Oligodendroglial cells in PMD often display insufficient differentiated phenotypes (Torii et al. 2014;Wolf et al. 2021). Differential effects of Rab7 subfamily members on oligodendroglial cell morphological differentiation may provide some hints for the potential target to treat oligodendroglial cell diseases such as PMD.

DNA Primer Nucleotide Sequences and PCR Techniques
Sense DNA primer for actin (internal control) ATG GAT GAC GAT ATC GCT GCG CTG GTC and antisense primer for actin (internal control) CTA GAA GCA CTT GCG GTG CAC GAT GGAG, sense primer for Rab7A ATG ACC TCT AGG AAG AAA GTG GTT GCTGA and antisense primer for Rab7A TCA ACA ACT GCA GCA GCT TTC TGC GG, and sense primer for Rab7B CAA GCA GTC ACT GCT ACG GTGT and antisense primer for Rab7B CCT TGT CCA GCT TGA TGA CCCC were used for the following reverse transcription-polymerase chain reaction (RT-PCR).
The cDNAs were prepared from Isogen (Nippon Gene, Tokyo, Japan)-extracted total RNA with the PrimeScript RT Master Mix kit (Takara Bio, Kyoto, Japan) in accordance with the manufacturer's instructions. PCR amplification from RT products was performed using Gflex DNA polymerase (Takara Bio) with 30 to 36 cycles, each consisting of a denaturation reaction at 98 °C (0.2 min), an annealing reaction at 56 to 65 °C (0.25 min) depending on the annealing temperature, and an extension reaction at 68 °C (0.5 min). The resultant PCR products were loaded onto 1 to 2% agarose gels (Nacalai Tesque).
For knockdown, cells were transfected with the respective synthesized 21-mer siRNAs (final concentration, 100 nM) with dTdT using the ScreenFect siRNA transfection kit (Fujifilm, Tokyo, Japan). The medium was replaced 4 h after transfection and cells were further cultured for experiments. Less than 5% of attached cells contained incorporated trypan blue (Nacalai Tesque) at 48 h after transfection Miyamoto et al. 2007;Nishino et al. 2022;Urai et al. 2018).
To induce differentiation, cells were cultured on polylysine (Nacalai Tesque)-coated cell culture dishes (Greiner Bio-One, Oberösterreich, Austria) in culture medium without FBS in 5% CO 2 at 37 °C Miyamoto et al. 2007;Nishino et al. 2022;Urai et al. 2018). At day 3 after the induction of differentiation, cells with no primary process were classified as category 1; cells with primary processes were classified as category 2; and cells with secondary processes branched from primary processes or with myelin membrane-like widespread processes were classified as category 3 Miyamoto et al. 2007;Nishino et al. 2022;Urai et al. 2018). Category 2 corresponded to these intermediate phenotypes. Cell morphologies were randomly captured using microscopic systems equipped with iNTER LENS (Micronet, Inc., Saitama, Japan) and depicted as the percentage in each category.
Their supernatants were denatured with a sample buffer (Nacalai Tesque). The samples were separated on pre-made sodium dodecylsulfate-polyacrylamide gels (Nacalai Tesque) . The electrophoretically separated proteins were transferred to PVDF membranes (Fujifilm) sandwiched between filter papers (Fujifilm), blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by HRP-conjugated secondary antibodies. A CanoScan LiDE400 image scanner (Canon, Tokyo, Japan) captured the peroxidase-reactive bands on X-ray films (Fujifilm) using CanoScan software (Canon). They were digitally scanned using UN-SCAN-IT software (Silk Scientific, Orem, UT, USA). Quantified immunoreactive bands (3 blots) were compared with the control's immunoreactive bands, which were set to 100%, using ImageJ software (NIH, Bethesda, MD, USA).

Statistical Analyses
Values are means ± standard deviation (SD) from separate experiments. Intergroup comparisons were made using the unpaired Student's t-test using Excel (Microsoft, Redmond, WA, USA). A one-way analysis of variance (ANOVA) was followed by a Tukey's multiple comparison test using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant when p < 0.05.

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

Rab7A Positively Affects Morphological Differentiation
In order to investigate whether Rab7A is involved in the regulation of oligodendroglial cell morphological differentiation, we transfected siRNA ( Figure S1) specific for Rab7A into FBD-102b cells. Knockdown of Rab7A but not control luciferase resulted in inhibiting morphological differentiation (Fig. 1 A and B). These changes were accomplished with decreased expression levels of differentiation and myelination marker proteins MBP and PLP1 (Fig. 2 A and B). In contrast, the levels of control actin proteins were comparable in Rab7A and control knockeddown cells, suggesting that Rab7A is involved in promoting morphological differentiation.

Rab7B Negatively Affects Morphological Differentiation
Next, we transfected siRNA ( Figure S1) specific for Rab7B into cells. Knockdown of Rab7B but not control luciferase resulted in promoting oligodendroglial cell morphological differentiation (Fig. 3 A and B). The knockdown also increased the expression levels of differentiation and myelination marker proteins but it did not affect the levels of control proteins (Fig. 4 A and B), indicating that Rab7B plays an inhibitory role in morphological differentiation.

Knockdown of Rab7B Recovers Tunicamycin-Induced Defective Morphological Differentiation
The results pertaining to the effects of Rab7B on morphological differentiation suggested the possibility that knockdown of Rab7B could help recover the inhibitory morphological differentiation in disease states such as conditions under ER stress (Torii et al. 2014;Wolf et al. 2021). We induced ER stress using tunicamycin (Morimura et al. 2014;Wilding et al. 2018).Tunicamycin actually inhibited morphological differentiation in a dose-dependent manner in FBD-102b cells ( Figure S2). Accompanied with this inhibition, tunicamycin upregulated ER stress signals, which have included increased phosphorylation levels of JNK and eIF2A. Phosphorylating JNK and eIF2A primarily act downstream of ER stress signaling (Hotamisligil and Davis 2016;Urano et al. 2000). In contrast, it did not affect the expression levels of JNK and eIF2A themselves.
As expected, under these experimental conditions, knockdown of Rab7B recovered tunicamycin-induced inhibition of morphological differentiation (Fig. 5 A and B) with increased expression levels of marker proteins (Fig. 6 A and  B), identifying Rab7B as an effective target for recovering ER stress-induced inhibition of morphological differentiation.
In contrast, knockdown of Rab7A failed to recover the tunicamycin-induced inhibitory effects of morphological differentiation ( Figure S3) as well as those of the expression levels of marker proteins ( Figure S4).

Knockdown of Rab7B Recovers Tunicamycin-Induced ER Stress Signaling
Finally, we asked whether Rab7B knockdown could recover ER stress signals. Knockdown of Rab7B in cells resulted in decreasing phosphorylation levels of JNK and eIF2A whereas the expression levels of JNK and eIF2A were comparable regardless of knockdown states (Fig. 7A-D). Taken together with the above results, Rab7B knockdown can be effective against tunicamycin-induced ER stress.

Discussion
Rab7A and Rab7B display ubiquitous expressions in a variety of cells and tissues and are directly involved in the regulation of the transporting system between late endosomes and lysosomes (Balderhaar and Ungermann 2013;Kuchitsu and Fukuda 2018;Stroupe 2018;Wang et al. 2011). It is thus thought that functional deficiency of Rab7 subfamily molecules is directly linked to deficiency of the intracellular transporting system between late endosomes and lysosomes to control protein states, such as their quality (Balderhaar and Ungermann 2013;Kuchitsu and Fukuda 2018;Stroupe 2018;Wang et al. 2011). It is natural for the deficiency to also be related to malfunction of autophagy in cell homeostasis (Balderhaar and Ungermann 2013;Kuchitsu and Fukuda 2018;Stroupe 2018;Wang et al. 2011). In addition to these organelle functional deficiencies, for example, there is often decreased lysosomal signaling related to the mechanistic target of rapamycin (mTOR) as a master regulator of basic cellular signaling pathways (Bar-Peled and Sabatini 2014; Condon and Sabatini 2019). It is therefore likely that Rab7A and Rab7B are important to many aspects of basic cellular functions.
In fact, some amino acid mutations of Rab7A result in progressive neuropathy in the peripheral nervous system. This is evident in autosomal-dominant Charcot-Marie-Tooth Fig. 1 Knockdown of Rab7A inhibits oligodendroglial cell morphological differentiation. A FBD-102b cells were transfected with an siRNA for control luciferase or Rab7A. Cells were allowed to differentiate ( +) or not to differentiate ( −) for 3 days. Magnified cell images from representative cell images (indicated by black lines) were also depicted in the figure. B As a classification of the state of differentiation, cells with no primary process were classified as cat-egory 1; cells with primary processes were classified as category 2; and cells with secondary processes branched from primary processes or with widespread membranes were classified as category 3. Cells belonging to each category were counted per field under the microscope and shown as the percentage. Following the induction of differentiation or control induction, cells at 3 days were statistically assessed (**p < 0.01; n = 10 fields [total 480 cells]) type 2B (CMT2B) disease, in which the common features are distal sensory loss, foot calluses, and poorly healing ulcers (Auer-Grumbach 2004;Cogli et al. 2009;Shy 2004;Verhoeven et al. 2006). Of interest, all mutations of Rab7A appear likely to generate constitutively activated Rab7A (Cogli et al. 2009;Yamauchi et al. 2010). To achieve cell physiological function, including transporting between late endosomes and lysosomes, Rab proteins generally require cycles between the active GTP-bound form and the inactive GDP-bound form (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995). The switching cycle is essential for transporting the related vesicles (Homma et al. 2021;Ng and Tang 2008;Pfeffer et al. 1995). It is thus presumed that the presence of GTP-locked Rab7A in cells blocks vesicle recycling; however, it remains unclear why GTP-locked Rab7A is specifically associated with triggering peripheral neuropathy. Additionally, it remains unknown whether mutations of Rab7B cause CMT2B and the other peripheral neuropathies. In this study, we describe for the first time that knockdown of Rab7A but not Rab7B in oligodendroglial cell line FBD-102b leads to inhibition of its morphological differentiation, indicating that Rab7A is required for differentiation. We previously reported that GTP-locked Rab7A inhibits neuronal cell morphological differentiation in N1E-115 cells, which are used as a neuronal differentiation model in the central nervous system (Hirose et al. 1998;Urai et al. 2018). It is likely that in the central nervous system cells, Rab7A plays key roles in morphological differentiation in both neuronal and glial cells. Further studies will allow us to determine whether Rab7A is actually responsible for central nervous system diseases.
In addition to the transporting system between late endosomes and lysosomes, Rab7B is believed to participate in regulating the transporting system in trans-Golgi networks (Borg et al. 2014;Distefano et al. 2015;Marubashi and Fukuda 2020;Progida et al. 2010;Vestre et al. 2021). Rab7B specifically regulates retrograde vesicle trafficking between late endosomes and trans-Golgi networks (Borg et al. 2014;Distefano et al. 2015;Marubashi and Fukuda 2020;Progida et al. 2010;Vestre et al. 2021). In the present study, we provide evidence that knockdown of Rab7B but not Rab7A in cells promotes oligodendroglial morphological differentiation, revealing that Rab7B is a negative regulator for differentiation. The reason for this may be that retrograde Fig. 2 Knockdown of Rab7A decreases oligodendroglial differentiation marker proteins. A, B Cells were transfected with an siRNA for control luciferase or Rab7A. After the induction of differentiation, cells were lysed, used for an immunoblotting using an antibody against PLP1, MBP, or control actin, and statistically assessed (*p < 0.05; n = 3 blots) ▸ Fig. 3 Knockdown of Rab7B greatly promotes morphological differentiation. A FBD-102b cells were transfected with an siRNA for control luciferase or Rab7B. Cells were allowed to differentiate or not to differentiate for 3 days. Magnified cell images from representative cell images (indicated by black lines) were also depicted in the fig-ure. B Following the induction of differentiation or control induction, cells at 3 days were statistically assessed (**p < 0.01 and *p < 0.05; n = 10 fields [total 520 cells]). Cells belonging to each differentiation category are shown as the percentage transporting between late endosomes and trans-Golgi networks can provide an unnecessary transporting pathway to oligodendroglial cells, although the precise cellular roles of this retrograde transporting system remain to be answered so far. Since mature differentiated myelin sheaths often grow to become more than 100-fold larger than the collective surface areas of the premyelinating plasma membranes, it is conceivable that intracellular systems transporting myelin membrane components are significantly activated and that anterograde transporting systems take precedence over retrograde ones. Alternatively, Rab7B has some specific roles in oligodendroglial cells. It is known that Rab7A binds to effectors such as RLIP (Rab-interacting lysosomal protein) and more than 100 binding partners (see the BioGRID website: https:// thebi ogrid. org/), possibly to elicit its function, although the interaction of Rab7B with its proteins has not been thoroughly investigated. In oligodendroglial cells, some effector proteins may specifically bind to Rab7A and others may bind only to Rab7B. To the best of our knowledge, the molecular mechanism in which Rab7B and Rab7A antagonistically regulates morphogenesis during differentiation has been unanticipated to date.
Although many of the oligodendroglial cell degenerative diseases (also called oligodendropathies) are serious, their therapeutic target molecules have been unknown. One of the oligodendropathies is PMD (Torii et al. 2014;Wolf et al. 2021). PMD is associated with various gene mutations, including amplification of the plp1 gene encoding major myelin plasma membrane protein, resulting in PLP1 accumulating in the ER. It is thought that the misfolded PLP1 proteins trigger ER stress, causing cell pathological phenotypes of oligodendropathies (Dhaunchak et al. 2011;Morimura et al. 2014;Torii et al. 2014;Wolf et al. 2021). One of the phenotypes results in the increase of undifferentiated and/ or hypomyelinating oligodendroglial cells. It is thus thought that decreased ER stress recovers these cell pathological phenotypes (Torii et al. 2014;Wolf et al. 2021;Wilding et al. 2018). Additionally, some chemicals to decrease ER stress reverse pathological phenotypes by ER stress inducers such as tunicamycin (Wilding et al.2018). It is known that tunicamycin mimics cellular disease states, as often observed in oligodendroglial cell diseases (Dhaunchak et al. 2011;Wrabetz et al. 2006) and many other neurological diseases (Barateiro Fig. 4 Knockdown of Rab7B greatly increases differentiation marker proteins. A, B Cells were transfected with an siRNA for control luciferase or Rab7B. After the induction of differentiation, cells were lysed and used for an immunoblotting using an antibody against PLP1, MBP, or control actin, and statistically assessed (*p < 0.05; n = 3 blots) ▸ et al. 2016; Simons and Nave 2015). Thus, we have established a hypothesis that knockdown of Rab7B may promote defective differentiation by tunicamycin-induced ER stress.
Knockdown of Rab7B indeed recovers tunicamycin-induced defective differentiation. The knockdown also decreases ER stress signaling. Knockdown of Rab7B recovers inhibition of morphological differentiation by tunicamycin-induced ER stress. A Cells were transfected with an siRNA for Rab7B and treated with tunicamycin (100 ng/ml). Cells were allowed to differentiate or not to differentiate for 3 days. Magnified cell images from representative cell images (indicated by black lines) were also depicted in the figure. B Following the induction of differentiation or control induction, cells at 3 days were statistically assessed (**p < 0.01 and *p < 0.05; n = 10 fields [total 512 cells]). Cells belonging to each differentiation category are shown as the percentage The last question in this study, whether Rab7B regulates ER stress, remains unanswered. On the other hand, Mateus et al. reported the close relationship between Rab7A and ER stress in human epithelial and epithelial-like cells (Mateus et al. 2018). Knockdown of Rab7A by the cognate iRNA or expression of dominantly inhibitory mutant constructs expands sheet-like ER structures to spread cell peripheries. Inhibition of Rab7A induces ER stress to cause enlargement of ER structures whereas re-expression of Rab7A in the experimental conditions decreases ER stress and rescues enlarging phenotypes of the ER. Rab7A appears likely to maintain cell homeostasis by controlling the ER. Loi et al. provided key evidence that endo-lysosomes positive to antigens of both Rab7, possibly Rab7A, and lysosomalassociated membrane protein 1 (LAMP1) engulf the excess ER (Loi et al. 2019). Although the ER engulfed by endolysosomes is not specified, they could engulf the ER with excess stress to maintain cell homeostasis. During these processes, Rab7A might critically mediate cell homeostasis centered to the ER.
Herein we demonstrate that Rab7A and Rab7B positively and negatively regulates oligodendroglial cell morphological differentiation, respectively, as established by changes in differentiation marker proteins. Knockdown of Rab7B rescues defective morphological differentiation by tunicamycin-induced ER stress, as established by changes in phosphorylation levels of ER stress-regulated kinase ( Figure S5). It is also possible that Rab7B can mediate naturally occurring ER stress under cell homeostasis oligodendroglial cells. Additional studies would promote our understanding not only of the detailed molecular mechanism by which Rab7A and Rab7B regulate morphological differentiation but also what type of target downstream molecule of Rab7A and Rab7B regulates differentiation and, in turn, myelination in cell line(s) other than FBD-102b cell line, primary cells, cocultures, and genetically modified mice. Further studies might allow us to clarify whether Rab7B is one of the therapeutic targets of PMD and the related oligodendroglial cell diseases. Studies in this line could lead to the development of target-specificmedicines, taking advantage of the common molecular and cellular pathological signaling pathway underlying diseases involving ER stress in the central nervous system. Fig. 6 Knockdown of Rab7B recovers decreased differentiation marker protein expression by tunicamycin-induced ER stress. A, B Cells were transfected with an siRNA for Rab7B and treated with tunicamycin (100 ng/ml). After the induction of differentiation, cells were lysed and used for an immunoblotting using an antibody against PLP1, MBP, or control actin, and statistically assessed (*p < 0.05; n = 3 blots) ▸ Fig. 7 Knockdown of Rab7B recovers tunicamycin-induced ER stress signaling. A, B Cells were transfected with an siRNA for Rab7B and treated with tunicamycin (100 ng/ml). After the induction of differentiation, cells were lysed and used for an immunoblotting using an antibody against pJNK or JNK and statistically assessed (**p < 0.01; n = 3 blots). C, D Cells were transfected with an siRNA for Rab7B and treated with tunicamycin (100 ng/ml). After the induction of differentiation, cells were lysed and used for an immunoblotting using an antibody against peIF2A or eIF2A and statistically assessed (*p < 0.05; n = 3 blots)