Knockdown of Rab9 Recovers Defective Morphological Differentiation Induced by Chemical ER Stress Inducer or PMD-Associated PLP1 Mutant Protein in FBD-102b Cells

Abstract

Small GTP-binding proteins of the Rab family regulate intracellular vesicle trafficking across many aspects of the transport system. Among these, Rab9 is recognized for its role in controlling the transport system not only around the trans-Golgi network but also around the late endosome. However, the specific functions across different cell types and tissues remain unclear. Here, for the first time, we report that Rab9 negatively regulates morphological changes in the FBD-102b cell line, an oligodendroglial precursor cell line undergoing morphological differentiation. The knockdown of Rab9 led to an increase in cell shape alterations characterized by widespread membrane extensions. These changes were accompanied by increased expression levels of oligodendroglial cell differentiation and myelination marker proteins. Notably, the knockdown of Rab9 was capable of recovering defective cell morphological changes induced by tunicamycin, an inducer of endoplasmic reticulum (ER) stress, which is one of the major causes of oligodendroglial cell diseases such as Pelizaeus-Merzbacher disease (PMD, currently known as hypomyelinating leukodystrophy type 1 [HLD1]). In addition, Rab9 knockdown recovered levels of ER stress marker proteins and differentiation markers. Similar results were obtained in the cases of dithiothreitol (DTT), another chemical ER stress inducer, as well as HLD1-associated proteolipid protein 1 (PLP1) mutant protein. These results indicate a unique role for Rab9 in oligodendroglial cell morphological changes, suggesting its potential as a therapeutic target for mitigating diseases such as HLD1 at the molecular and cellular levels.

Keywords: ER stress; Rab9; hypomyelinating leukodystrophy; morphogenesis; oligodendrocyte.

Figure 1

Knockdown of Rab9 promotes cell morphological changes. (A) FBD-102b cells were transfected with control luciferase siRNA (siLuc) or Rab9 siRNA (siRab9). Following the induction of differentiation, cell morphologies were photographed and cells with differentiated oligodendroglial cell-like widespread membranes were statistically depicted at day 0 or 3 (*** p < 0.001; n = 10 fields). Typical cell morphologies with differentiated oligodendroglial cell-like widespread membranes were surrounded by white dotted lines. (B) Cell lysates at day 3 following the induction of differentiation were immunoblotted with the respective antibodies against PLP1, MBP, and internal control actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots).

Figure 2

Knockdown of Rab9 recovers phenotypes induced by tunicamycin. (A) FBD-102b cells were transfected with luciferase siRNA (siLuc) or Rab9 siRNA (siRab9). Following the induction of differentiation in the presence of 100 ng/mL of tunicamycin, cell morphologies were photographed and cells with differentiated oligodendroglial cell-like widespread membranes were statistically depicted at day 0 or 3 (*** p < 0.001; n = 10 fields). Typical cell morphologies with differentiated oligodendroglial cell-like widespread membranes were surrounded by white dotted lines. (B) Cell lysates at day 3 were immunoblotted with the respective antibodies against PLP1, MBP, and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots).

Figure 3

Knockdown of Rab9 decreases tunicamycin-induced ER stress signaling. (A,B) FBD-102b cells were transfected with luciferase siRNA (siLuc) or Rab9 siRNA (siRab9). Following the induction of differentiation in the presence of 100 ng/mL of tunicamycin, cell lysates at day 3 were immunoblotted with the respective antibodies against HSPA5 and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots). (C,D) Following the induction of differentiation in the presence of 100 ng/mL of tunicamycin, cell lysates were immunoblotted with the respective antibodies against CHOP and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots). (E,F) Following the induction of differentiation in the presence of 100 ng/mL of tunicamycin, cell lysates were immunoblotted with the respective antibodies against phosphorylated eIF2a (peIF2a), eIF2a, and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots).

Figure 4

Knockdown of Rab9 recovers phenotypes induced by DTT. (A) FBD-102b cells were transfected with luciferase siRNA (siLuc) or Rab9 siRNA (siRab9). Following the induction of differentiation in the presence of 1 mM DTT, cell morphologies were photographed and cells with differentiated oligodendroglial cell-like widespread membranes were statistically depicted at day 0 or 3 (*** p < 0.001; n = 10 fields). Typical cell morphologies with differentiated oligodendroglial cell-like widespread membranes were surrounded by white dotted lines. (B) Cell lysates at day 3 were immunoblotted with the respective antibodies against PLP1, MBP, and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots).

Figure 5

Knockdown of Rab9 decreases DTT-induced ER stress signaling. (A,B) FBD-102b cells were transfected with luciferase siRNA (siLuc) or Rab9 siRNA (siRab9). Following the induction of differentiation in the presence of 1 mM DTT, cell lysates at day 3 were immunoblotted with the respective antibodies against HSPA5 and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots). (C,D) Following the induction of differentiation in the presence of 1 mM DTT, cell lysates were immunoblotted with the respective antibodies against CHOP and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots). (E,F) Following the induction of differentiation in the presence of 1 mM DTT, cell lysates were immunoblotted with the respective antibodies against phosphorylated eIF2a (peIF2a), eIF2a, and actin and statistically depicted as a percentage comparison (* p < 0.05; n = 3 blots).