Frontotemporal dementia and/or amyotrophic lateral sclerosis type 7 (FTD/ALS7) is an autosomal dominant neurodegenerative disorder characterized by the onset of ALS and/or FTD mainly in adulthood. Patients with some types of mutations, including the Thr104Asn (T104N) mutation of charged multivesicular body protein 2B (CHMP2B), have predominantly ALS phenotypes, whereas patients with other mutations have predominantly FTD phenotypes. A few patients with further other mutations have both phenotypes approximately equally; however, the reason why phenotypes differ depending on the position of the mutation is unknown. CHMP2B composes one part of the endosomal sorting complexes required for transport (ESCRT), specifically ESCRT-III, in the cytoplasm. We describe here, for the first time, that CHMP2B with the T104N mutation inhibits neuronal process elongation in the N1E-115 cell line, a model of neuronal differentiation. The inhibitory phenotype was accompanied by changes in marker protein expression. It is noteworthy that CHMP2B with the T104N mutation but not its wild-type was preferentially accumulated in the Golgi body. Of the four major Golgi stress signaling pathways currently known, the pathway through Arf4, as the small GTPase, was specifically upregulated in cells expressing CHMP2B with the T104N mutation. Conversely, knockdown of Arf4 with the cognate small interfering (si)RNA recovered the neuronal process elongation inhibited by the T104N mutation. These results suggest that the T104N mutation of CHMP2B inhibits neuronal morphological differentiation by triggering Golgi stress signaling, revealing a possible therapeutic molecular target for recovering potential molecular and cellular phenotypes underlying FTD/ALS7.

A number of recent discoveries suggest close links between anxiety- or probable relief-like behaviors and intracellular signaling molecules controlling morphogenesis in glial cells such as oligodendrocytes (oligodendroglial cells) in the brain, which have protective effects on neuronal cells. In the former behaviors, their intracellular signaling molecules include small GTPase members, some of which mediate cell morphological changes. Rnd2 is one such member, belonging to the Rho family of small GTPases. Despite the currently known functions of Rnd2, the precise roles of Rnd2 in cell morphogenesis and related functions in health and disease states remain to be elucidated. Herein we show that signaling through anxiety-related loss of function of the rnd2 gene is related to the regulation of oligodendroglial cell morphological differentiation in the FBD-102b cell line, which is often utilized as oligodendroglial cell differentiation model. Knockdown of Rnd2 with the clustered regularly interspaced palindromic repeats (CRISPR)/CasRx system or RNA interference has been shown to inhibit morphological differentiation. Similarly, knockdown of Prag1 and Fyn kinase, signaling molecules acting downstream of Rnd2, also blunts differentiation. Rnd2 or Prag1 knockdown also decreases Fyn phosphorylation, which is critical for its activation and for oligodendroglial cell differentiation and myelination. Of note, hesperetin, a citrus flavonoid with protective effects on oligodendroglial cells as well as neuronal ones, can recover the defective differentiation induced by the knockdown of Rnd2/Prag1/Fyn. These results suggest that signaling through Rnd2/Prag1/Fyn is directly associated with normal oligodendroglial cell morphological differentiation. Deficiency of the signaling cascade is recovered by hesperetin, presenting one potential molecular construction underlying anxiety and possible therapeutic targets.