Mutant-specific dysfunction of RHOBTB2 impairs mitochondrial function and Na⁺/K⁺-ATPase levels in a cell model

RHOBTB2 is an atypical Rho GTPase implicated in developmental and epileptic encephalopathy, yet its pathogenic mechanisms remain poorly understood. In this study, we established a cell model in which RHOBTB2 expression was induced by a doxycycline-inducible system to investigate the functional consequences of disease-associated RHOBTB2 mutants. Protein expression and localization analyses revealed mutant-specific behaviors: GTPase-domain mutants such as D92H and W217C showed no significant difference in RHOBTB2 protein levels compared with WT, while hotspot mutants (R461H, R485C, R489Q) exhibited increased RHOBTB2 protein levels with nuclear and mitochondrial accumulation. RNA-seq analysis revealed that cells expressing each mutant showed distinct transcriptomic changes. Notably, induction of the R489Q mutant caused a robust downregulation of ion channel-related genes, supporting its potential role in disrupting neuronal excitability. Furthermore, expression of mutant RHOBTB2 led to a reduction in Na⁺/K⁺-ATPase protein levels via a lysosome-dependent degradation pathway. This effect was particularly prominent in GTPase-domain mutants (D92H and W217C), suggesting a mechanistic link between mutant RHOBTB2 and impaired ion homeostasis. Moreover, the R489Q and W217C mutants impaired mitochondrial respiration, whereas other mutants did not show detectable mitochondrial dysfunction. Importantly, the Y284D cancer-associated mutant did not share these phenotypes, highlighting the diversity of functional outcomes across different mutation sites. The observed dysregulation of ion transport pathways and mitochondrial impairment in some mutants may represent key mechanisms underlying seizure susceptibility and other neurological manifestations in RHOBTB2-associated encephalopathies.