Altered dimerization of certain riboflavin transporter 2 mutants: a possible source of UPR, altered calcium signalling and mitochondrial derangements in RTD2

Riboflavin transporter deficiency Type 2 (RTD2, OMIM #614707), formerly known as Brown-Vialetto-Van Laere Syndrome 2 (BVVLS 2), is a rare autosomal recessive neurodegenerative disorder caused by biallelic variants in the SLC52A2 gene, encoding for riboflavin transporter 2 (RFVT2). This transporter plays a critical role in flavin cofactor delivery, particularly in the brain. Clinically, RTD2 presents with progressive hearing loss, optic atrophy, muscle weakness, respiratory issues, and pontobulbar palsy. Current treatment involves high-dose riboflavin and other supplements. In this study we explored the molecular mechanisms behind RTD2, focusing on the dimerization of RFVT2 and the associated cellular stress mechanisms in patient-specific models. We demonstrated that RFVT2 exists as a homodimer and that pathogenic variants significantly impair its dimerization, which may contribute to the induction of ER stress. This hypothesis was supported by elevated levels of BiP, an ER stress marker, in patient iPSC-derived motor neurons. Similar findings were confirmed in patient-derived fibroblasts, where we also observed mitochondrial dysfunction and disrupted calcium signaling. Interestingly, no significant changes in FAD content were detected in both cell models, suggesting that proteotoxic stress may be a crucial pathogenic mechanism in RTD2, even in the absence of signs of FAD deficiency. FAD autofluorescence and FLIM measurements reinforce the occurrence of mitochondrial dysfunction in patient MNs. These findings provide insight into the pathogenic mechanisms of RTD2, highlighting the critical role of RFVT2 misfolding, ER stress, and mitochondrial dysfunction in this neurodegenerative disorder.