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The cytoskeletal network plays a crucial role in the differentiation, morphogenesis, function and homeostasis of the nervous tissue, so that alterations in any of its components may lead to neurodegenerative diseases. Riboflavin transporter deficiency (RTD), a childhood-onset disorder characterized by degeneration of motor neurons (MNs), is caused by biallelic mutations in genes encoding the human riboflavin (RF) transporters. In a patient-specific induced pluripotent stem cells (iPSCs) model of RTD, we recently demonstrated altered cell-cell contacts, energy dysmetabolism and redox imbalance. The present study focuses on cytoskeletal composition and dynamics associated to RTD, utilizing patients' iPSCs and derived MNs. Abnormal expression and distribution of α- and β-tubulin (α- and β-TUB), as well as imbalanced tyrosination of α-TUB, accompanied by an impaired ability to re-polymerize after nocodazole treatment, were found in RTD patient-derived iPSCs. Following differentiation, MNs showed consistent changes in TUB content, which was associated with abnormal morphofunctional features, such as neurite length and Ca2+ homeostasis, suggesting impaired differentiation. Beneficial effects of RF supplementation, alone or in combination with the antioxidant molecule N-acetyl cystine (NAC), were assessed. RF administration resulted in partially improved cytoskeletal features in patients' iPSCs and MNs, suggesting that redundancy of transporters may rescue cell functionality in the presence of adequate concentrations of the vitamin. Moreover, supplementation with NAC was demonstrated to be effective in restoring all the considered parameters, when used in combination with RF, thus supporting the therapeutic use of both compounds., Summary: This study deals with pathomechanisms underlying riboflavin transporter deficiency, a rare recessively inherited early-onset neurodegenerative condition. Using patient-derived iPSCs, we report on cytoskeletal abnormalities, which are reverted by combined riboflavin/antioxidant treatment.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated iPSC lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells dramatically impacting cell redox status. In the present work, we extend our study to motor neurons (MNs), i.e., the cell type mostly affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis, following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker, and βIII Tubulin, as neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by Focused Ion Beam/Scanning Electron Microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient specific RTD models, and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs, emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies, aimed at correcting mitochondrial defects, based on the use of antioxidants.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated induced pluripotent stem cell (iPSC) lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells, dramatically impacting cell redox status. This work extends our study to motor neurons (MNs), i.e., the cell type most affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis (following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker), and &beta, III-Tubulin, as a neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by focused ion beam/scanning electron microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient-specific RTD models and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs&mdash, emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies aimed at correcting mitochondrial defects, based on the use of antioxidants.