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Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown – Vialetto disease that is partially rescued by riboflavin

View Article: PubMed Central - PubMed

ABSTRACT

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown–Vialetto–Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell–selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy–lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

No MeSH data available.


Mitochondrial function and autophagy in BBVL MNs before and after B2 treatment.Respiratory chain activity (A) and β-oxidation activity (B) analysis in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. No defect in respiratory chain and β-oxidation activity was observed in BVVL cells. The enzymatic activity of respiratory complexes I–IV (cI–IV) and acetyl CoA carboxylases (P CoA, P CoA, and B CoA) was determined and normalized to citrate synthase activity. Values are expressed as nmoles/min/mg protein. (C) Western blot analysis of MFN2 and DRP1 in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. The MFN2/DRP1 ratio was reduced in BVVL-MNs relative to WT-MNs (*P < 0.05, ANOVA). (D) LC3BII/I and p62 expression in WT-MNs, BVVL2-MNs, and BVVL2-MNs+B2. The LC3B-II/I ratio was reduced whereas p62 expression was increased in BVVL-MNs with respect to WT (***P < 0.0001, *P < 0.05, ANOVA). After riboflavin treatment, LC3B-II/I ratio and p62 expression showed a shift towards a WT pattern. (E) Western blot analysis of Bnip3 and Parkin in WT-MNs, BVVL-MNs, and BVVL-MNs+B2 (**P < 0.001, *P < 0.05, ANOVA). The levels of both proteins were reduced in BVVL-MNs with respect to WT-MNs.
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f5: Mitochondrial function and autophagy in BBVL MNs before and after B2 treatment.Respiratory chain activity (A) and β-oxidation activity (B) analysis in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. No defect in respiratory chain and β-oxidation activity was observed in BVVL cells. The enzymatic activity of respiratory complexes I–IV (cI–IV) and acetyl CoA carboxylases (P CoA, P CoA, and B CoA) was determined and normalized to citrate synthase activity. Values are expressed as nmoles/min/mg protein. (C) Western blot analysis of MFN2 and DRP1 in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. The MFN2/DRP1 ratio was reduced in BVVL-MNs relative to WT-MNs (*P < 0.05, ANOVA). (D) LC3BII/I and p62 expression in WT-MNs, BVVL2-MNs, and BVVL2-MNs+B2. The LC3B-II/I ratio was reduced whereas p62 expression was increased in BVVL-MNs with respect to WT (***P < 0.0001, *P < 0.05, ANOVA). After riboflavin treatment, LC3B-II/I ratio and p62 expression showed a shift towards a WT pattern. (E) Western blot analysis of Bnip3 and Parkin in WT-MNs, BVVL-MNs, and BVVL-MNs+B2 (**P < 0.001, *P < 0.05, ANOVA). The levels of both proteins were reduced in BVVL-MNs with respect to WT-MNs.

Mentions: First, we assessed for the presence of an OXPHOS or fatty acid β-oxidation impairment in BVVL-MNs, as observed in MADD. Mitochondrial respiration and β-oxidation were not different in BVVL-MNs (with or without riboflavin treatment) with respect to control cells (Fig. 5A,B).


Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown – Vialetto disease that is partially rescued by riboflavin
Mitochondrial function and autophagy in BBVL MNs before and after B2 treatment.Respiratory chain activity (A) and β-oxidation activity (B) analysis in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. No defect in respiratory chain and β-oxidation activity was observed in BVVL cells. The enzymatic activity of respiratory complexes I–IV (cI–IV) and acetyl CoA carboxylases (P CoA, P CoA, and B CoA) was determined and normalized to citrate synthase activity. Values are expressed as nmoles/min/mg protein. (C) Western blot analysis of MFN2 and DRP1 in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. The MFN2/DRP1 ratio was reduced in BVVL-MNs relative to WT-MNs (*P < 0.05, ANOVA). (D) LC3BII/I and p62 expression in WT-MNs, BVVL2-MNs, and BVVL2-MNs+B2. The LC3B-II/I ratio was reduced whereas p62 expression was increased in BVVL-MNs with respect to WT (***P < 0.0001, *P < 0.05, ANOVA). After riboflavin treatment, LC3B-II/I ratio and p62 expression showed a shift towards a WT pattern. (E) Western blot analysis of Bnip3 and Parkin in WT-MNs, BVVL-MNs, and BVVL-MNs+B2 (**P < 0.001, *P < 0.05, ANOVA). The levels of both proteins were reduced in BVVL-MNs with respect to WT-MNs.
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Related In: Results  -  Collection

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f5: Mitochondrial function and autophagy in BBVL MNs before and after B2 treatment.Respiratory chain activity (A) and β-oxidation activity (B) analysis in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. No defect in respiratory chain and β-oxidation activity was observed in BVVL cells. The enzymatic activity of respiratory complexes I–IV (cI–IV) and acetyl CoA carboxylases (P CoA, P CoA, and B CoA) was determined and normalized to citrate synthase activity. Values are expressed as nmoles/min/mg protein. (C) Western blot analysis of MFN2 and DRP1 in BVVL-MNs, BVVL-MNs+B2, and WT-MNs. The MFN2/DRP1 ratio was reduced in BVVL-MNs relative to WT-MNs (*P < 0.05, ANOVA). (D) LC3BII/I and p62 expression in WT-MNs, BVVL2-MNs, and BVVL2-MNs+B2. The LC3B-II/I ratio was reduced whereas p62 expression was increased in BVVL-MNs with respect to WT (***P < 0.0001, *P < 0.05, ANOVA). After riboflavin treatment, LC3B-II/I ratio and p62 expression showed a shift towards a WT pattern. (E) Western blot analysis of Bnip3 and Parkin in WT-MNs, BVVL-MNs, and BVVL-MNs+B2 (**P < 0.001, *P < 0.05, ANOVA). The levels of both proteins were reduced in BVVL-MNs with respect to WT-MNs.
Mentions: First, we assessed for the presence of an OXPHOS or fatty acid β-oxidation impairment in BVVL-MNs, as observed in MADD. Mitochondrial respiration and β-oxidation were not different in BVVL-MNs (with or without riboflavin treatment) with respect to control cells (Fig. 5A,B).

View Article: PubMed Central - PubMed

ABSTRACT

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown&ndash;Vialetto&ndash;Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell&ndash;selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy&ndash;lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

No MeSH data available.