Fig 2: MNRR1 activation using tizoxanide enhances mitochondrial function, mitophagy, and protects from LPS-induced inflammation in MELAS patient fibroblasts (A): Equal amounts of lysates from MELAS patient fibroblast (MF 1, 2, or 3) lysates treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h were separated on an SDS-PAGE gel and probed for LC3B, PGC1α, MNRR1, MTCO2, TOM20, and phospho-ubiquitin (S65) levels. GAPDH or Tubulin was probed as a loading control. (B): Oxygen consumption measured from MELAS patient fibroblasts treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h (n = 4 biological replicates, mean ± SE). (C): Mitochondrial ATP rate measured in MELAS patient fibroblasts treated with vehicle (DMSO) or tizoxanide (10 μM) for 48 h (n = 4 biological replicates, mean ± SE). (D): Heatmap representation of ROS and TNFα transcript levels from MELAS patient fibroblasts treated with vehicle (DMSO), LPS (DMSO + 500 ng/mL LPS), or LPS plus tizoxanide (10 μM tizoxanide + 500 ng/mL LPS) for 24 h (n = 4 biological replicates). Values for each sample are shown. * indicates p < 0.05.

Fig 3: CSDE1 associates with the mitochondrial import receptor TOM20 and mitochondrial protein-encoding transcripts. (a) Identification of TOM20-associated proteins. HEK293T cells overexpressing FLAG-tagged TOM20 were subjected to immunoprecipitation (IP) using an anti-FLAG antibody. Proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining. Proteins identified by mass spectrometry, including CSDE1, are listed on the right. (b) Mass spectrometric peptide coverage of CSDE1. Black bars indicate the positions of unique peptides identified by mass spectrometry mapped onto the CSDE1 amino acid sequence (54.6% sequence coverage). (c) Reciprocal co-immunoprecipitation of CSDE1 and TOM20. Lysates from HEK293T cells expressing HA-tagged CSDE1 were immunoprecipitated with an anti-HA antibody. Co-immunoprecipitated endogenous TOM20 was detected by Western blotting (WB). TCL, total cell lysate. (d,e) Gene Set Enrichment Analysis (GSEA) of CSDE1-associated transcripts. GSEA was performed using the CSDE1 RIP-seq ranked gene list derived from naïve mouse L4/5 DRG tissue (GSE301321). The dot plots show representative enriched Gene Ontology (GO) categories after redundancy reduction in (d) Biological Process (BP) and (e) Cellular Component (CC). The x-axis indicates normalized enrichment score (NES), dot size represents GeneCount (the number of genes in the core enrichment set), and color indicates GeneRatio (GeneCount/setSize). Full results are provided in Table S2.

Fig 4: Characterization of the CSDE1-TOM20 association and mitochondrial localization of CSDE1. (a) Schematic representation of full-length CSDE1 and the HA-tagged truncation mutants used to map the TOM20-associated region. The constructs encompass the N-terminal (CSD 1–3), middle (CSD 3–6), and C-terminal (CSD 7–9) cold shock domains. (b) Domain mapping of the CSDE1-TOM20 association. HEK293T cells expressing the indicated HA-tagged CSDE1 fragments were subjected to immunoprecipitation (IP) with an anti-HA antibody. Co-immunoprecipitated endogenous TOM20 was analyzed by Western blotting. TCL, total cell lysate. (c) RNA-independent association between CSDE1 and TOM20. Co-immunoprecipitation was performed in HEK293T cell lysates treated with or without RNase A/T1. Retention of the CSDE1-TOM20 association was assessed by Western blotting (top), and RNA degradation was confirmed by agarose gel electrophoresis (bottom). (d) Subcellular fractionation analysis of endogenous CSDE1. Cytosolic (Cyt) and mitochondrial (Mito) fractions were isolated from HEK293T cells, embryonic dorsal root ganglia (eDRG), and embryonic mouse whole brains. Immunoblotting was performed for CSDE1 and markers for mitochondria (NDUFS1, MT-CO1, UQCRFS1, COXIV, Cyt c) and the cytosol (α-Tubulin, GAPDH). WCL, whole-cell lysate. (e) Quantification of mitochondrial-fraction CSDE1 levels. The proportion of CSDE1 detected in the mitochondrial fraction relative to the whole-cell lysate was quantified based on the results shown in (d). Data are presented as mean ± SD (n.s., not significant). (f) In situ visualization of the CSDE1–TOM20 association. Proximity ligation assay (PLA) shows endogenous proximity between CSDE1 and TOM20 (green puncta). Cells were counterstained with DAPI (nuclei, blue) and TUJ1 (neuronal marker, gray). A negative control without primary antibodies is shown to confirm signal specificity (top panels). Scale bar, 10 µm.