Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), yet it's unclear how this allele mediates risk. APOE4 carriers experience reduced mobility and faster decline in muscle strength, suggesting skeletal muscle involvement. Mitochondria are critical for muscle function and although we have reported defects in muscle mitochondrial respiration during early cognitive decline, APOE4-mediated effects on muscle mitochondria are unknown. Here, we sought to determine the impact of APOE4 on skeletal muscle bioenergetics using young, male and female APOE3 (control) and APOE4 targeted replacement mice (n = 8 per genotype/sex combination). We examined the proteome, mitochondrial respiration, fiber size, and fiber-type distribution in skeletal muscle. We found that APOE4 alters mitochondrial pathway expression in young mouse muscle in a sex-dependent manner without affecting respiration and fiber size or composition relative to APOE3. In both sexes, the expression of mitochondrial pathways involved in electron transport, ATP synthesis, and heat production by uncoupling proteins and mitochondrial dysfunction significantly differed between APOE4 and APOE3 muscle. For pathways with predicted direction of activation, electron transport and oxidative phosphorylation were upregulated while mitochondrial dysfunction and sirtuin signaling were downregulated in female APOE4 vs. APOE3 muscle. In males, sulfur amino acid metabolism was upregulated in APOE4 vs. APOE3 muscle. This work highlights early involvement of skeletal muscle in a mouse model of APOE4-linked AD, which may contribute to AD pathogenesis or serve as a biomarker for brain health.

Defect of GTPBP3, the human mitochondrial tRNA-modifying enzyme, can lead to Combined Oxidative Phosphorylation Deficiency 23 (COXPD23). Up to now, about 20 different variants of the GTPBP3 gene have been reported; however, genotype-phenotype analysis has rarely been described. Here, we reported a 9-year-old boy with COXPD23 who presented with hyperlactatemia, hypertrophic cardiomyopathy, seizures, feeding difficulties, intellectual disability and motor developmental delay, and abnormal visual development. Biallelic pathogenic variants of the GTPBP3 gene were identified in this boy, one novel variant c.1102dupC (p. Arg368Profs*22) inherited from the mother and the other known variant c.689A>C (p. Gln230Pro) inherited from father. We curated 18 COXPD23 patients with GTPBP3 variants to investigate the genotype-phenotype correlation. We found that hyperlactatemia and cardiomyopathy were critical clinical features in COXPD23 and the average onset age was 1.7 years (3 months of age for the homozygote). Clinical classification of COXPD23 for the two types, severe and mild, was well described in this study. We observed arrhythmia and congestive heart failure frequently in the severe type with early childhood mortality, while developmental delay was mainly observed in the mild type. The proportion of homozygous variants (71.4%) significantly differed from that of compound heterozygous variants (18.1%) in the severe type. Compared with the variants in gnomAD, the proportion of LOFVs in GTPBP3 was higher in COXPD23 patients (48.6% versus 8.9%, p < 0.0001 ****), and 31% of them were frameshift variants, showing the LOF mechanism of GTPBP3. Additionally, the variants in patients were significantly enriched in the TrmE-type G domain, indicating that the G domain was crucial for GTPBP3 protein function. The TrmE-type G domain contained several significant motifs involved in the binding of guanine nucleotides and Mg2+, the hydrolysis of GTP, and the regulation of the functional status of GTPases. In conclusion, we reported a mild COXPD23 case with typical GTPBP3-related symptoms, including seizures and abnormal visual development seldom observed previously. Our study provides novel insight into understanding the clinical diagnosis and genetic counseling of patients with COXPD23 by exploring the genetic pathogenesis and genotype-phenotype correlation of COXPD23.

Liver fibrosis is a pathological wound-healing response caused by chronic liver damage. Mitochondria regulate hepatic energy metabolism and oxidative stress. Accumulating evidence has revealed that increased mitochondrial oxidative stress contributes to the activation of fibrogenesis. However, the roles and underlying mechanisms of mitochondrial oxidative stress in liver fibrosis remain unknown. In this study, C57BL/6 mice were used to establish a model of liver fibrosis via oral gavage with CCl4 treatment for 8 weeks. Furthermore, intervention experiments were achieved by CCl4 combined with the intraperitoneal injection of mitoquinone mesylate (mitoQ). We demonstrated that the chronic CCl4 exposure resulted in severe hepatic fibrogenesis and significantly promoted the production of reactive oxygen species (ROS) and mitochondrial abnormalities. Besides, JNK/YAP pathway was also activated. By contrast, the administration of mitoQ markedly inhibited the expression of pro-fibrogenic transforming growth factor-β as well as type I collagen. The antifibrotic effects of mitoQ were also confirmed by hematoxylin and eosin staining and Sirius red staining. Moreover, mitoQ substantially reduced CCl4-induced mitochondrial damage and the release of ROS. Further studies suggested that this protection against liver fibrosis was mechanistically related to the inhibition of phosphorylation of JNK and the nuclear translocation of YAP. In conclusion, these findings revealed that mitoQ attenuated liver fibrosis by inhibiting ROS production and the JNK/YAP signaling pathway. Selective targeting JNK/YAP may serve as a therapeutic strategy for retarding progression of chronic liver disease.

A recent epigenome-wide association study of genes associated with type 2 diabetics (T2D), used integrative cross-omics analysis to identify 22 abnormally methylated CpG sites associated with insulin and glucose metabolism. Here, in this epigenetic analysis we preliminarily determine whether the same CpG sites identified in T2D also apply to type 1 diabetes (T1D). We then determine whether BCG vaccination could correct the abnormal methylation patterns, considering that the two diseases share metabolic derangements. T1D (n = 13) and control (n = 8) subjects were studied at baseline and then T1D subjects studied yearly for 3 years after receiving BCG vaccinations in a clinical trial. In this biomarker analysis, methylation patterns were evaluated on CD4+ T-lymphocytes from baseline and yearly blood samples using the human Illumina Methylation EPIC Bead Chip. Methylation analysis combined with mRNA analysis using RNAseq. Broad but not complete overlap was observed between T1D and T2D in CpG sites with abnormal methylation. And in the three-year observation period after BCG vaccinations, the majority of the abnormal methylation sites were corrected in vivo. Genes of particular interest were related to oxidative phosphorylation (CPT1A, LETM1, ABCG1), to the histone lysine demethylase gene (KDM2B), and mTOR signaling through the DDIT4 gene. The highlighted CpG sites for both KDM2B and DDIT4 genes were hypomethylated at baseline compared to controls; BCG vaccination corrected the defect by hypermethylation. Glycolysis is regulated by methylation of genes. This study unexpectedly identified both KDM2B and DDIT4 as genes controlling BCG-driven re-methylation of histones, and the activation of the mTOR pathway for facilitated glucose transport respectively. The BCG effect at the gene level was confirmed by reciprocal mRNA changes. The DDIT4 gene with known inhibitory role of mTOR was re-methylated after BCG, a step likely to allow improved glucose transport. BCGs driven methylation of KDM2B's site should halt augmented histone activity, a step known to allow cytokine activation and increased glycolysis.

Deletions in mitochondrial DNA (mtDNA) are an important cause of human disease and their accumulation has been implicated in the ageing process. As mtDNA is a high copy number genome, the coexistence of deleted and wild-type mtDNA molecules within a single cell defines heteroplasmy. When deleted mtDNA molecules, driven by intracellular clonal expansion, reach a sufficiently high level, a biochemical defect emerges, contributing to the appearance and progression of clinical pathology. Consequently, it is relevant to determine the heteroplasmy levels within individual cells to understand the mechanism of clonal expansion. Heteroplasmy is reflected in a mosaic distribution of cytochrome c oxidase (COX)-deficient muscle fibers. We applied droplet digital PCR (ddPCR) to single muscle fibers collected by laser-capture microdissection (LCM) from muscle biopsies of patients with different paradigms of mitochondrial disease, characterized by the accumulation of single or multiple mtDNA deletions. By combining these two sensitive approaches, ddPCR and LCM, we document different models of clonal expansion in patients with single and multiple mtDNA deletions, implicating different mechanisms and time points for the development of COX deficiency in these molecularly distinct mitochondrial cytopathies.