The induction of mitochondrial biogenesis could potentially alleviate mitochondrial and muscle disease. We show here that dimethyl fumarate (DMF) dose-dependently induces mitochondrial biogenesis and function dosed to cells in vitro, and also dosed in vivo to mice and humans. The induction of mitochondrial gene expression is more dependent on DMF's target Nrf2 than hydroxycarboxylic acid receptor 2 (HCAR2). Thus, DMF induces mitochondrial biogenesis primarily through its action on Nrf2, and is the first drug demonstrated to increase mitochondrial biogenesis with in vivo human dosing. This is the first demonstration that mitochondrial biogenesis is deficient in Multiple Sclerosis patients, which could have implications for MS pathophysiology and therapy. The observation that DMF stimulates mitochondrial biogenesis, gene expression and function suggests that it could be considered for mitochondrial disease therapy and/or therapy in muscle disease in which mitochondrial function is important.

Inherited deficiency in the mitochondrial protein frataxin (FXN) causes the rare disease Friedreich's ataxia (FA), for which there is no successful treatment. We identified a redox deficiency in FA cells and used this to model the disease. We screened a 1600-compound library to identify existing drugs, which could be of therapeutic benefit. We identified the topical anesthetic dyclonine as protective. Dyclonine increased FXN transcript and FXN protein dose-dependently in FA cells and brains of animal models. Dyclonine also rescued FXN-dependent enzyme deficiencies in the iron–sulfur enzymes, aconitase and succinate dehydrogenase. Dyclonine induces the Nrf2 [nuclear factor (erythroid-derived 2)-like 2] transcription factor, which we show binds an upstream response element in the FXN locus. Additionally, dyclonine also inhibited the activity of histone methyltransferase G9a, known to methylate histone H3K9 to silence FA chromatin. Chronic dosing in a FA mouse model prevented a performance decline in balance beam studies. A human clinical proof-of-concept study was completed in eight FA patients dosed twice daily using a 1% dyclonine rinse for 1 week. Six of the eight patients showed an increase in buccal cell FXN levels, and fold induction was significantly correlated with disease severity. Dyclonine represents a novel therapeutic strategy that can potentially be repurposed for the treatment of FA.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by inherited deficiency of the mitochondrial protein Frataxin (FXN), which has no approved therapy and is an area in which biomarkers are needed for clinical development. Here, we investigated the consequences of FXN deficiency in patient-derived FRDA fibroblast cell models, the FRDA mouse model KIKO, and in whole blood collected from patients with FRDA. We observed decreased mitochondrial copy number in all the three FRDA models tested: cells, mice and patient blood. In addition, we observed 40% residual mitochondrial gene expression in FRDA patient blood. These deficiencies of mitochondrial biogenesis in FRDA cells and patient blood are significantly correlated with FXN expression, consistent with the idea that the decreased mitochondrial biogenesis is a consequence of FXN deficiency. The observations appear relevant to the FRDA pathophysiological mechanism, as FXN-dependent deficiency in mitochondrial biogenesis and consequent mitochondrial bioenergetic defect could contribute to the neurodegenerative process. The observations may also have translational potential, as mitochondrial biogenesis could now be followed as a clinical biomarker of FRDA as a correlate of disease severity, progression, and therapeutic effect. Also, mitochondrial copy number in blood is objective, scalar and more investigator-independent than clinical-neurological patient rating scales. Thus, FXN deficiency causes mitochondrial deficiency in FRDA cells, the KIKO mouse model, and in whole blood of patients with FRDA, and this deficiency could potentially be used in clinical trial design.

Friedreich’s Ataxia (FA) is an inherited neurodegenerative disorder resulting from decreased expression of the mitochondrial protein frataxin, for which there is no approved therapy. High throughput screening of clinically used drugs identified Dimethyl fumarate (DMF) as protective in FA patient cells. Here we demonstrate that DMF significantly increases frataxin gene ( FXN ) expression in FA cell model, FA mouse model and in DMF treated humans. DMF also rescues mitochondrial biogenesis deficiency in FA-patient derived cell model. We further examined the mechanism of DMF's frataxin induction in FA patient cells. It has been shown that transcription-inhibitory R-loops form at GAA expansion mutations, thus decreasing FXN expression. In FA patient cells, we demonstrate that DMF significantly increases transcription initiation. As a potential consequence, we observe significant reduction in both R-loop formation and transcriptional pausing thereby significantly increasing FXN expression. Lastly, DMF dosed Multiple Sclerosis (MS) patients showed significant increase in FXN expression by ~85%. Since inherited deficiency in FXN is the primary cause of FA, and DMF is demonstrated to increase FXN expression in humans, DMF could be considered for Friedreich's therapy.

Declines in mitochondrial mass are thought to be a hallmark of mammalian aging, and a ketogenic diet (KD) may prevent the age-related decreases in mitochondrial content. The objective of this study was to investigate the impact of a KD on markers of mitochondrial mass. Mice were fed an isocaloric control diet (CD) or KD from 12 months of age. Tissues were collected after 1 month and 14 months of intervention, and a panel of commonly used markers of mitochondrial mass (mitochondrial enzyme activities and levels, mitochondrial to nuclear DNA ratio, and cardiolipin content) were measured. Our results showed that a KD stimulated activities of marker mitochondrial enzymes including citrate synthase, Complex I, and Complex IV in hindlimb muscle in aged mice. KD also increased the activity of citrate synthase and prevented an age-related decrease in Complex IV activity in aged brain. No other markers were increased in these tissues. Furthermore, the impacts of a KD on liver and kidney were mixed with no pattern indicative of a change in mitochondrial mass. In conclusion, results of the present study suggest that a KD induces tissue-specific changes in mitochondrial enzyme activities, or structure, rather than global changes in mitochondrial mass across tissues.

BackgroundWhile an association between full mutation CGG-repeat expansions of the Fragile X Mental Retardation 1 (FMR1) gene and connective tissue problems are clearly described, problems in fragile X premutation carriers (fXPCs) CGG-repeat range (55–200 repeats) of the FMR1 gene may be overlooked.ObjectiveTo report five FMR1 fXPCs cases with the hypermobile Ehlers-Danlos syndrome (hEDS) phenotype.MethodsWe collected medical histories and FMR1 molecular measures from five cases who presented with joint hypermobility and loose connective tissue and met inclusion criteria for hEDS.ResultsFive cases were female and ranged between 16 and 49 years. The range of CGG-repeat allele sizes ranged from 66 to 150 repeats. All had symptoms of hEDS since early childhood. Commonalities in molecular pathogenesis and coexisting conditions between the fXPCs and hEDS are also presented. The premutation can lead to a reduction of fragile X mental retardation protein, which is crucial in maintaining functions of the extracellular matrix-related proteins, particularly matrix metallopeptidase 9 and elastin. Moreover, elevated FMR1 messenger RNA causes sequestration of proteins, which results in RNA toxicity.ConclusionBoth hEDS phenotype and premutation involvement may co-occur because of related commonalities in pathogenesis.

Recent advances in neurobiology have provided several molecular entrees for targeted treatments for Fragile X syndrome (FXS). However, the efficacy of these treatments has been demonstrated mainly in animal models and has not been consistently predictive of targeted drugs’ response in the preponderance of human clinical trials. Because of the heterogeneity of FXS at various levels, including the molecular level, phenotypic manifestation, and drug response, it is critically important to identify biomarkers that can help in patient stratification and prediction of therapeutic efficacy. The primary objective of this study was to assess the ability of molecular biomarkers to predict phenotypic subgroups, symptom severity, and treatment response to metformin in clinically treated patients with FXS. We specifically tested a triplex protein array comprising of hexokinase 1 (HK1), RAS (all isoforms), and Matrix Metalloproteinase 9 (MMP9) that we previously demonstrated were dysregulated in the FXS mouse model and in blood samples from patient with FXS. Seventeen participants with FXS, 12 males and 5 females, treated clinically with metformin were included in this study. The disruption in expression abundance of these proteins was normalized and associated with significant self-reported improvement in clinical phenotypes (CGI-I in addition to BMI) in a subset of participants with FXS. Our preliminary findings suggest that these proteins are of strong molecular relevance to the FXS pathology that could make them useful molecular biomarkers for this syndrome.

Autism spectrum disorder (ASD) is an early onset, developmental disorder whose genetic cause is heterogeneous and complex. In total, 70% of ASD cases are due to an unknown etiology. Among the monogenic causes of ASD, fragile X syndrome (FXS) accounts for 2–4% of ASD cases, and 60% of individuals with FXS present with ASD. Epigenetic changes, specifically DNA methylation, which modulates gene expression levels, play a significant role in the pathogenesis of both disorders. Thus, in this study, using the Human Methylation EPIC Bead Chip, we examined the global DNA methylation profiles of biological samples derived from 57 age-matched male participants (2–6 years old), including 23 subjects with ASD, 23 subjects with FXS with ASD (FXSA) and 11 typical developing (TD) children. After controlling for technical variation and white blood cell composition, using the conservatory threshold of the false discovery rate (FDR ≤ 0.05), in the three comparison groups, TD vs. AD, TD vs. FXSA and ASD vs. FXSA, we identified 156, 79 and 3100 differentially methylated sites (DMS), and 14, 13 and 263 differential methylation regions (DMRs). Interestingly, several genes differentially methylated among the three groups were among those listed in the SFARI Gene database, including the PAK2, GTF2I and FOXP1 genes important for brain development. Further, enrichment analyses identified pathways involved in several functions, including synaptic plasticity. Our preliminary study identified a significant role of altered DNA methylation in the pathology of ASD and FXS, suggesting that the characterization of a DNA methylation signature may help to unravel the pathogenicity of FXS and ASD and may help the development of an improved diagnostic classification of children with ASD and FXSA. In addition, it may pave the way for developing therapeutic interventions that could reverse the altered methylome profile in children with neurodevelopmental disorders.