TMEM65 regulates NCLX-dependent mitochondrial calcium efflux.

The balance between mitochondrial calcium (mCa2+) uptake and efflux regulates ATP production, but if perturbed causes energy starvation or mCa2+ overload and cell death. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of mCa2+ efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic mCa2+ overload. However, the mechanisms that regulate NCLX activity remain largely unknown. We used proximity biotinylation proteomic screening to identify the NCLX interactome and define novel regulators of NCLX function. Here, we discover the mitochondrial inner membrane protein, TMEM65, as an NCLX-proximal protein that potently enhances sodium (Na+)-dependent mCa2+ efflux. Mechanistically, acute pharmacologic NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in mCa2+ efflux. Further, loss-of-function studies show that TMEM65 is required for Na+-dependent mCa2+ efflux. Co-fractionation and in silico structural modeling of TMEM65 and NCLX suggest these two proteins exist in a common macromolecular complex in which TMEM65 directly stimulates NCLX function. In line with these findings, knockdown of Tmem65 in mice promotes mCa2+ overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. We further demonstrate that TMEM65 deletion causes excessive mitochondrial permeability transition, whereas TMEM65 overexpression protects against necrotic cell death during cellular Ca2+ stress. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent mCa2+ efflux, causing pathogenic mCa2+ overload, cell death and organ-level dysfunction, and that gain of TMEM65 function mitigates these effects. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent mCa2+ efflux and suggest modulation of TMEM65 as a novel strategy for the therapeutic control of mCa2+ homeostasis.

Glutamine uptake and catabolism is required for myofibroblast formation and persistence.

BACKGROUND: Fibrosis and extracellular matrix remodeling are mediated by resident cardiac fibroblasts (CFs). In response to injury, fibroblasts activate, differentiating into specialized synthetic and contractile myofibroblasts producing copious extracellular matrix proteins (e.g., collagens). Myofibroblast persistence in chronic diseases, such as HF, leads to progressive cardiac dysfunction and maladaptive remodeling. We recently reported that an increase in αKG (alpha-ketoglutarate) bioavailability, which contributes to enhanced αKG-dependent lysine demethylase activity and chromatin remodeling, is required for myofibroblast formation. Therefore, we aimed to determine the substrates and metabolic pathways contributing to αKG biosynthesis and their requirement for myofibroblast formation. METHODS: Stable isotope metabolomics identified glutaminolysis as a key metabolic pathway required for αKG biosynthesis and myofibroblast formation, therefore we tested the effects of pharmacologic inhibition (CB-839) or genetic deletion of glutaminase (Gls1-/-) on myofibroblast formation in both murine and human cardiac fibroblasts. We employed immunofluorescence staining, functional gel contraction, western blotting, and bioenergetic assays to determine the myofibroblast phenotype. RESULTS: Carbon tracing indicated enhanced glutaminolysis mediating increased αKG abundance. Pharmacological and genetic inhibition of glutaminolysis prevented myofibroblast formation indicated by a reduction in αSMA+ cells, collagen gel contraction, collagen abundance, and the bioenergetic response. Inhibition of glutaminolysis also prevented TGFß-mediated histone demethylation and supplementation with cell-permeable αKG rescued the myofibroblast phenotype. Importantly, inhibition of glutaminolysis was sufficient to prevent myofibroblast formation in CFs isolated from the human failing heart. CONCLUSIONS: These results define glutaminolysis as necessary for myofibroblast formation and persistence, providing substantial rationale to evaluate several new therapeutic targets to treat cardiac fibrosis.

Mitochondria in Pathological Cardiac Remodeling.

Adverse cardiac remodeling is often precipitated by chronic stress or injury inflicted upon the heart during the progression of cardiovascular diseases. Mitochondria play an important role in the cardiomyocyte response to stress by serving as a signaling hub for changes in cellular energetics, redox balance, contractile function, and cell death. Cardiac remodeling involves alterations to mitochondrial form and function that are either compensatory to maintain contractility or maladaptive, which promotes heart failure progression. In this mini-review, we focus on three mitochondrial processes that contribution to cardiac remodeling: Ca2+ signaling, mitochondrial dynamics, and mitochondrial metabolism.

Myofibroblasts and Fibrosis: Mitochondrial and Metabolic Control of Cellular Differentiation.

Cardiac fibrosis is mediated by the activation of resident cardiac fibroblasts, which differentiate into myofibroblasts in response to injury or stress. Although myofibroblast formation is a physiological response to acute injury, such as myocardial infarction, myofibroblast persistence, as occurs in heart failure, contributes to maladaptive remodeling and progressive functional decline. Although traditional pathways of activation, such as TGFß (transforming growth factor ß) and AngII (angiotensin II), have been well characterized, less understood are the alterations in mitochondrial function and cellular metabolism that are necessary to initiate and sustain myofibroblast formation and function. In this review, we highlight recent reports detailing the mitochondrial and metabolic mechanisms that contribute to myofibroblast differentiation, persistence, and function with the hope of identifying novel therapeutic targets to treat, and potentially reverse, tissue organ fibrosis.

Association of Extracellular Vesicle Biomarkers With Alzheimer Disease in the Baltimore Longitudinal Study of Aging.

IMPORTANCE: Blood biomarkers able to diagnose Alzheimer disease (AD) at the preclinical stage would enable trial enrollment when the disease is potentially reversible. Plasma neuronal-enriched extracellular vesicles (nEVs) of patients with AD were reported to exhibit elevated levels of phosphorylated (p) tau, Aß42, and phosphorylated insulin receptor substrate 1 (IRS-1). OBJECTIVE: To validate nEV biomarkers as AD predictors. DESIGN, SETTING, PARTICIPANTS: This case-control study included longitudinal plasma samples from cognitively normal participants in the Baltimore Longitudinal Study of Aging (BLSA) cohort who developed AD up to January 2015 and age- and sex-matched controls who remained cognitively normal over a similar length of follow-up. Repeated samples were blindly analyzed over 1 year from participants with clinical AD and controls from the Johns Hopkins Alzheimer Disease Research Center (JHADRC). Data were collected from September 2016 to January 2018. Analyses were conducted in March 2019. MAIN OUTCOMES AND MEASURES: Neuronal-enriched extracellular vesicles were immunoprecipitated; tau, Aß42, and IRS-1 biomarkers were quantified by immunoassays; and nEV concentration and diameter were determined by nanoparticle tracking analysis. Levels and longitudinal trajectories of nEV biomarkers between participants with future AD and control participants were compared. RESULTS: Overall, 887 longitudinal plasma samples from 128 BLSA participants who eventually developed AD and 222 age and sex-matched controls who remained cognitively normal were analyzed. Participants were followed up (from earliest sample to AD symptom onset) for a mean (SD) of 3.5 (2.31) years (range, 0-9.73 years). Overall, 161 participants were included in the training set, and 80 were in the test set. Participants in the BLSA cohort with future AD (mean [SD] age, 79.09 [7.02] years; 68 women [53.13%]) had longitudinally higher p-tau181, p-tau231, pSer312-IRS-1, pY-IRS-1, and nEV diameter than controls (mean [SD] age, 76.2 [7.36] years; 110 women [50.45%]) but had similar Aß42, total tau, TSG101, and nEV concentration. In the training BLSA set, a model combining preclinical longitudinal data achieved 89.6% area under curve (AUC), 81.8% sensitivity, and 85.8% specificity for predicting AD. The model was validated in the test BLSA set (80% AUC, 55.6% sensitivity, 88.7% specificity). Preclinical levels of nEV biomarkers were associated with cognitive performance. In addition, 128 repeated samples over 1 year from 64 JHADRC participants with clinical AD and controls were analyzed. In the JHADRC cohort (35 participants with AD: mean [SD] age, 74.03 [8.73] years; 18 women [51.43%] and 29 controls: mean [SD] age, 72.14 [7.86] years; 23 women [79.31%]), nEV biomarkers achieved discrimination with 98.9% AUC, 100% sensitivity, and 94.7% specificity in the training set and 76.7% AUC, 91.7% sensitivity, and 60% specificity in the test set. CONCLUSIONS AND RELEVANCE: We validated nEV biomarker candidates and further demonstrated that their preclinical longitudinal trajectories can predict AD diagnosis. These findings motivate further development of nEV biomarkers toward a clinical blood test for AD.

Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes.

Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimer's and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum, or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EVs enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner.

Spontaneous ATM Gene Reversion in A-T iPSC to Produce an Isogenic Cell Line.

A spontaneously reverted iPSC line was identified from an A-T subject with heterozygous ATM truncation mutations. The reverted iPSC line expressed ATM protein and was capable of radiation-induced phosphorylation of CHK2 and H2A.X. Genome-wide SNP analysis confirmed a match to source T cells and also to a distinct, non-reverted iPSC line from the same subject. Rearranged T cell receptor sequences predict that the iPSC culture originated as several independently reprogrammed cells that resolved into a single major clone, suggesting that gene correction likely occurred early in the reprogramming process. Gene expression analysis comparing ATM(-/-) iPSC lines to unrelated ATM(+/-) cells identifies a large number of differences, but comparing only the isogenic pair of A-T iPSC lines reveals that the primary pathway affected by loss of ATM is a diminished expression of p53-related mRNAs. Gene reversion in culture, although likely a rare event, provided a novel, reverted cell line for studying ATM function.

Frataxin levels in peripheral tissue in Friedreich ataxia.

OBJECTIVE: Friedreich ataxia (FRDA) is an autosomal recessive ataxia resulting from mutations in the frataxin gene (FXN). Such mutations, usually expanded guanine-adenine-adenine (GAA) repeats, give rise to decreased levels of frataxin protein in both affected and unaffected tissues. The goal was to understand the relationship of frataxin levels in peripheral tissues to disease status. METHODS: Frataxin levels were measured in buccal cells and blood, and analyzed in relation to disease features. Site-directed mutant frataxin was also transfected into human embryonic kidney cells to model results from specific point mutations. RESULTS: There was no evidence for change in frataxin levels over time with repeated measures analysis, although linear regression analysis of cross-sectional data predicted a small increase over decades. GAA repeat length predicted frataxin levels in both tissues, and frataxin levels themselves predicted neurological ratings (accounting for age). Compound heterozygous patients for a GAA expansion and a point mutation in FXN generally had lower levels of frataxin than those homozygous for the presence of two GAA repeat expansions, though levels varied dramatically between tissues in some compound heterozygotes for point mutations. The G130V mutation led to decreased levels of frataxin in vitro as well as in vivo, while the R165C mutation produced normal immunoreactive levels of frataxin both in vitro and in vivo. Start codon mutations led to low levels of frataxin in buccal cells but preserved immunoreactive frataxin levels in blood. INTERPRETATION: The present data show that peripheral frataxin levels reflect disease features in FRDA, but emphasize the need for interpretation of such levels in the context of specific mutations.