High fat diet ameliorates mitochondrial cardiomyopathy in CHCHD10 mutant mice.

Mutations in CHCHD10, a mitochondrial protein with undefined functions, are associated with autosomal dominant mitochondrial diseases. Chchd10 knock-in mice harboring a heterozygous S55L mutation (equivalent to human pathogenic S59L) develop a fatal mitochondrial cardiomyopathy caused by CHCHD10 aggregation and proteotoxic mitochondrial integrated stress response (mtISR). In mutant hearts, mtISR is accompanied by a metabolic rewiring characterized by increased reliance on glycolysis rather than fatty acid oxidation. To counteract this metabolic rewiring, heterozygous S55L mice were subjected to chronic high-fat diet (HFD) to decrease insulin sensitivity and glucose uptake and enhance fatty acid utilization in the heart. HFD ameliorated the ventricular dysfunction of mutant hearts and significantly extended the survival of mutant female mice affected by severe pregnancy-induced cardiomyopathy. Gene expression profiles confirmed that HFD increased fatty acid utilization and ameliorated cardiomyopathy markers. Importantly, HFD also decreased accumulation of aggregated CHCHD10 in the S55L heart, suggesting activation of quality control mechanisms. Overall, our findings indicate that metabolic therapy can be effective in mitochondrial cardiomyopathies associated with proteotoxic stress.

Sex-dependent effects of the uncompetitive N-methyl-D-aspartate receptor antagonist REL-1017 in G93A-SOD1 amyotrophic lateral sclerosis mice.

Introduction: The pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the demise of motor neurons has been linked to excitotoxicity caused by excessive calcium influx via N-methyl-D-aspartate receptors (NMDARs), suggesting that uncompetitive NMDAR antagonism could be a strategy to attenuate motor neuron degeneration. REL-1017, the dextro-isomer of racemic methadone, is a low-affinity uncompetitive NMDAR antagonist. Importantly, in humans REL-1017 has shown excellent tolerability in clinical trials for major depression. Methods: Here, we tested if REL-1017 improves the disease phenotypes in the G93A SOD1 mouse, a well-established model of familial ALS, by examining survival and motor functions, as well as the expression of genes and proteins involved in neuroplasticity. Results: We found a sex-dependent effect of REL-1017 in G93A SOD1 mice. A delay of ALS symptom onset, assessed as 10%-decrease of body weight (p < 0.01 vs. control untreated mice) and an extension of lifespan (p < 0.001 vs. control untreated mice) was observed in male G93A SOD1 mice. Female G93A SOD1 mice treated with REL-1017 showed an improvement of muscle strength (p < 0.01 vs. control untreated mice). Both males and females treated with REL-1017 showed a decrease in hind limb clasping. Sex-dependent effects of REL-1017 were also detected in molecular markers of neuronal plasticity (PSD95 and SYN1) in the spinal cord and in the GluN1 NMDAR subunit in quadricep muscles. Conclusion: In conclusion, this study provides preclinical in vivo evidence supporting the clinical evaluation of REL-1017 in ALS.

Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.

High fat diet ameliorates mitochondrial cardiomyopathy in CHCHD10 mutant mice.

Mutations in CHCHD10 , a mitochondrial protein with undefined functions, are associated with autosomal dominant mitochondrial diseases. Chchd10 knock-in mice harboring a heterozygous S55L mutation (equivalent to human pathogenic S59L) develop a fatal mitochondrial cardiomyopathy caused by CHCHD10 aggregation and proteotoxic mitochondrial integrated stress response (mtISR). In mutant hearts, mtISR is accompanied by a metabolic rewiring characterized by increased reliance on glycolysis rather than fatty acid oxidation. To counteract this metabolic rewiring, heterozygous S55L mice were subjected to chronic high fat diet (HFD) to decrease insulin sensitivity and glucose uptake and enhance fatty acid utilization in the heart. HFD ameliorated the ventricular dysfunction of mutant hearts and significantly extended the survival of mutant female mice affected by severe pregnancy-induced cardiomyopathy. Gene expression profiles confirmed that HFD increased fatty acid utilization and ameliorated cardiomyopathy markers. Importantly, HFD also decreased accumulation of aggregated CHCHD10 in the S55L heart, suggesting activation of quality control mechanisms. Overall, our findings indicate that metabolic therapy can be effective in mitochondrial cardiomyopathies associated with proteotoxic stress.

Effects of PB-TURSO on the transcriptional and metabolic landscape of sporadic ALS fibroblasts.

OBJECTIVE: ALS is a rapidly progressive, fatal disorder caused by motor neuron degeneration, for which there is a great unmet therapeutic need. AMX0035, a combination of sodium phenylbutyrate (PB) and taurursodiol (TUDCA, TURSO), has shown promising results in early ALS clinical trials, but its mechanisms of action remain to be elucidated. Therefore, our goal was to obtain an unbiased landscape of the molecular effects of AMX0035 in ALS patient-derived cells. METHODS: We investigated the transcriptomic and metabolomic profiles of primary skin fibroblasts from sporadic ALS patients and healthy controls (n = 12/group) treated with PB, TUDCA, or PB-TUDCA combination (Combo). Data were evaluated with multiple approaches including differential gene expression and metabolite abundance, Gene Ontology and metabolic pathway analysis, weighted gene co-expression correlation analysis (WGCNA), and combined multiomics integrated analysis. RESULTS: Combo changed many more genes and metabolites than either PB or TUDCA individually. Most changes were unique to Combo and affected the expression of genes involved in nucleocytoplasmic transport, unfolded protein response, mitochondrial function, RNA metabolism, and innate immunity. WGCNA showed significant correlations between ALS gene expression modules and clinical parameters that were abolished by Combo treatment. INTERPRETATION: This study is the first to explore the molecular effects of Combo in ALS patient-derived cells. It shows that Combo has a greater and distinct impact compared with the individual compounds and provides clues to drug targets and mechanisms of action, which may underlie the benefits of this investigational drug combination.

Mutant CHCHD10 causes an extensive metabolic rewiring that precedes OXPHOS dysfunction in a murine model of mitochondrial cardiomyopathy.

Mitochondrial cardiomyopathies are fatal diseases, with no effective treatment. Alterations of heart mitochondrial function activate the mitochondrial integrated stress response (ISRmt), a transcriptional program affecting cell metabolism, mitochondrial biogenesis, and proteostasis. In humans, mutations in CHCHD10, a mitochondrial protein with unknown function, were recently associated with dominant multi-system mitochondrial diseases, whose pathogenic mechanisms remain to be elucidated. Here, in CHCHD10 knockin mutant mice, we identify an extensive cardiac metabolic rewiring triggered by proteotoxic ISRmt. The stress response arises early on, before the onset of bioenergetic impairments, triggering a switch from oxidative to glycolytic metabolism, enhancement of transsulfuration and one carbon (1C) metabolism, and widespread metabolic imbalance. In parallel, increased NADPH oxidases elicit antioxidant responses, leading to heme depletion. As the disease progresses, the adaptive metabolic stress response fails, resulting in fatal cardiomyopathy. Our findings suggest that early interventions to counteract metabolic imbalance could ameliorate mitochondrial cardiomyopathy associated with proteotoxic ISRmt.

Modulation of the IGF1R-MTOR pathway attenuates motor neuron toxicity of human ALS SOD1G93A astrocytes.

ALS (amyotrophic lateral sclerosis), the most common motor neuron disease, causes muscle denervation and rapidly fatal paralysis. While motor neurons are the most affected cells in ALS, studies on the pathophysiology of the disease have highlighted the importance of non-cell autonomous mechanisms, which implicate astrocytes and other glial cells. In ALS, subsets of reactive astrocytes lose their physiological functions and become toxic for motor neurons, thereby contributing to disease pathogenesis. Evidence of astrocyte contribution to disease pathogenesis are well established in cellular and animal models of familial ALS linked to mutant SOD1, where astrocytes promote motor neuron cell death. The mechanism underlying astrocytes reactivity in conditions of CNS injury have been shown to involve the MTOR pathway. However, the role of this conserved metabolic signaling pathway, and the potential therapeutic effects of its modulation, have not been investigated in ALS astrocytes. Here, we show elevated activation of the MTOR pathway in human-derived astrocytes harboring mutant SOD1, which results in inhibition of macroautophagy/autophagy, increased cell proliferation, and enhanced astrocyte reactivity. We demonstrate that MTOR pathway activation in mutant SOD1 astrocytes is due to post-transcriptional upregulation of the IGF1R (insulin like growth factor 1 receptor), an upstream positive modulator of the MTOR pathway. Importantly, inhibition of the IGF1R-MTOR pathway decreases cell proliferation and reactivity of mutant SOD1 astrocytes, and attenuates their toxicity to motor neurons. These results suggest that modulation of astrocytic IGF1R-MTOR pathway could be a viable therapeutic strategy in SOD1 ALS and potentially other neurological diseases.Abbreviations: ACM: astrocyte conditioned medium; AKT: AKT serine/threonine kinase; ALS: amyotrophic lateral sclerosis; BrdU: thymidine analog 5-bromo-2'-deoxyuridine; CNS: central nervous system; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GFAP: glial fibrillary acidic protein; IGF1R: insulin like growth factor 1 receptor; INSR: insulin receptor; iPSA: iPSC-derived astrocytes; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta;MTOR: mechanistic target of rapamycin kinase; NES: nestin; PPK1: 3-phosphoinositide dependent protein kinase 1; PI: propidium iodide; PPP: picropodophyllotoxin; PTEN: phosphatase and tensin homolog; S100B/S100ß: S100 calcium binding protein B; SLC1A3/ EAAT1: solute carrier family 1 member 3; SMI-32: antibody to nonphosphorylated NEFH; SOD1: superoxide dismutase 1; TUBB3: tubulin beta 3 class III; ULK1: unc-51 like autophagy activating kinase 1.

Accelerated transsulfuration metabolically defines a discrete subclass of amyotrophic lateral sclerosis patients.

Amyotrophic lateral sclerosis is a disease characterized by progressive paralysis and death. Most ALS-cases are sporadic (sALS) and patient heterogeneity poses challenges for effective therapies. Applying metabolite profiling on 77-sALS patient-derived-fibroblasts and 43-controls, we found ~25% of sALS cases (termed sALS-1) are characterized by transsulfuration pathway upregulation, where methionine-derived-homocysteine is channeled into cysteine for glutathione synthesis. sALS-1 fibroblasts selectively exhibited a growth defect under oxidative conditions, fully-rescued by N-acetylcysteine (NAC). [U13C]-glucose tracing showed transsulfuration pathway activation with accelerated glucose flux into the Krebs cycle. We established a four-metabolite support vector machine model predicting sALS-1 metabotype with 97.5% accuracy. Both sALS-1 metabotype and growth phenotype were validated in an independent cohort of sALS cases. Importantly, plasma metabolite profiling identified a system-wide cysteine metabolism perturbation as a hallmark of sALS-1. Findings reveal that sALS patients can be stratified into distinct metabotypes with differential sensitivity to metabolic stress, providing novel insights for personalized therapy.

Glial mitochondrial function and dysfunction in health and neurodegeneration.

Mitochondria play essential metabolic roles in neural cells. Mitochondrial dysfunction has profound effects on the brain. In primary mitochondrial diseases, mutations that impair specific oxidative phosphorylation (OXPHOS) proteins or OXPHOS assembly factors lead to isolated biochemical defects and a heterogeneous group of clinical phenotypes, including mitochondrial encephalopathies. A broader defect of OXPHOS function, due to mutations in proteins involved in mitochondrial DNA maintenance, mitochondrial biogenesis, or mitochondrial tRNAs can also underlie severe mitochondrial encephalopathies. While primary mitochondrial dysfunction causes rare genetic forms of neurological disorders, secondary mitochondrial dysfunction is involved in the pathophysiology of some of the most common neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Many studies have investigated mitochondrial function and dysfunction in bulk central nervous system (CNS) tissue. However, the interpretation of these studies has been often complicated by the extreme cellular heterogeneity of the CNS, which includes many different types of neurons and glial cells. Because neurons are especially dependent on OXPHOS for ATP generation, mitochondrial dysfunction is thought to be directly involved in cell autonomous neuronal demise. Despite being metabolically more flexible than neurons, glial mitochondria also play an essential role in the function of the CNS, and have adapted specific metabolic and mitochondrial features to support their diversity of functions. This review analyzes our current understanding and the gaps in knowledge of mitochondrial properties of glia and how they affect neuronal functions, in health and disease.

Nrf2 signaling links ER oxidative protein folding and calcium homeostasis in health and disease.

We report a signaling pathway linking two fundamental functions of the ER, oxidative protein folding, and intracellular calcium regulation. Cells sense ER oxidative protein folding through H2O2, which induces Nrf2 nuclear translocation. Nrf2 regulates the expression of GPx8, an ER glutathione peroxidase that modulates ER calcium levels. Because ER protein folding is dependent on calcium, this pathway functions as rheostat of ER calcium levels. Protein misfolding and calcium dysregulation contribute to the pathophysiology of many diseases, including amyotrophic lateral sclerosis, in which astrocytic calcium dysregulation participates in causing motor neuron death. In human-derived astrocytes harboring mutant SOD1 causative of familial amyotrophic lateral sclerosis, we show that impaired ER redox signaling decreases Nrf2 nuclear translocation, resulting in ER calcium overload and increased calcium-dependent cell secretion, leading to motor neuron death. Nrf2 activation in SOD1 mutant astrocytes with dimethyl fumarate restores calcium homeostasis and ameliorates motor neuron death. These results highlight a regulatory mechanism of intracellular calcium homeostasis by ER redox signaling and suggest that this mechanism could be a therapeutic target in SOD1 mutant astrocytes.

Distal denervation in the SOD1 knockout mouse correlates with loss of mitochondria at the motor nerve terminal.

Impairment of mitochondrial transport has long been implicated in the pathogenesis of neuropathy and neurodegeneration. However, the role of mitochondria in stabilizing motor nerve terminals at neuromuscular junction (NMJ) remains unclear. We previously demonstrated that mice lacking the antioxidant enzyme, superoxide dismutase-1 (Sod1-/-), develop progressive NMJ denervation. This was rescued by expression of SOD1 exclusively in the mitochondrial intermembrane space (MitoSOD1/Sod1-/-), suggesting that oxidative stress within mitochondria drives denervation in these animals. However, we also observed reduced mitochondrial density in Sod1-/- motor axons in vitro. To investigate the relationship between mitochondrial density and NMJ innervation in vivo, we crossed Sod1-/- mice with the fluorescent reporter strains Thy1-YFP and Thy1-mitoCFP. We identified an age-dependent loss of mitochondria at motor nerve terminals in Sod1-/- mice, that closely correlated with NMJ denervation, and was rescued by MitoSOD1 expression. To test whether augmenting mitochondrial transport rescues Sod1-/- axons, we generated transgenic mice overexpressing the mitochondrial cargo adaptor, Miro1. This led to a partial rescue of mitochondrial density at motor nerve terminals by 12 months of age, but was insufficient to prevent denervation. These findings suggest that loss of mitochondria in the distal motor axon may contribute to denervation in Sod1-/- mice, perhaps via loss of key mitochondrial functions such as calcium buffering and/or energy production.

ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

Parkin is a disease modifier in the mutant SOD1 mouse model of ALS.

Mutant Cu/Zn superoxide dismutase (SOD1) causes mitochondrial alterations that contribute to motor neuron demise in amyotrophic lateral sclerosis (ALS). When mitochondria are damaged, cells activate mitochondria quality control (MQC) mechanisms leading to mitophagy. Here, we show that in the spinal cord of G93A mutant SOD1 transgenic mice (SOD1-G93A mice), the autophagy receptor p62 is recruited to mitochondria and mitophagy is activated. Furthermore, the mitochondrial ubiquitin ligase Parkin and mitochondrial dynamics proteins, such as Miro1, and Mfn2, which are ubiquitinated by Parkin, and the mitochondrial biogenesis regulator PGC1α are depleted. Unexpectedly, Parkin genetic ablation delays disease progression and prolongs survival in SOD1-G93A mice, as it slows down motor neuron loss and muscle denervation and attenuates the depletion of mitochondrial dynamics proteins and PGC1α. Our results indicate that Parkin is a disease modifier in ALS, because chronic Parkin-mediated MQC activation depletes mitochondrial dynamics-related proteins, inhibits mitochondrial biogenesis, and worsens mitochondrial dysfunction.

Proteinopathies and OXPHOS dysfunction in neurodegenerative diseases.

Mitochondria participate in essential processes in the nervous system such as energy and intermediate metabolism, calcium homeostasis, and apoptosis. Major neurodegenerative diseases are characterized pathologically by accumulation of misfolded proteins as a result of gene mutations or abnormal protein homeostasis. Misfolded proteins associate with mitochondria, forming oligomeric and fibrillary aggregates. As mitochondrial dysfunction, particularly of the oxidative phosphorylation system (OXPHOS), occurs in neurodegeneration, it is postulated that such defects are caused by the accumulation of misfolded proteins. However, this hypothesis and the pathological role of proteinopathies in mitochondria remain elusive. In this study, we critically review the proposed mechanisms whereby exemplary misfolded proteins associate with mitochondria and their consequences on OXPHOS.

Fibroblast bioenergetics to classify amyotrophic lateral sclerosis patients.

BACKGROUND: The objective of this study was to investigate cellular bioenergetics in primary skin fibroblasts derived from patients with amyotrophic lateral sclerosis (ALS) and to determine if they can be used as classifiers for patient stratification. METHODS: We assembled a collection of unprecedented size of fibroblasts from patients with sporadic ALS (sALS, n = 171), primary lateral sclerosis (PLS, n = 34), ALS/PLS with C9orf72 mutations (n = 13), and healthy controls (n = 91). In search for novel ALS classifiers, we performed extensive studies of fibroblast bioenergetics, including mitochondrial membrane potential, respiration, glycolysis, and ATP content. Next, we developed a machine learning approach to determine whether fibroblast bioenergetic features could be used to stratify patients. RESULTS: Compared to controls, sALS and PLS fibroblasts had higher average mitochondrial membrane potential, respiration, and glycolysis, suggesting that they were in a hypermetabolic state. Only membrane potential was elevated in C9Orf72 lines. ATP steady state levels did not correlate with respiration and glycolysis in sALS and PLS lines. Based on bioenergetic profiles, a support vector machine (SVM) was trained to classify sALS and PLS with 99% specificity and 70% sensitivity. CONCLUSIONS: sALS, PLS, and C9Orf72 fibroblasts share hypermetabolic features, while presenting differences of bioenergetics. The absence of correlation between energy metabolism activation and ATP levels in sALS and PLS fibroblasts suggests that in these cells hypermetabolism is a mechanism to adapt to energy dissipation. Results from SVM support the use of metabolic characteristics of ALS fibroblasts and multivariate analysis to develop classifiers for patient stratification.

Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo.

BACKGROUND: Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43A315T mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. METHODS: In the brain of TDP-43A315T mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. RESULTS: We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43A315T peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43A315T mouse brain and cells expressing A315T mutant TDP-43. CONCLUSIONS: While alterations of mitochondrial morphology and dynamics in TDP-43 mutant neurons are well established, the present study did not demonstrate oxidative phosphorylation defects in TDP-43 mutants, in vitro and in vivo. On the other hand, the increase in mitochondrial calcium uptake in A315T TDP-43 mutants was an intriguing finding, which needs to be investigated further to understand its mechanisms and potential pathogenic implications.

Alterations in voltage-sensing of the mitochondrial permeability transition pore in ANT1-deficient cells.

The probability of mitochondrial permeability transition (mPT) pore opening is inversely related to the magnitude of the proton electrochemical gradient. The module conferring sensitivity of the pore to this gradient has not been identified. We investigated mPT's voltage-sensing properties elicited by calcimycin or H2O2 in human fibroblasts exhibiting partial or complete lack of ANT1 and in C2C12 myotubes with knocked-down ANT1 expression. mPT onset was assessed by measuring in situ mitochondrial volume using the 'thinness ratio' and the 'cobalt-calcein' technique. De-energization hastened calcimycin-induced swelling in control and partially-expressing ANT1 fibroblasts, but not in cells lacking ANT1, despite greater losses of mitochondrial membrane potential. Matrix Ca(2+) levels measured by X-rhod-1 or mitochondrially-targeted ratiometric biosensor 4mtD3cpv, or ADP-ATP exchange rates did not differ among cell types. ANT1-null fibroblasts were also resistant to H2O2-induced mitochondrial swelling. Permeabilized C2C12 myotubes with knocked-down ANT1 exhibited higher calcium uptake capacity and voltage-thresholds of mPT opening inferred from cytochrome c release, but intact cells showed no differences in calcimycin-induced onset of mPT, irrespective of energization and ANT1 expression, albeit the number of cells undergoing mPT increased less significantly upon chemically-induced hypoxia than control cells. We conclude that ANT1 confers sensitivity of the pore to the electrochemical gradient.

Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis.

Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS.

Bioenergetic markers in skin fibroblasts of sporadic amyotrophic lateral sclerosis and progressive lateral sclerosis patients.

Energy metabolism could influence amyotrophic lateral sclerosis (ALS) and progressive lateral sclerosis (PLS) pathogenesis and the response to therapy. We developed a novel assay to simultaneously assess mitochondrial content and membrane potential in patients' skin fibroblasts. In ALS and PLS fibroblasts, membrane potential was increased and mitochondrial content decreased, relative to healthy controls. In ALS higher mitochondrial membrane potential correlated with age at diagnosis, and in PLS it correlated with disease severity. These unprecedented findings in ALS and PLS fibroblasts could shed new light onto disease pathogenesis and help in developing biomarkers to predict disease evolution and the individual response to therapy in motor neuron diseases.