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. CHCHD10
S55L
mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10
S55L
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 CHCHD10
S55L
-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10
S55L
mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.
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.
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.
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.
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.
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.
•Skin fibroblast profiling identifies a distinct sporadic ALS case metabotype (sALS-1).•ALS-1 cells are defined by perturbed sulfur metabolism and glucose hypermetabolism.•ALS-1 cells display oxidative stress and increased use of sulfur for GSH synthesis.•ALS-1 biomarkers allow plasma profiling for facile recognition of ALS-1 cases.•The 25% of sporadic ALS cases that are ALS-1 may benefit from new targeted therapies.
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-43
mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43.
In the brain of TDP-43
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.
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-43
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-43
mouse brain and cells expressing A315T mutant TDP-43.
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.
The brain’s neural computations rely on the differing cellular architectures and intrinsic properties of excitatory and inhibitory neurons, and disruptions in the balance of excitation (E) versus ...inhibition (I) can lead to neuropathological conditions such as autism and epilepsy. Metabolic perturbations are a common driver of E/I imbalance, but it remains unclear whether these two neuron types are differentially sensitive to metabolic lesions. Here, we characterized differences in presynaptic bioenergetic regulation between excitatory and inhibitory nerve terminals using genetically encoded indicators expressed in primary dissociated neuronal cultures. Our experiments showed that inhibitory nerve terminals sustain higher ATP levels than excitatory nerve terminals with increased mitochondrial metabolism. Mitochondria in inhibitory neurons additionally play a greater role in buffering presynaptic Ca2+, and inhibitory mitochondrial Ca2+ handling is differentially regulated by TMEM65- mediated acceleration of mitochondrial Ca2+ extrusion after bursts of activity. In excitatory neurons, expression of the Ca2+ activated mitochondrial aspartate/glutamate carrier AGC1 is differentially important. We then investigated whether differential reliance on mitochondrial metabolism could explain some of the mechanism of the ketogenic diet’s therapeutic effect in drug-resistant epilepsy. We showed that nerve terminals can use the ketone β-hydroxybutyrate (BHB) to fuel synaptic transmission for about 20 minutes of moderate activity, identifying a cell autonomous mechanism of ketone metabolism. Active inhibitory neurons generate ATP more rapidly from pyruvate than from BHB, but we saw no difference between these fuels in excitatory neurons. These results suggest that differences in excitatory and inhibitory neuron metabolism of BHB are not responsible for the therapeutic effect of the ketogenic diet, though overall differences in mitochondrial metabolism may mediate a differential benefit in inhibitory neurons. Finally, we found that Ca2+ dependent upregulation of mitochondrial metabolism is needed to fuel neurotransmission in both inhibitory and excitatory neurons fueled by mitochondria. Overall, these experiments identify a differential reliance on mitochondrial function in two major neuron types.
Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease with limited therapeutic options. Biomarkers are needed for early disease detection, clinical trial design, and personalized ...medicine. Early evidence suggests that specific morphometric features in ALS primary skin fibroblasts may be used as biomarkers; however, this hypothesis has not been rigorously tested in conclusively large fibroblast populations. Here, we imaged ALS-relevant organelles (mitochondria, endoplasmic reticulum, lysosomes) and proteins (TAR DNA-binding protein 43, Ras GTPase-activating protein-binding protein 1, heat-shock protein 60) at baseline and under stress perturbations and tested their predictive power on a total set of 443 human fibroblast lines from ALS and healthy individuals. Machine learning approaches were able to confidently predict stress perturbation states (ROC-AUC ∼0.99) but not disease groups or clinical features (ROC-AUC 0.58–0.64). Our findings indicate that multivariate models using patient-derived fibroblast morphometry can accurately predict different stressors but are insufficient to develop viable ALS biomarkers.
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 H
O
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.
Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 ...(SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.
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