Direct observation of the interconversion of normal and toxic forms of α-synuclein.

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.

Phospholipase iPLA2ß averts ferroptosis by eliminating a redox lipid death signal.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to the accumulation of 15-hydroperoxy (Hp)-arachidonoyl-phosphatidylethanolamine (15-HpETE-PE), generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, phosphatidylethanolamine (PE)-binding protein (PEBP)1. As the Ca2+-independent phospholipase A2ß (iPLA2ß, PLA2G6 or PNPLA9 gene) can preferentially hydrolyze peroxidized phospholipids, it may eliminate the ferroptotic 15-HpETE-PE death signal. Here, we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA2ß averts ferroptosis, whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson's disease (PD)-associated mutation (fPDR747W) and found selectively decreased 15-HpETE-PE-hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-Cas9-engineered Pnpla9R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein-mutant SncaA53T mice, with decreased iPLA2ß expression and a PD-relevant phenotype. Thus, iPLA2ß is a new ferroptosis regulator, and its mutations may be implicated in PD pathogenesis.

Elevated 4R-tau in astrocytes from asymptomatic carriers of the MAPT 10+16 intronic mutation.

The microtubule-associated protein tau gene (MAPT) 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers which, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau transcript and protein ratios. These elevated levels of 4R-tau in astrocytes implicate glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of pre-clinical tauopathies.

Inorganic Polyphosphate and F0F1-ATP Synthase of Mammalian Mitochondria.

Inorganic polyphosphate is a polymer which plays multiple important roles in yeast and bacteria. In higher organisms the role of polyP has been intensively studied in last decades and involvements of this polymer in signal transduction, cell death mechanisms, energy production, and many other processes were demonstrated. In contrast to yeast and bacteria, where enzymes responsible for synthesis and hydrolysis of polyP were identified, in mammalian cells polyP clearly plays important role in physiology and pathology but enzymes responsible for synthesis of polyP or consumption of this polymer are still not identified. Here, we discuss the role of mitochondrial F0F1-ATP synthase in polyP synthesis with results, which confirm this proposal. We also discuss the role of other enzymes which may play important roles in polyP metabolism.

Age-related changes in the energy of human mesenchymal stem cells.

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells.

Maturation and phenotype of pathophysiological neuronal excitability of human cells in tau-related dementia.

Frontotemporal dementia and parkinsonism (FTDP-17) caused by the 10+16 splice-site mutation in the gene encoding microtubule-associated protein tau (MAPT) provides an established platform to model tau-related dementia in vitro Neurons derived from human induced pluripotent stem cells (iPSCs) have been shown to recapitulate the neurodevelopmental profile of tau pathology during in vitro corticogenesis, as in the adult human brain. However, the neurophysiological phenotype of these cells has remained unknown, leaving unanswered questions regarding the functional relevance and the gnostic power of this disease model. In this study, we used electrophysiology to explore the membrane properties and intrinsic excitability of the generated neurons and found that human cells mature by ∼150 days of neurogenesis to become compatible with matured cortical neurons. In earlier FTDP-17, however, neurons exhibited a depolarized resting membrane potential associated with increased resistance and reduced voltage-gated Na+- and K+-channel-mediated conductance. Expression of the Nav1.6 protein was reduced in FTDP-17. These effects led to reduced cell capability of induced firing and changed the action potential waveform in FTDP-17. The revealed neuropathology might thus contribute to the clinicopathological profile of the disease. This sheds new light on the significance of human in vitro models of dementia.

Mitochondrial ROS control neuronal excitability and cell fate in frontotemporal dementia.

INTRODUCTION: The second most common form of early-onset dementia-frontotemporal dementia (FTD)-is often characterized by the aggregation of the microtubule-associated protein tau. Here we studied the mechanism of tau-induced neuronal dysfunction in neurons with the FTD-related 10+16 MAPT mutation. METHODS: Live imaging, electrophysiology, and redox proteomics were used in 10+16 induced pluripotent stem cell-derived neurons and a model of tau spreading in primary cultures. RESULTS: Overproduction of mitochondrial reactive oxygen species (ROS) in 10+16 neurons alters the trafficking of specific glutamate receptor subunits via redox regulation. Increased surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors containing GluA1 and NR2B subunits leads to impaired glutamatergic signaling, calcium overload, and excitotoxicity. Mitochondrial antioxidants restore the altered response and prevent neuronal death. Importantly, extracellular 4R tau induces the same pathological response in healthy neurons, thus proposing a mechanism for disease propagation. DISCUSSION: These results demonstrate mitochondrial ROS modulate glutamatergic signaling in FTD, and suggest a new therapeutic strategy.

Mitochondria and lipid peroxidation in the mechanism of neurodegeneration: Finding ways for prevention.

The world's population aging progression renders age-related neurodegenerative diseases to be one of the biggest unsolved problems of modern society. Despite the progress in studying the development of pathology, finding ways for modifying neurodegenerative disorders remains a high priority. One common feature of neurodegenerative diseases is mitochondrial dysfunction and overproduction of reactive oxygen species, resulting in oxidative stress. Although lipid peroxidation is one of the markers for oxidative stress, it also plays an important role in cell physiology, including activation of phospholipases and stimulation of signaling cascades. Excessive lipid peroxidation is a hallmark for most neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and many other neurological conditions. The products of lipid peroxidation have been shown to be the trigger for necrotic, apoptotic, and more specifically for oxidative stress-related, that is, ferroptosis and neuronal cell death. Here we discuss the involvement of lipid peroxidation in the mechanism of neuronal loss and some novel therapeutic directions to oppose it.

Activation of RAGE leads to the release of glutamate from astrocytes and stimulates calcium signal in neurons.

The receptor for advanced glycation end products (RAGE) is a signal receptor first shown to be activated by advanced glycation end products, but also by a variety of signal molecules, including pathological advanced oxidation protein products and ß-amyloid. However, most of the RAGE activators have multiple intracellular targets, making it difficult to unravel the exact pathway of RAGE activation. Here, we show that the cell-impermeable RAGE fragment sequence (60-76) of the V-domain of the receptor is able to activate RAGE present on the plasma membrane of neurons and, preferentially, astrocytes. This leads to the exocytosis of vesicular glutamate transporter vesicles and the release of glutamate from astrocytes, which stimulate NMDA and AMPA/kainate receptors, resulting in calcium signals predominantly in neurons. Thus, we show a specific mechanism of RAGE activation by the RAGE fragment and propose a mechanism by which RAGE activation can contribute to the neuronal-astrocytic communication in physiology and pathology.

Variability of mitochondrial energy balance across brain regions.

Brain is not homogenous and neurons from various brain regions are known to have different vulnerabilities to mitochondrial mutations and mitochondrial toxins. However, it is not clear if this vulnerability is connected to different energy metabolism in specific brain regions. Here, using live-cell imaging, we compared mitochondrial membrane potential and nicotinamide adenine dinucleotide (NADH) redox balance in acute rat brain slices in different brain regions and further detailed the mitochondrial metabolism in primary neurons and astrocytes from rat cortex, midbrain and cerebellum. We have found that mitochondrial membrane potential is higher in brain slices from the hippocampus and brain stem. In primary co-cultures, mitochondrial membrane potential in astrocytes was lower than in neurons, whereas in midbrain cells it was higher than in cortex and cerebellum. The rate of NADH production and mitochondrial NADH pool were highest in acute slices from midbrain and midbrain primary neurons and astrocytes. Although the level of adenosine tri phosphate (ATP) was similar among primary neurons and astrocytes from cortex, midbrain and cerebellum, the rate of ATP consumption was highest in midbrain cells that lead to faster neuronal and astrocytic collapse in response to inhibitors of ATP production. Thus, midbrain neurons and astrocytes have a higher metabolic rate and ATP consumption that makes them more vulnerable to energy deprivation.

Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.

Inorganic polyphosphate is produced and hydrolyzed in F0F1-ATP synthase of mammalian mitochondria.

Inorganic polyphosphate (polyP) is a polymer present in all living organisms. Although polyP is found to be involved in a variety of functions in cells of higher organisms, the enzyme responsible for polyP production and consumption has not yet been identified. Here, we studied the effect of polyP on mitochondrial respiration, oxidative phosphorylation and activity of F0F1-ATPsynthase. We have found that polyP activates mitochondrial respiration which does not coupled with ATP production (V2) but inhibits ADP-dependent respiration (V3). Moreover, PolyP can stimulate F0F1-ATPase activity in the presence of ATP and, importantly, can be hydrolyzed in this enzyme instead of ATP. Furthermore, PolyP can be produced in mitochondria in the presence of substrates for respiration and phosphate by the F0F1-ATPsynthase. Thus, polyP is an energy molecule in mammalian cells which can be produced and hydrolyzed in the mitochondrial F0F1-ATPsynthase.

Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors.

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the CNS. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity, which is accountable for neuronal death in acute neurological disorders, including stroke and trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of PolyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that preincubation of primary neurons with PolyP reduced glutamate-induced and AMPA-induced but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices, PolyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of PolyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Preincubation of cortical neurons with PolyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by PolyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.SIGNIFICANCE STATEMENT One of the oldest polymers in the evolution of living matter is the inorganic polyphosphate (PolyP). It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signaling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signaling induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors. The effect of PolyP on glutamate-induced and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of PolyP to restrict the glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease.

Mutations in the small heat shock protein Hsp27, encoded by the HSPB1 gene, have been shown to cause Charcot Marie Tooth Disease type 2 (CMT-2) or distal hereditary motor neuropathy (dHMN). Protein aggregation and axonal transport deficits have been implicated in the disease. In this study, we conducted analysis of bidirectional movements of mitochondria in primary motor neuron axons expressing wild type and mutant Hsp27. We found significantly slower retrograde transport of mitochondria in Ser135Phe, Pro39Leu and Arg140Gly mutant Hsp27 expressing motor neurons than in wild type Hsp27 neurons, although anterograde movement velocities remained normal. Retrograde transport of other important cargoes, such as the p75 neurotrophic factor receptor was minimally altered in mutant Hsp27 neurons, implicating that axonal transport deficits primarily affect mitochondria and the axonal transport machinery itself is less affected. Investigation of mitochondrial function revealed a decrease in mitochondrial membrane potential in mutant Hsp27 expressing motor axons, as well as a reduction in mitochondrial complex 1 activity, increased vulnerability of mitochondria to mitochondrial stressors, leading to elevated superoxide release and reduced mitochondrial glutathione (GSH) levels, although cytosolic GSH remained normal. This mitochondrial redox imbalance in mutant Hsp27 motor neurons is likely to cause low level of oxidative stress, which in turn will contribute to, and indeed may be the underlying cause of the deficits in mitochondrial axonal transport. Together, these findings suggest that the mitochondrial abnormalities in mutant Hsp27-induced neuropathies may be a primary cause of pathology, leading to further deficits in the mitochondrial axonal transport and onset of disease.

Cellular mechanisms of complex I-associated pathology.

Mitochondria control vitally important functions in cells, including energy production, cell signalling and regulation of cell death. Considering this, any alteration in mitochondrial metabolism would lead to cellular dysfunction and the development of a disease. A large proportion of disorders associated with mitochondria are induced by mutations or chemical inhibition of the mitochondrial complex I - the entry point to the electron transport chain. Subunits of the enzyme NADH: ubiquinone oxidoreductase, are encoded by both nuclear and mitochondrial DNA and mutations in these genes lead to cardio and muscular pathologies and diseases of the central nervous system. Despite such a clear involvement of complex I deficiency in numerous disorders, the molecular and cellular mechanisms leading to the development of pathology are not very clear. In this review, we summarise how lack of activity of complex I could differentially change mitochondrial and cellular functions and how these changes could lead to a pathology, following discrete routes.

Role of DJ-1 in the mechanism of pathogenesis of Parkinson's disease.

DJ-1 protein has multiple specific mechanisms to protect dopaminergic neurons against neurodegeneration in Parkinson's disease. Wild type DJ-1 can acts as oxidative stress sensor and as an antioxidant. DJ-1 exhibits the properties of molecular chaperone, protease, glyoxalase, transcriptional regulator that protects mitochondria from oxidative stress. DJ-1 increases the expression of two mitochondrial uncoupling proteins (UCP 4 and UCP5), that decrease mitochondrial membrane potential and leads to the suppression of ROS production, optimizes of a number of mitochondrial functions, and is regarded as protection for the neuronal cell survival. We discuss also the stabilizing interaction of DJ-1 with the mitochondrial Bcl-xL protein, which regulates the activity of (Inositol trisphosphate receptor) IP3R, prevents the cytochrome c release from mitochondria and inhibits the apoptosis activation. Upon oxidative stress DJ-1 is able to regulate various transcription factors including nuclear factor Nrf2, PI3K/PKB, and p53 signal pathways. Stress-activated transcription factor Nrf2 regulates the pathways to protect cells against oxidative stress and metabolic pathways initiating the NADPH and ATP production. DJ-1 induces the Nrf2 dissociation from its inhibitor Keap1 (Kelch-like ECH-associated protein 1), promoting Nrf2 nuclear translocation and binding to antioxidant response elements. DJ-1 is shown to be a co-activator of the transcription factor NF-kB. Under nitrosative stress, DJ-1 may regulate PI3K/PKB signaling through PTEN transnitrosylation, which leads to inhibition of phosphatase activity. DJ-1 has a complex modulating effect on the p53 pathway: one side DJ-1 directly binds to p53 to restore its transcriptional activity and on the other hand DJ-1 can stimulate deacylation and suppress p53 transcriptional activity. The ability of the DJ-1 to induce activation of different transcriptional factors and change redox balance protect neurons against aggregation of α-synuclein and oligomer-induced neurodegeneration.

KEAP1 inhibition is neuroprotective and suppresses the development of epilepsy.

Hippocampal sclerosis is a common acquired disease that is a major cause of drug-resistant epilepsy. A mechanism that has been proposed to lead from brain insult to hippocampal sclerosis is the excessive generation of reactive oxygen species, and consequent mitochondrial failure. Here we use a novel strategy to increase endogenous antioxidant defences using RTA 408, which we show activates nuclear factor erythroid 2-related factor 2 (Nrf2, encoded by NFE2L2) through inhibition of kelch like ECH associated protein 1 (KEAP1) through its primary sensor C151. Activation of Nrf2 with RTA 408 inhibited reactive oxygen species production, mitochondrial depolarization and cell death in an in vitro model of seizure-like activity. RTA 408 given after status epilepticus in vivo increased ATP, prevented neuronal death, and dramatically reduced (by 94%) the frequency of late spontaneous seizures for at least 4 months following status epilepticus. Thus, acute KEAP1 inhibition following status epilepticus exerts a neuroprotective and disease-modifying effect, supporting the hypothesis that reactive oxygen species generation is a key event in the development of epilepsy.

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading.

The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies.