Publications by authors named "Elizabeth A Jonas"

57 Publications

Presynaptic Kv3 channels are required for fast and slow endocytosis of synaptic vesicles.

Neuron 2021 03 27;109(6):938-946.e5. Epub 2021 Jan 27.

National Institute of Neurological Disorders and Stroke, 35 Convent Dr., Bethesda, MD 20892, USA. Electronic address:

Since their discovery decades ago, the primary physiological and pathological effects of potassium channels have been attributed to their ion conductance, which sets membrane potential and repolarizes action potentials. For example, Kv3 family channels regulate neurotransmitter release by repolarizing action potentials. Here we report a surprising but crucial function independent of potassium conductance: by organizing the F-actin cytoskeleton in mouse nerve terminals, the Kv3.3 protein facilitates slow endocytosis, rapid endocytosis, vesicle mobilization to the readily releasable pool, and recovery of synaptic depression during repetitive firing. A channel mutation that causes spinocerebellar ataxia inhibits endocytosis, vesicle mobilization, and synaptic transmission during repetitive firing by disrupting the ability of the channel to nucleate F-actin. These results unmask novel functions of potassium channels in endocytosis and vesicle mobilization crucial for sustaining synaptic transmission during repetitive firing. Potassium channel mutations that impair these "non-conducting" functions may thus contribute to generation of diverse neurological disorders.
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http://dx.doi.org/10.1016/j.neuron.2021.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979485PMC
March 2021

Mitochondria: powerhouses of presynaptic plasticity.

J Physiol 2021 Mar 18;599(5):1363-1364. Epub 2021 Jan 18.

Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, CT, USA.

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http://dx.doi.org/10.1113/JP281040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942974PMC
March 2021

ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome.

Cell 2020 Sep 13;182(5):1170-1185.e9. Epub 2020 Aug 13.

Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, CT 06511, USA; Marine Biological Laboratory, Woods Hole, MA 02543, USA. Electronic address:

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1 mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1 mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase β subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.
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http://dx.doi.org/10.1016/j.cell.2020.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484101PMC
September 2020

Oxidative stress battles neuronal Bcl-xL in a fight to the death.

Neural Regen Res 2021 Jan;16(1):12-15

Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT, USA.

Bcl-xL is a pro-survival protein of the Bcl2 family found in the mitochondrial membrane. Bcl-xL supports growth, development, and maturation of neurons, and it also prevents neuronal death during neurotoxic stimulation. This article reviews the mechanisms and upstream signaling that regulate the activity and abundance of Bcl-xL. Our team and others have reported that oxidative stress is a key regulator of intracellular Bcl-xL balance in neurons. Oxidative stress regulates synthesis, degradation, and activity of Bcl-xL and therefore neuronal function. During apoptosis, pro-apoptotic Bcl2 proteins such as Bax and Bak translocate to and oligomerize in the mitochondrial membrane. Formation of oligomers causes release of cytochrome c and activation of caspases that lead to neuronal death. Bcl-xL binds directly to pro-apoptotic Bcl2 proteins to block apoptotic signaling. Although anti-apoptotic roles of Bcl-xL have been well documented, an increasing number of studies in recent decades show that protein binding partners of Bcl-xL are not limited to Bcl2 proteins. Bcl-xL forms a complex with FFo ATP synthase, DJ-1, DRP1, IP3R, and the ryanodine receptor. These proteins support physiological processes in neurons such as growth and development and prevent neuronal damage by regulating mitochondrial ATP production, synapse formation, synaptic vesicle recycling, neurotransmission, and calcium signaling. However, under conditions of oxidative stress, Bcl-xL undergoes proteolytic cleavage thus lowering the abundance of functional Bcl-xL in neurons. Additionally, oxidative stress alters formation of Bcl-xL-mediated multiprotein complexes by regulating post-translational phosphorylation. Finally, oxidative stress regulates transcription factors that target the Bcl-x gene and alter accessibility of microRNA to mRNA influencing mRNA levels of Bcl-xL. In this review, we discussed how Bcl-xL supports the normal physiology of neurons, and how oxidative stress contributes to pathology by manipulating the dynamics of Bcl-xL production, degradation, and activity.
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http://dx.doi.org/10.4103/1673-5374.286946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818872PMC
January 2021

Inefficient thermogenic mitochondrial respiration due to futile proton leak in a mouse model of fragile X syndrome.

FASEB J 2020 06 20;34(6):7404-7426. Epub 2020 Apr 20.

Department of Anesthesiology, Columbia University Medical Center, New York, NY, USA.

Fragile X syndrome (FXS) is the leading known inherited intellectual disability and the most common genetic cause of autism. The full mutation results in transcriptional silencing of the Fmr1 gene and loss of fragile X mental retardation protein (FMRP) expression. Defects in neuroenergetic capacity are known to cause a variety of neurodevelopmental disorders. Thus, we explored the integrity of forebrain mitochondria in Fmr1 knockout mice during the peak of synaptogenesis. We found inefficient thermogenic respiration due to futile proton leak in Fmr1 KO mitochondria caused by coenzyme Q (CoQ) deficiency and an open cyclosporine-sensitive channel. Repletion of mitochondrial CoQ within the Fmr1 KO forebrain closed the channel, blocked the pathological proton leak, restored rates of protein synthesis during synaptogenesis, and normalized the key phenotypic features later in life. The findings demonstrate that FMRP deficiency results in inefficient oxidative phosphorylation during the neurodevelopment and suggest that dysfunctional mitochondria may contribute to the FXS phenotype.
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http://dx.doi.org/10.1096/fj.202000283RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692004PMC
June 2020

Alpha-Tocotrienol Prevents Oxidative Stress-Mediated Post-Translational Cleavage of Bcl-xL in Primary Hippocampal Neurons.

Int J Mol Sci 2019 Dec 28;21(1). Epub 2019 Dec 28.

Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT 06511, USA.

B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic member of the Bcl2 family of proteins, which supports neurite outgrowth and neurotransmission by improving mitochondrial function. During excitotoxic stimulation, however, Bcl-xL undergoes post-translational cleavage to ∆N-Bcl-xL, and accumulation of ∆N-Bcl-xL causes mitochondrial dysfunction and neuronal death. In this study, we hypothesized that the generation of reactive oxygen species (ROS) during excitotoxicity leads to formation of ∆N-Bcl-xL. We further proposed that the application of an antioxidant with neuroprotective properties such as α-tocotrienol (TCT) will prevent ∆N-Bcl-xL-induced mitochondrial dysfunction via its antioxidant properties. Primary hippocampal neurons were treated with α-TCT, glutamate, or a combination of both. Glutamate challenge significantly increased cytosolic and mitochondrial ROS and ∆N-Bcl-xL levels. ∆N-Bcl-xL accumulation was accompanied by intracellular ATP depletion, loss of mitochondrial membrane potential, and cell death. α-TCT prevented loss of mitochondrial membrane potential in hippocampal neurons overexpressing ∆N-Bcl-xL, suggesting that ∆N-Bcl-xL caused the loss of mitochondrial function under excitotoxic conditions. Our data suggest that production of ROS is an important cause of ∆N-Bcl-xL formation and that preventing ROS production may be an effective strategy to prevent ∆N-Bcl-xL-mediated mitochondrial dysfunction and thus promote neuronal survival.
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http://dx.doi.org/10.3390/ijms21010220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982044PMC
December 2019

A mitochondrial megachannel resides in monomeric FF ATP synthase.

Nat Commun 2019 12 20;10(1):5823. Epub 2019 Dec 20.

Section of Endocrinology, Department of Internal Medicine, Yale University, New Haven, CT, USA.

Purified mitochondrial ATP synthase has been shown to form Ca-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86 angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.
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http://dx.doi.org/10.1038/s41467-019-13766-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925261PMC
December 2019

Dopamine fuels its own release.

Nat Neurosci 2020 01;23(1):1-2

Department of Internal Medicine (Endocrinology), Yale University School of Medicine, New Haven, CT, USA.

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http://dx.doi.org/10.1038/s41593-019-0563-4DOI Listing
January 2020

Parkinson's disease protein DJ-1 regulates ATP synthase protein components to increase neuronal process outgrowth.

Cell Death Dis 2019 06 13;10(6):469. Epub 2019 Jun 13.

Department of Internal Medicine (Endocrinology), Yale University, New Haven, CT, USA.

Familial Parkinson's disease (PD) protein DJ-1 mutations are linked to early onset PD. We have found that DJ-1 binds directly to the FF ATP synthase β subunit. DJ-1's interaction with the β subunit decreased mitochondrial uncoupling and enhanced ATP production efficiency while in contrast mutations in DJ-1 or DJ-1 knockout increased mitochondrial uncoupling, and depolarized neuronal mitochondria. In mesencephalic DJ-1 KO cultures, there was a progressive loss of neuronal process extension. This was ameliorated by a pharmacological reagent, dexpramipexole, that binds to ATP synthase, closing a mitochondrial inner membrane leak and enhancing ATP synthase efficiency. ATP synthase c-subunit can form an uncoupling channel; we measured, therefore, ATP synthase F (β subunit) and c-subunit protein levels. We found that ATP synthase β subunit protein level in the DJ-1 KO neurons was approximately half that found in their wild-type counterparts, comprising a severe defect in ATP synthase stoichiometry and unmasking c-subunit. We suggest that DJ-1 enhances dopaminergic cell metabolism and growth by its regulation of ATP synthase protein components.
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http://dx.doi.org/10.1038/s41419-019-1679-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565618PMC
June 2019

The mitochondrial metabolic function of is modulated by .

FASEB J 2019 08 29;33(8):8925-8934. Epub 2019 Apr 29.

Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom.

Mitochondrial metabolic plasticity is a key adaptive mechanism in response to changes in cellular metabolic demand. Changes in mitochondrial metabolic efficiency have been linked to pathophysiological conditions, including cancer, neurodegeneration, and obesity. The ubiquitously expressed (Parkinsonism-associated deglycase) is known as a Parkinson's disease gene and an oncogene. The pleiotropic functions of include reactive oxygen species scavenging, RNA binding, chaperone activity, endocytosis, and modulation of major signaling pathways involved in cell survival and metabolism. Nevertheless, how these functions are linked to the role of in mitochondrial plasticity is not fully understood. In this study, we describe an interaction between and that regulates the localization of , in a hypoxia-dependent manner, either to the cytosol or to mitochondria. This interaction acts as a modulator of mitochondrial metabolic efficiency and a switch between glycolysis and oxidative phosphorylation. Modulation of this novel molecular mechanism of mitochondrial metabolic efficiency is potentially involved in the neuroprotective function of as well as its role in proliferation of cancer cells.-Weinert, M., Millet, A., Jonas, E. A., Alavian, K. N. The mitochondrial metabolic function of is modulated by .
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http://dx.doi.org/10.1096/fj.201802754RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988861PMC
August 2019

ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore.

Cell Rep 2019 01;26(1):11-17.e2

College of Dentistry, Department of Basic Sciences, New York University, New York, NY 10010, USA. Electronic address:

Permeability transition (PT) is an increase in mitochondrial inner membrane permeability that can lead to a disruption of mitochondrial function and cell death. PT is responsible for tissue damage in stroke and myocardial infarction. It is caused by the opening of a large conductance (∼1.5 nS) channel, the mitochondrial PT pore (mPTP). We directly tested the role of the c-subunit of ATP synthase in mPTP formation by measuring channel activity in c-subunit knockout mitochondria. We found that the classic mPTP conductance was lacking in c-subunit knockout mitochondria, but channels sensitive to the PT inhibitor cyclosporine A could be recorded. These channels had a significantly lower conductance compared with the cyclosporine A-sensitive channels detected in parental cells and were sensitive to the ATP/ADP translocase inhibitor bongkrekic acid. We propose that, in the absence of the c-subunit, mPTP cannot be formed, and a distinct cyclosporine A-sensitive low-conductance channel emerges.
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http://dx.doi.org/10.1016/j.celrep.2018.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521848PMC
January 2019

Nutritional Regulators of Bcl-xL in the Brain.

Molecules 2018 Nov 19;23(11). Epub 2018 Nov 19.

Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT 06520, USA.

B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic Bcl-2 protein found in the mitochondrial membrane. Bcl-xL is reported to support normal brain development and protects neurons against toxic stimulation during pathological process via its roles in regulation of mitochondrial functions. Despite promising evidence showing neuroprotective properties of Bcl-xL, commonly applied molecular approaches such as genetic manipulation may not be readily applicable for human subjects. Therefore, findings at the bench may be slow to be translated into treatments for disease. Currently, there is no FDA approved application that specifically targets Bcl-xL and treats brain-associated pathology in humans. In this review, we will discuss naturally occurring nutrients that may exhibit regulatory effects on Bcl-xL expression or activity, thus potentially providing affordable, readily-applicable, easy, and safe strategies to protect the brain.
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http://dx.doi.org/10.3390/molecules23113019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278276PMC
November 2018

Cardiac metabolic effects of K1.2 channel deletion and evidence for its mitochondrial localization.

FASEB J 2018 Jun 4:fj201800139R. Epub 2018 Jun 4.

Department of Anesthesiology and Perioperative Medicine, University of Rochester Medical Center, Rochester, New York, USA.

Controversy surrounds the molecular identity of mitochondrial K channels that are important for protection against cardiac ischemia-reperfusion injury. Although K1.2 (sodium-activated potassium channel encoded by Kcn2) is necessary for cardioprotection by volatile anesthetics, electrophysiological evidence for a channel of this type in mitochondria is lacking. The endogenous physiological role of a potential mito-K1.2 channel is also unclear. In this study, single channel patch-clamp of 27 independent cardiac mitochondrial inner membrane (mitoplast) preparations from wild-type (WT) mice yielded 6 channels matching the known ion sensitivity, ion selectivity, pharmacology, and conductance properties of K1.2 (slope conductance, 138 ± 1 pS). However, similar experiments on 40 preparations from Kcnt2 mice yielded no such channels. The K opener bithionol uncoupled respiration in WT but not Kcnt2 cardiomyocytes. Furthermore, when oxidizing only fat as substrate, Kcnt2 cardiomyocytes and hearts were less responsive to increases in energetic demand. Kcnt2 mice also had elevated body fat, but no baseline differences in the cardiac metabolome. These data support the existence of a cardiac mitochondrial K1.2 channel, and a role for cardiac K1.2 in regulating metabolism under conditions of high energetic demand.-Smith, C. O., Wang, Y. T., Nadtochiy, S. M., Miller, J. H., Jonas, E. A., Dirksen, R. T., Nehrke, K., Brookes, P. S. Cardiac metabolic effects of K1.2 channel deletion and evidence for its mitochondrial localization.
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http://dx.doi.org/10.1096/fj.201800139RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181635PMC
June 2018

BDNF signaling: Harnessing stress to battle mood disorder.

Proc Natl Acad Sci U S A 2018 04 28;115(15):3742-3744. Epub 2018 Mar 28.

Department of Internal Medicine, Yale University, New Haven, CT 06520

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http://dx.doi.org/10.1073/pnas.1803645115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899500PMC
April 2018

ΔN-Bcl-xL, a therapeutic target for neuroprotection.

Neural Regen Res 2017 Nov;12(11):1791-1794

Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT, USA.

The B-cell lymphoma-extra large (Bcl-xL) is a mitochondrial anti-apoptotic protein that plays a role in neuroprotection. However, during excitotoxic stimulation, Bcl-xL undergoes caspase-dependent cleavage and produces a fragmented form, ΔN-Bcl-xL. Accumulation of ΔN-Bcl-xL is associated with mitochondrial dysfunction and neuronal death. Therefore, strategies to inhibit the activity or formation of ΔN-Bcl-xL protect the brain against excitotoxic injuries. Our team found that the pharmacological inhibitor ABT-737 exerts dose dependent effects in primary neurons. When primary hippocampal neurons were treated with 1 μM ABT-737, glutamate-mediated mitochondrial damage and neuronal death were exacerbated, whereas 10 nM ABT-737, a 100-fold lower concentration, protected mitochondrial function and enhanced neuronal viability against glutamate toxicity. In addition, we suggested acute vs. prolonged formation of ΔN-Bcl-xL may have different effects on mitochondrial or neuronal functions. Unlike acute production of ΔN-Bcl-xL by glutamate, overexpression of ΔN-Bcl-xL did not cause drastic changes in neuronal viability. We predicted that neurons undergo adaptation and may activate altered metabolism to compensate for ΔN-Bcl-xL-mediated mitochondrial dysfunction. Although the detailed mechanism of ABT-mediated neurotoxicity neuroprotection is still unclear, our study shows that the mitochondrial membrane protein ΔN-Bcl-xL is a central target for interventions.
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http://dx.doi.org/10.4103/1673-5374.219033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745825PMC
November 2017

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Cell Death Differ 2018 03 11;25(3):542-572. Epub 2017 Dec 11.

Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.
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http://dx.doi.org/10.1038/s41418-017-0020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864235PMC
March 2018

Phylogenetic Profiling of Mitochondrial Proteins and Integration Analysis of Bacterial Transcription Units Suggest Evolution of F1Fo ATP Synthase from Multiple Modules.

J Mol Evol 2017 12 24;85(5-6):219-233. Epub 2017 Nov 24.

Division of Brain Sciences, Department of Medicine, Imperial College London, E508, Burlington Danes Hammersmith Hospital, DuCane Road, London, W12 0NN, UK.

ATP synthase is a complex universal enzyme responsible for ATP synthesis across all kingdoms of life. The F-type ATP synthase has been suggested to have evolved from two functionally independent, catalytic (F1) and membrane bound (Fo), ancestral modules. While the modular evolution of the synthase is supported by studies indicating independent assembly of the two subunits, the presence of intermediate assembly products suggests a more complex evolutionary process. We analyzed the phylogenetic profiles of the human mitochondrial proteins and bacterial transcription units to gain additional insight into the evolution of the F-type ATP synthase complex. In this study, we report the presence of intermediary modules based on the phylogenetic profiles of the human mitochondrial proteins. The two main intermediary modules comprise the αβ hexamer in the F1 and the c-subunit ring in the Fo. A comprehensive analysis of bacterial transcription units of F1Fo ATP synthase revealed that while a long and constant order of F1Fo ATP synthase genes exists in a majority of bacterial genomes, highly conserved combinations of separate transcription units are present among certain bacterial classes and phyla. Based on our findings, we propose a model that includes the involvement of multiple modules in the evolution of F1Fo ATP synthase. The central and peripheral stalk subunits provide a link for the integration of the F1/Fo modules.
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http://dx.doi.org/10.1007/s00239-017-9819-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709465PMC
December 2017

Inhibition of Bcl-xL prevents pro-death actions of ΔN-Bcl-xL at the mitochondrial inner membrane during glutamate excitotoxicity.

Cell Death Differ 2017 11 4;24(11):1963-1974. Epub 2017 Aug 4.

Department Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, CT 06511, USA.

ABT-737 is a pharmacological inhibitor of the anti-apoptotic activity of B-cell lymphoma-extra large (Bcl-xL) protein; it promotes apoptosis of cancer cells by occupying the BH3-binding pocket. We have shown previously that ABT-737 lowers cell metabolic efficiency by inhibiting ATP synthase activity. However, we also found that ABT-737 protects rodent brain from ischemic injury in vivo by inhibiting formation of the pro-apoptotic, cleaved form of Bcl-xL, ΔN-Bcl-xL. We now report that a high concentration of ABT-737 (1 μM), or a more selective Bcl-xL inhibitor WEHI-539 (5 μM) enhances glutamate-induced neurotoxicity while a low concentration of ABT-737 (10 nM) or WEHI-539 (10 nM) is neuroprotective. High ABT-737 markedly increased ΔN-Bcl-xL formation, aggravated glutamate-induced death and resulted in the loss of mitochondrial membrane potential and decline in ATP production. Although the usual cause of death by ABT-737 is thought to be related to activation of Bax at the outer mitochondrial membrane due to sequestration of Bcl-xL, we now find that low ABT-737 not only prevents Bax activation, but it also inhibits the decline in mitochondrial potential produced by glutamate toxicity or by direct application of ΔN-Bcl-xL to mitochondria. Loss of mitochondrial inner membrane potential is also prevented by cyclosporine A, implicating the mitochondrial permeability transition pore in death aggravated by ΔN-Bcl-xL. In keeping with this, we find that glutamate/ΔN-Bcl-xL-induced neuronal death is attenuated by depletion of the ATP synthase c-subunit. C-subunit depletion prevented depolarization of mitochondrial membranes in ΔN-Bcl-xL expressing cells and substantially prevented the morphological change in neurites associated with glutamate/ΔN-Bcl-xL insult. Our findings suggest that low ABT-737 or WEHI-539 promotes survival during glutamate toxicity by preventing the effect of ΔN-Bcl-xL on mitochondrial inner membrane depolarization, highlighting ΔN-Bcl-xL as an important therapeutic target in injured brain.
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http://dx.doi.org/10.1038/cdd.2017.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635221PMC
November 2017

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis.

Stroke 2017 04 1;48(4):1033-1043. Epub 2017 Mar 1.

From the Burke Medical Research Institute, White Plains, New York (M.Z., S.S.K., Y.C., R.R.R.); Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York (M.Z., S.S.K., Y.C., T.A.M., R.R.R.); Host Defense Discovery Performance Unit, Infectious Diseases Therapy Area Unit (P.J.G.) and Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area (J.B., J.F.), GlaxoSmithKline, Collegeville, PA; Laboratory of Neuroendocrinology, The Rockefeller University, New York (T.A.M.); and Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT (E.A.J.).

Background And Purpose: Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options.

Methods: We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy.

Results: Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro.

Conclusions: Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.
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http://dx.doi.org/10.1161/STROKEAHA.116.015609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613764PMC
April 2017

Erratum to: The Mitochondrial Permeability Transition Pore and ATP Synthase.

Handb Exp Pharmacol 2017 ;240:489

Division of Cardiology, Department of Pediatrics, University of Rochester Medical Center, 601 Elmwood Ave., Box 631, Rochester, 14642, NY, USA.

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http://dx.doi.org/10.1007/164_2016_87DOI Listing
January 2017

The Mitochondrial Permeability Transition Pore and ATP Synthase.

Handb Exp Pharmacol 2017 ;240:21-46

Division of Cardiology, Department of Pediatrics, University of Rochester Medical Center, 601 Elmwood Ave., Box 631, Rochester, 14642, NY, USA.

Mitochondrial ATP generation by oxidative phosphorylation combines the stepwise oxidation by the electron transport chain (ETC) of the reducing equivalents NADH and FADH with the generation of ATP by the ATP synthase. Recent studies show that the ATP synthase is not only essential for the generation of ATP but may also contribute to the formation of the mitochondrial permeability transition pore (PTP). We present a model, in which the PTP is located within the c-subunit ring in the F subunit of the ATP synthase. Opening of the PTP was long associated with uncoupling of the ETC and the initiation of programmed cell death. More recently, it was shown that PTP opening may serve a physiologic role: it can transiently open to regulate mitochondrial signaling in mature cells, and it is open in the embryonic mouse heart. This review will discuss how the ATP synthase paradoxically lies at the center of both ATP generation and cell death.
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http://dx.doi.org/10.1007/164_2016_5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439278PMC
December 2017

Physiological roles of the mitochondrial permeability transition pore.

J Bioenerg Biomembr 2017 Feb 11;49(1):13-25. Epub 2016 Feb 11.

Department Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT, USA.

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the FF ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.
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http://dx.doi.org/10.1007/s10863-016-9652-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981558PMC
February 2017

Decreased SGK1 Expression and Function Contributes to Behavioral Deficits Induced by Traumatic Stress.

PLoS Biol 2015 Oct 27;13(10):e1002282. Epub 2015 Oct 27.

Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, United States of America; Abraham Ribicoff Research Facilities, Connecticut Mental Health Center, New Haven, Connecticut, United States of America; VA National Center for PTSD, VA Connecticut Healthcare System, West Haven, Connecticut, United States of America.

Exposure to extreme stress can trigger the development of major depressive disorder (MDD) as well as post-traumatic stress disorder (PTSD). The molecular mechanisms underlying the structural and functional alterations within corticolimbic brain regions, including the prefrontal cortex (PFC) and amygdala of individuals subjected to traumatic stress, remain unknown. In this study, we show that serum and glucocorticoid regulated kinase 1 (SGK1) expression is down-regulated in the postmortem PFC of PTSD subjects. Furthermore, we demonstrate that inhibition of SGK1 in the rat medial PFC results in helplessness- and anhedonic-like behaviors in rodent models. These behavioral changes are accompanied by abnormal dendritic spine morphology and synaptic dysfunction. Together, the results are consistent with the possibility that altered SGK1 signaling contributes to the behavioral and morphological phenotypes associated with traumatic stress pathophysiology.
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http://dx.doi.org/10.1371/journal.pbio.1002282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623974PMC
October 2015

Cell death disguised: The mitochondrial permeability transition pore as the c-subunit of the F(1)F(O) ATP synthase.

Pharmacol Res 2015 Sep 5;99:382-92. Epub 2015 May 5.

Division of Brain Sciences, Department of Medicine, Imperial College London, UK.

Ion transport across the mitochondrial inner and outer membranes is central to mitochondrial function, including regulation of oxidative phosphorylation and cell death. Although essential for ATP production by mitochondria, recent findings have confirmed that the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane and cell death. This review will discuss recent advances in understanding the molecular components of mPTP, its regulatory mechanisms and how these contribute directly to its physiological as well as pathological roles.
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http://dx.doi.org/10.1016/j.phrs.2015.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4567435PMC
September 2015

Fluorescent Measurement of Synaptic Activity Using SynaptopHluorin in Isolated Hippocampal Neurons.

Bio Protoc 2014 Dec;4(23)

Department of Internal Medicine (Endocrinology), School of Medicine, Yale University, New Haven, USA; Department of Neurobiology, School of Medicine, Yale University, New Haven, USA.

This protocol comprises the entire process of fluorescent measurement of vesicle recycling using the probe SynaptopHluorin, a pH-dependent GFP variant whose fluorescence increases at the synapse upon vesicle release due to fluorescence quenching in acidic vesicles. This technique provides a genetic tool to monitor synaptic vesicle recycling in real time in cultured hippocampal neurons.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4950945PMC
http://dx.doi.org/10.21769/bioprotoc.1304DOI Listing
December 2014

Mitochondrial membrane protein Bcl-xL, a regulator of adult neuronal growth and synaptic plasticity: multiple functions beyond apoptosis.

Neural Regen Res 2014 Oct;9(19):1706-7

Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT, USA.

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http://dx.doi.org/10.4103/1673-5374.143413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238157PMC
October 2014

The mitochondrial complex V-associated large-conductance inner membrane current is regulated by cyclosporine and dexpramipexole.

Mol Pharmacol 2015 Jan 20;87(1):1-8. Epub 2014 Oct 20.

Department of Internal Medicine (K.N.A., P.Z., S.S., H.L., E.A.J.) and Department of Neurobiology (E.A.J.), Yale University School of Medicine, New Haven, Connecticut; Division of Brain Sciences, Department of Medicine, Imperial College London, London, United Kingdom (K.N.A.); Department of Neuroscience, Imaging and Clinical Sciences, University G.d'Annunzio of Chieti-Pescara, Chieti-Pescara, Italy (L.B.); Knopp Biosciences LLC, Pittsburgh, Pennsylvania (S.I.D., A.P.S., V.K.G.); and Biocurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts (P.J.S.S.)

Inefficiency of oxidative phosphorylation can result from futile leak conductance through the inner mitochondrial membrane. Stress or injury may exacerbate this leak conductance, putting cells, and particularly neurons, at risk of dysfunction and even death when energy demand exceeds cellular energy production. Using a novel method, we have recently described an ion conductance consistent with mitochondrial permeability transition pore (mPTP) within the c-subunit of the ATP synthase. Excitotoxicity, reactive oxygen species-producing stimuli, or elevated mitochondrial matrix calcium opens the channel, which is inhibited by cyclosporine A and ATP/ADP. Here we show that ATP and the neuroprotective drug dexpramipexole (DEX) inhibited an ion conductance consistent with this c-subunit channel (mPTP) in brain-derived submitochondrial vesicles (SMVs) enriched for F1FO ATP synthase (complex V). Treatment of SMVs with urea denatured extramembrane components of complex V, eliminated DEX- but not ATP-mediated current inhibition, and reduced binding of [(14)C]DEX. Direct effects of DEX on the synthesis and hydrolysis of ATP by complex V suggest that interaction of the compound with its target results in functional conformational changes in the enzyme complex. [(14)C]DEX bound specifically to purified recombinant b and oligomycin sensitivity-conferring protein subunits of the mitochondrial F1FO ATP synthase. Previous data indicate that DEX increased the efficiency of energy production in cells, including neurons. Taken together, these studies suggest that modulation of a complex V-associated inner mitochondrial membrane current is metabolically important and may represent an avenue for the development of new therapeutics for neurodegenerative disorders.
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http://dx.doi.org/10.1124/mol.114.095661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279080PMC
January 2015

Bcl-xL in neuroprotection and plasticity.

Front Physiol 2014 17;5:355. Epub 2014 Sep 17.

Division of Brain Sciences, Department of Medicine, Imperial College London London, UK.

Accepted features of neurodegenerative disease include mitochondrial and protein folding dysfunction and activation of pro-death factors. Neurons that experience high metabolic demand or those found in organisms with genetic mutations in proteins that control cell stress may be more susceptible to aging and neurodegenerative disease. In neurons, events that normally promote growth, synapse formation, and plasticity are also often deployed to control neurotoxicity. Such protective strategies are coordinated by master stress-fighting proteins. One such specialized protein is the anti-cell death Bcl-2 family member Bcl-xL, whose myriad death-protecting functions include enhancement of bioenergetic efficiency, prevention of mitochondrial permeability transition channel activity, protection from mitochondrial outer membrane permeabilization (MOMP) to pro-apoptotic factors, and improvement in the rate of vesicular trafficking. Synapse formation and normal neuronal activity provide protection from neuronal death. Therefore, Bcl-xL brings about synapse formation as a neuroprotective strategy. In this review we will consider how this multi-functional master regulator protein uses many strategies to enhance synaptic and neuronal function and thus counteracts neurodegenerative stimuli.
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http://dx.doi.org/10.3389/fphys.2014.00355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166110PMC
October 2014

An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore.

Proc Natl Acad Sci U S A 2014 Jul 16;111(29):10580-5. Epub 2014 Jun 16.

Section of Endocrinology, Department of Internal Medicine, Yale University, New Haven, CT 06520-8020;

Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca(2+)) dysregulation followed by rapid loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the FO of the F1FO ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca(2+) enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F1, providing a mechanism for mPTP opening. In contrast, recombinant F1 beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca(2+)-induced IMM depolarization and inhibits Ca(2+) and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.
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http://dx.doi.org/10.1073/pnas.1401591111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115574PMC
July 2014

Impaired import: how huntingtin harms.

Nat Neurosci 2014 Jun;17(6):747-9

Departments of Internal Medicine and Neurobiology, Yale University, New Haven, Connecticut, USA.

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http://dx.doi.org/10.1038/nn.3726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448962PMC
June 2014