Mitochondrial uncoupling protein-4 regulates calcium homeostasis and sensitivity to store depletion-induced apoptosis in neural cells

An increase in the cytoplasmic-free Ca(2+) concentration mediates cellular responses to environmental signals that influence a range of processes, including gene expression, motility, secretion of hormones and neurotransmitters, changes in energy metabolism, and apoptosis. Mitochondria play important roles in cellular Ca(2+) homeostasis and signaling, but the roles of specific mitochondrial proteins in these processes are unknown. Uncoupling proteins (UCPs) are a family of proteins located in the inner mitochondrial membrane that can dissociate oxidative phosphorylation from respiration, thereby promoting heat production and decreasing oxyradical production. Here we show that UCP4, a neuronal UCP, influences store-operated Ca(2+) entry, a process in which depletion of endoplasmic reticulum Ca(2+) stores triggers Ca(2+) influx through plasma membrane "store-operated" channels. PC12 neural cells expressing human UCP4 exhibit reduced Ca(2+) entry in response to thapsigargin-induced endoplasmic reticulum Ca(2+) store depletion. The elevations of cytoplasmic and intramitochondrial Ca(2+) concentrations and mitochondrial oxidative stress induced by thapsigargin were attenuated in cells expressing UCP4. The stabilization of Ca(2+) homeostasis and preservation of mitochondrial function by UCP4 was correlated with reduced mitochondrial reactive oxygen species generation, oxidative stress, and Gadd153 up-regulation and increased resistance of the cells to death. Reduced Ca(2+)-dependent cytosolic phospholipase A2 activation and oxidative metabolism of arachidonic acid also contributed to the stabilization of mitochondrial function in cells expressing human UCP4. These findings demonstrate that UCP4 can regulate cellular Ca(2+) homeostasis, suggesting that UCPs may play roles in modulating Ca(2+) signaling in physiological and pathological conditions.     

Mitochondrial dysfunction in epilepsy

Mitochondrial dysfunction has been identified as one potential cause of epileptic seizures. Impaired mitochondrial function has been reported for the seizure focus of patients with temporal lobe epilepsy and Ammon's horn sclerosis and of adult and immature animal models of epilepsy. Since mitochondrial oxidative phosphorylation provides the major source of ATP in neurons and mitochondria participate in cellular Ca(2+) homeostasis and generation of reactive oxygen species, their dysfunction strongly affects neuronal excitability and synaptic transmission. Therefore, mitochondrial dysfunction is proposed to be highly relevant for seizure generation. Additionally, mitochondrial dysfunction is known to trigger neuronal cell death, which is a prominent feature of therapy-resistant epilepsy. For this reason mitochondria have to be considered as promising targets for neuroprotective strategies in epilepsy.     

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death

J. Neurochem. (2012) 122, 1118-1128. ABSTRACT: P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.     

线粒体功能的检测方法

线粒体为细胞内的能量工厂,对细胞的增殖、分化、存活以及稳态的维持起着重要的调节作用。线粒体功能障碍与机体生长、发育异常、认知发生障碍以及多种器官病变密切相关。线粒体形态、结构和功能的检测对于了解线粒体的稳态以及功能状态有着重要意义,线粒体的功能状态与线粒体膜电位、线粒体膜通道、线粒体Ca2+浓度、ATP生成、呼吸链复合体活性、活性氧生成以及DNA突变密切相关。本文就线粒体形态、结构和功能的检测方法及其研究进展进行了综述。     

Neuronal and glial calcium signaling in Alzheimer's disease

Cognitive impairment and emotional disturbances in Alzheimer's disease (AD) result from the degeneration of synapses and death of neurons in the limbic system and associated regions of the cerebral cortex. An alteration in the proteolytic processing of the amyloid precursor protein (APP) results in increased production and accumulation of amyloid beta-peptide (Abeta) in the brain. Abeta has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and apoptosis by a mechanism involving disruption of cellular calcium homeostasis. By inducing membrane lipid peroxidation and generation of the aldehyde 4-hydroxynonenal, Abeta impairs the function of membrane ion-motive ATPases and glucose and glutamate transporters, and can enhance calcium influx through voltage-dependent and ligand-gated calcium channels. Reduced levels of a secreted form of APP which normally regulates synaptic plasticity and cell survival may also promote disruption of synaptic calcium homeostasis in AD. Some cases of inherited AD are caused by mutations in presenilins 1 and 2 which perturb endoplasmic reticulum (ER) calcium homeostasis such that greater amounts of calcium are released upon stimulation, possibly as the result of alterations in IP(3) and ryanodine receptor channels, Ca(2+)-ATPases and the ER stress protein Herp. Abnormalities in calcium regulation in astrocytes, oligodendrocytes, and microglia have also been documented in studies of experimental models of AD, suggesting contributions of these alterations to neuronal dysfunction and cell death in AD. Collectively, the available data show that perturbed cellular calcium homeostasis plays a prominent role in the pathogenesis of AD, suggesting potential benefits of preventative and therapeutic strategies that stabilize cellular calcium homeostasis.     

Mitochondrial dysfunction and glutamate excitotoxicity studied in primary neuronal cultures

Primary dissociated neuronal cultures have been intensively exploited for the past 15 years as model systems to investigate excitotoxic neuronal degeneration. Even this simplified system contains a complex web of interactions between calcium homeostasis, ATP production and the generation and detoxification of reactive oxygen species. There is increasing realization that the mitochondrion occupies the center stage in these processes. This review covers the normal bioenergetics of the cultured neuron, the ways in which mitochondrial dysfunction impacts upon the ability of the neuron to withstand excitotoxic stress, the nature of the stresses imposed by NMDA receptor activation and possible molecular mechanisms of excitotoxic cell death.     

Contributions of Bcl-xL to acute and long term changes in bioenergetics during neuronal plasticity

Mitochondria manufacture and release metabolites and manage calcium during neuronal activity and synaptic transmission, but whether long term alterations in mitochondrial function contribute to the neuronal plasticity underlying changes in organism behavior patterns is still poorly understood. Although normal neuronal plasticity may determine learning, in contrast a persistent decline in synaptic strength or neuronal excitability may portend neurite retraction and eventual somatic death. Anti-death proteins such as Bcl-xL not only provide neuroprotection at the neuronal soma during cell death stimuli, but also appear to enhance neurotransmitter release and synaptic growth and development. It is proposed that Bcl-xL performs these functions through its ability to regulate mitochondrial release of bioenergetic metabolites and calcium, and through its ability to rapidly alter mitochondrial positioning and morphology. Bcl-xL also interacts with proteins that directly alter synaptic vesicle recycling. Bcl-xL translocates acutely to sub-cellular membranes during neuronal activity to achieve changes in synaptic efficacy. After stressful stimuli, pro-apoptotic cleaved delta N Bcl-xL (ΔN Bcl-xL) induces mitochondrial ion channel activity leading to synaptic depression and this is regulated by caspase activation. During physiological states of decreased synaptic stimulation, loss of mitochondrial Bcl-xL and low level caspase activation occur prior to the onset of long term decline in synaptic efficacy. The degree to which Bcl-xL changes mitochondrial membrane permeability may control the direction of change in synaptic strength. The small molecule Bcl-xL inhibitor ABT-737 has been useful in defining the role of Bcl-xL in synaptic processes. Bcl-xL is crucial to the normal health of neurons and synapses and its malfunction may contribute to neurodegenerative disease. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Mitochondria and neuroplasticity

The production of neurons from neural progenitor cells, the growth of axons and dendrites and the formation and reorganization of synapses are examples of neuroplasticity. These processes are regulated by cell-autonomous and intercellular (paracrine and endocrine) programs that mediate responses of neural cells to environmental input. Mitochondria are highly mobile and move within and between subcellular compartments involved in neuroplasticity (synaptic terminals, dendrites, cell body and the axon). By generating energy (ATP and NAD⁺), and regulating subcellular Ca²⁺ and redox homoeostasis, mitochondria may play important roles in controlling fundamental processes in neuroplasticity, including neural differentiation, neurite outgrowth, neurotransmitter release and dendritic remodelling. Particularly intriguing is emerging data suggesting that mitochondria emit molecular signals (e.g. reactive oxygen species, proteins and lipid mediators) that can act locally or travel to distant targets including the nucleus. Disturbances in mitochondrial functions and signalling may play roles in impaired neuroplasticity and neuronal degeneration in Alzheimer''s disease, Parkinson''s disease, psychiatric disorders and stroke.     

Prostate Apoptosis Response-4 Production in Synaptic Compartments Following Apoptotic and Excitotoxic Insults

Synapses are often located at great distances from the cell body and so must be capable of transducing signals into both local and distant responses. Although progress has been made in understanding biochemical cascades involved in neuronal death during development of the nervous system and in various neurodegenerative disorders, it is not known whether such cascades function locally in synaptic compartments. Prostate apoptosis response-4 (Par-4) is a leucine zipper and death domain-containing protein that plays a role in neuronal apoptosis. We now report that Par-4 levels are rapidly increased in cortical synaptosomes and in dendrites of hippocampal neurons in culture and in vivo, following exposure to apoptotic or excitotoxic insults. Par-4 expression is regulated at the translational level within synaptic compartments. Par-4 antisense treatment suppressed mitochondrial dysfunction and caspase activation in synaptosomes and prevented death of cultured hippocampal neurons following exposure to excitotoxic and apoptotic insults. Local translational regulation of death-related proteins in synaptic compartments may play a role in programmed cell death, adaptive remodeling of synapses, and neurodegenerative disorders.     

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.     

The role of synaptic microRNAs in Alzheimer's disease

Structurally and functionally active synapses are essential for neurotransmission and for maintaining normal synaptic and cognitive functions. Researchers have found that synaptic dysfunction is associated with the onset and progression of neurodegenerative diseases, such as Alzheimer's disease (AD), and synaptic dysfunction is even one of the main physiological hallmarks of AD. MiRNAs are present in small, subcellular compartments of the neuron such as neural dendrites, synaptic vesicles, and synaptosomes are known as synaptic miRNAs. Synaptic miRNAs involved in governing multiple synaptic functions that lead to healthy brain functioning and synaptic activity. However, the precise role of synaptic miRNAs has not been determined in AD progression. This review emphasizes the presence of miRNAs at the synapse, synaptic compartments and roles of miRNAs in multiple synaptic functions. We focused on synaptic miRNAs alteration in AD, and how the modulation of miRNAs effect the synaptic functions in AD. We also discussed the impact of synaptic miRNAs in AD progression concerning the synaptic ATP production, mitochondrial function, and synaptic activity.     

Cardiolipin in Central Nervous System Physiology and Pathology

Cardiolipin, an anionic phospholipid found primarily in the inner mitochondrial membrane, has many well-defined roles within the peripheral tissues, including the maintenance of mitochondrial membrane fluidity and the regulation of mitochondrial functions. Within the central nervous system (CNS), cardiolipin is found within both neuronal and non-neuronal glial cells, where it regulates metabolic processes, supports mitochondrial functions, and promotes brain cell viability. Furthermore, cardiolipin has been shown to act as an elimination signal and participate in programmed cell death by apoptosis of both neurons and glia. Since cardiolipin is associated with regulating brain homeostasis, the modification of its structure, or even a decrease in the overall levels of cardiolipin, can result in mitochondrial dysfunction, which is a characteristic feature of many diseases. In this review, we outline the various functions of cardiolipin within the cells of the CNS, including neurons, astrocytes, microglia, and oligodendrocytes. In addition, we discuss the role cardiolipin may play in the pathogenesis of the neurodegenerative disorders Alzheimer’s disease and Parkinson’s disease, as well as traumatic brain injury.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.     

Mitochondrial uncoupling proteins in the CNS: in support of function and survival

Mitochondrial uncoupling mediated by uncoupling protein 1 (UCP1) is classically associated with non-shivering thermogenesis by brown fat. Recent evidence indicates that UCP family proteins are also present in selected neurons. Unlike UCP1, these proteins (UCP2, UCP4 and BMCP1/UCP5) are not constitutive uncouplers and are not crucial for non-shivering thermogenesis. However, they can be activated by free radicals and free fatty acids, and their activity has a profound influence on neuronal function. By regulating mitochondrial biogenesis, calcium flux, free radical production and local temperature, neuronal UCPs can directly influence neurotransmission, synaptic plasticity and neurodegenerative processes. Insights into the regulation and function of these proteins offer unsuspected avenues for a better understanding of synaptic transmission and neurodegeneration.     

Peroxisome proliferator-activated receptor alpha (PPARalpha) activators, bezafibrate and Wy-14,643, increase uncoupling protein-3 mRNA levels without modifying the mitochondrial membrane potential in primary culture of rat preadipocytes

Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which act as pores for H(+) ions, dissipating the electrochemical gradient that develops during mitochondrial respiration at the expense of ATP synthesis. We have studied the effects of two fibrates, bezafibrate and Wy-14,643, on UCP-3 and UCP-2 mRNA levels in primary monolayer cultures of rat adipocytes and undifferentiated preadipocytes. Treatment with both PPARalpha activators for 24 h up-regulated UCP-3 mRNA levels. Thus, bezafibrate treatment resulted in an 8-fold induction in UCP-3 mRNA levels in preadipocytes compared with the 3.5-fold induction observed in adipocytes. Differences in the induction of UCP-3 between these cells correlated well with the higher expression of PPARalpha and RXRalpha mRNA values in preadipocytes compared to adipocytes. Wy-14,643 caused similar effects on UCP-3 mRNA expression. In contrast to UCP-3, UCP-2 mRNA levels were only slightly modified by bezafibrate in adipocytes. The induction in UCP-3 expression was not accompanied by changes in the mitochondrial membrane potential of rat primary preadipocytes after bezafibrate or Wy-14,643 treatment. Since it has been proposed that UCP-3 could be involved in the regulation of the use of fatty acids as fuel substrates, the UCP-3 induction achieved after bezafibrate and Wy-14, 643 treatment may indicate a higher oxidation of fatty acids, limiting their availability to be stored as triglycerides. This change may result in a reduced rate of conversion of preadipocytes to adipocytes, which directly affects fat depots.     

Mitochondria and the regulation of free radical damage in the eye

Neuronal cell death can be determined by the overall level of reactive oxygen species (ROS) resulting from the combination of extrinsic sources and intrinsic production as a byproduct of oxidative phosphorylation. Key controllers of the intrinsic production of ROS are the mitochondrial uncoupling proteins (UCPs). By allowing a controlled leak of protons across the inner mitochondrial membrane activation of these proteins can decrease ROS and promote cell survival. In both primate models of Parkinson’s disease and mouse models of seizures, increased activity of UCP2 significantly increased neuronal cells survival. In the retina UCP2 is expressed in many neurons and glial cells, but was not detected in rod photoreceptors. Retinal ganglion cell survival following excitotoxic damage was much greater in animals overexpressing UCP2. Traditional Chinese medicines, such as an extract of Cistanche tubulosa, may provide benefit by altering mitochondrial metabolism.     

Control of Mitochondrial pH by Uncoupling Protein 4 in Astrocytes Promotes Neuronal Survival

Brain activity is energetically costly and requires a steady and highly regulated flow of energy equivalents between neural cells. It is believed that a substantial share of cerebral glucose, the major source of energy of the brain, will preferentially be metabolized in astrocytes via aerobic glycolysis. The aim of this study was to evaluate whether uncoupling proteins (UCPs), located in the inner membrane of mitochondria, play a role in setting up the metabolic response pattern of astrocytes. UCPs are believed to mediate the transmembrane transfer of protons, resulting in the uncoupling of oxidative phosphorylation from ATP production. UCPs are therefore potentially important regulators of energy fluxes. The main UCP isoforms expressed in the brain are UCP2, UCP4, and UCP5. We examined in particular the role of UCP4 in neuron-astrocyte metabolic coupling and measured a range of functional metabolic parameters including mitochondrial electrical potential and pH, reactive oxygen species production, NAD/NADH ratio, ATP/ADP ratio, CO₂ and lactate production, and oxygen consumption rate. In brief, we found that UCP4 regulates the intramitochondrial pH of astrocytes, which acidifies as a consequence of glutamate uptake, with the main consequence of reducing efficiency of mitochondrial ATP production. The diminished ATP production is effectively compensated by enhancement of glycolysis. This nonoxidative production of energy is not associated with deleterious H₂O₂ production. We show that astrocytes expressing more UCP4 produced more lactate, which is used as an energy source by neurons, and had the ability to enhance neuronal survival.     

IDH2 deficiency promotes mitochondrial dysfunction and dopaminergic neurotoxicity: implications for Parkinson’s disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and its pathogenesis is under intense investigation. Substantial evidence indicates that mitochondrial dysfunction and oxidative stress play central roles in the pathophysiology of PD, through activation of mitochondria-dependent apoptotic molecular pathways. Several mitochondrial internal regulating factors act to maintain mitochondrial function. However, the mechanism by which these internal regulating factors contribute to mitochondrial dysfunction in PD remains elusive. One of these factors, mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2), has been implicated in the regulation of mitochondrial redox balance and reduction of oxidative stress-induced cell injury. Here we report that IDH2 regulates mitochondrial dysfunction and cell death in MPP⁺/MPTP-induced DA neuronal cells, and in a mouse model of PD. Down-regulation of IDH2 increased DA neuron sensitivity to MPP⁺; lowered IDH2 levels facilitated induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Deficient IDH2 also promoted loss of DA SNpc neurons in an MPTP mouse model of PD. Interestingly, Mito-TEMPO, a mitochondrial ROS-specific scavenger, protected degeneration of SNpc DA neurons in the MPTP model of PD. These findings demonstrate that IDH2 contributes to degeneration of the DA neuron in the neurotoxin model of PD and establish IDH2 as a molecular target of potential therapeutic significance for this disabling neurological illness.     

Protective effects of R-alpha-lipoic acid and acetyl-L-carnitine in MIN6 and isolated rat islet cells chronically exposed to oleic acid

Mitochondrial dysfunction due to oxidative stress and concomitant impaired beta-cell function may play a key role in type 2 diabetes. Preventing and/or ameliorating oxidative mitochondrial dysfunction with mitochondria-specific nutrients may have preventive or therapeutic potential. In the present study, the oxidative mechanism of mitochondrial dysfunction in pancreatic beta-cells exposed to sublethal levels of oleic acid (OA) and the protective effects of mitochondrial nutrients [R-alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC)] were investigated. Chronic exposure (72 h) of insulinoma MIN6 cells to OA (0.2-0.8 mM) increased intracellular oxidant formation, decreased mitochondrial membrane potential (MMP), enhanced uncoupling protein-2 (UCP-2) mRNA and protein expression, and consequently, decreased glucose-induced ATP production and suppressed glucose-stimulated insulin secretion. Pretreatment with LA and/or ALC reduced oxidant formation, increased MMP, regulated UCP-2 mRNA and protein expression, increased glucose-induced ATP production, and restored glucose-stimulated insulin secretion. The key findings on ATP production and insulin secretion were verified with isolated rat islets. These results suggest that mitochondrial dysfunction is involved in OA-induced pancreatic beta-cell dysfunction and that pretreatment with mitochondrial protective nutrients could be an effective strategy to prevent beta-cell dysfunction.