Altered Calcium Homeostasis in Cells Transformed by Mitochondria from Individuals with Parkinson's Disease

Abstract: Parkinson's disease may be linked to defects in mitochondrial function. Mitochondrially transformed cells (cybrids) were created from Parkinson's disease patients or disease-free controls. Parkinson's disease cybrids had 26% less complex I activity, but maintained comparable basal calcium and energy levels. Parkinson's disease cybrids recovered from a carbachol-induced increase in cytosolic calcium 53% more slowly than controls even with lanthanum and thapsigargin blockade. Inhibition of complex I with the Parkinson's disease-inducing metabolite 1-methyl-4-phenylpyridinium (MPP⁺) similarly reduced the rate of recovery after carbachol. This MPP⁺-induced reduction in recovery rates was much more pronounced in control cybrids than in Parkinson's disease cybrids. Parkinson's disease cybrids had less carbonyl cyanide m -chlorophenylhydrazone-releasable calcium. Bypassing complex I with succinate partially restored Parkinson's disease cybrid, and MPP⁺ suppressed control cybrid recovery rates. The subtle alteration in calcium homeostasis of Parkinson's disease cybrids may reflect an increased susceptibility to cell death under circumstances not ordinarily toxic.     

Mitochondrial Dysfunction in Neurodegeneration

Numerous toxins are known to interfere with mitochondrial respiratory chain function. Use has been made of these in the development of pesticides and herbicides, and accidental use in man has led to the development of animal models for human disease. The propensity for mitochondrial toxins to induce neuronal cell death may well reflect not only their metabolic pathways but also the sensitivity of neurons to inhibition of oxidative phosphorylation. Thus, the accidental exposure of humans to l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine and to 3-nitropropionic acid has led to primate models of Parkinson's disease and Huntington's disease, respectively. These models were made all the more remarkable when identical biochemical deficiencies were identified in relevant areas of humans suffering from the respective idiopathic diseases. The place of complex I deficiency in Parkinson's disease remains undetermined, but there is recent evidence to suggest that, in some cases at least, it may play a primary role. The complex II/III deficiency in Huntington's disease is likely to be secondary and induced by other pathogenetic factors. The potential to intervene in the cascade of reactions involving mitochondrial dysfunction and cell death offers prospects for the development of new treatment strategies either for neuroprotection in prophylaxis or rescue.     

Cell death mechanisms in neurodegeneration

Progressive cell loss in specific neuronal populations often associated with typical cytoskeletal protein aggregations is a pathological hallmark of neurodegenerative disorders, but the nature, time course and molecular causes of cell death and their relation to cytoskeletal pathologies are still unresolved. Apoptosis or alternative pathways of cell death have been discussed in Alzheimer's disease and other neurodegenerative disorders. Apoptotic DNA fragmentation in human brain as a sign of neuronal injury is found too frequent as to account for continous neuron loss in these slowly progressive processes. Morphological studies revealed extremely rare apoptotic neuronal death in Alzheimer's disease but yielded mixed results for Parkinson's disease and other neurodegenerative disorders. Based on recent data in human brain, as well as in animal and cell culture models, a picture is beginning to emerge suggesting that, in addition to apoptosis, other forms of programmed cell death may participate in neurodegeneration. Better understanding of the molecular players will further elucidate the mechanisms of cell death in these disorders and their relations to cytoskeletal abnormalities. Susceptible cell populations in a proapoptotic environment show increased vulnerability towards multiple noxious factors discussed in the pathogenesis of neurodegeneration. In conclusion, although many in vivo and in vitro data are in favor of apoptosis involvement in neurodegenerative processes, there is considerable evidence that very complex events may contribute to neuronal death with possible repair mechanisms, the elucidation of which may prove useful for future prevention and therapy of neurodegenerative disorders.     

Involvement of Free Radicals in Excitotoxicity In Vivo

Abstract: Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N-tert -butyl-α-(2-sulfophenyl)-nitrone (S-PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N -methyl- d -aspartate (NMDA), kainic acid, and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). In a similar manner, striatal lesions produced by 1-methyl-4-phenylpyridinium (MPP⁺), malonate, and 3-acetylpyridine were significantly attenuated by either S-PBN or α-phenyl- N-tert -butylnitrone (PBN) treatment. Administration of S-PBN in combination with the NMDA antagonist MK-801 produced additive effects against malonate and 3-acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (^•OH) as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S-PBN, consistent with a free radical scavenging effect. S-PBN had no effects on malonate-induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

Calcium and neurodegeneration

When properly controlled, Ca2+ fluxes across the plasma membrane and between intracellular compartments play critical roles in fundamental functions of neurons, including the regulation of neurite outgrowth and synaptogenesis, synaptic transmission and plasticity, and cell survival. During aging, and particularly in neurodegenerative disorders, cellular Ca2+-regulating systems are compromised resulting in synaptic dysfunction, impaired plasticity and neuronal degeneration. Oxidative stress, perturbed energy metabolism and aggregation of disease-related proteins (amyloid beta-peptide, alpha-synuclein, huntingtin, etc.) adversely affect Ca2+ homeostasis by mechanisms that have been elucidated recently. Alterations of Ca2+-regulating proteins in the plasma membrane (ligand- and voltage-gated Ca2+ channels, ion-motive ATPases, and glucose and glutamate transporters), endoplasmic reticulum (presenilin-1, Herp, and ryanodine and inositol triphosphate receptors), and mitochondria (electron transport chain proteins, Bcl-2 family members, and uncoupling proteins) are implicated in age-related neuronal dysfunction and disease. The adverse effects of aging on neuronal Ca2+ regulation are subject to modification by genetic (mutations in presenilins, alpha-synuclein, huntingtin, or Cu/Zn-superoxide dismutase; apolipoprotein E isotype, etc.) and environmental (dietary energy intake, exercise, exposure to toxins, etc.) factors that may cause or affect the risk of neurodegenerative disease. A better understanding of the cellular and molecular mechanisms that promote or prevent disturbances in cellular Ca2+ homeostasis during aging may lead to novel approaches for therapeutic intervention in neurological disorders such as Alzheimer's and Parkinson's diseases and stroke.     

Mitochondrial calcium function and dysfunction in the central nervous system

The ability of isolated brain mitochondria to accumulate, store and release calcium has been extensively characterized. Extrapolation to the intact neuron led to predictions that the in situ mitochondria would reversibly accumulate Ca²⁺ when the concentration of the cation in the vicinity of the mitochondria rose above the ‘set-point’ at which uptake and efflux were in balance, storing Ca²⁺ as a complex with phosphate, and slowly releasing the cation when plasma membrane ion pumps lowered the cytoplasmic free Ca²⁺. Excessive accumulation of the cation was predicted to lead to activation of the permeability transition, with catastrophic consequences for the neuron. Each of these predictions has been confirmed with intact neurons, and there is convincing evidence for the permeability transition in cellular Ca²⁺ overload associated with glutamate excitotoxicity and stroke, while the neurodegenerative disease in which possible defects in mitochondrial Ca²⁺ handling have been most intensively investigated is Huntington's Disease. In this brief review evidence that mitochondrial Ca²⁺ transport is relevant to neuronal survival in these conditions will be discussed.     

Proteolytic mechanisms in necrotic cell death and neurodegeneration

Programmed neuronal cell death is required during development to achieve the accurate wiring of the nervous system. However, genetic or accidental factors can lead to the premature, non-programmed death of neurons during adult life. Inappropriate death of cells in the nervous system is the cause of multiple neurodegenerative disorders. Pathological neuronal death can occur by apoptosis, by necrosis or by a combination of both. Necrotic cell death underlies the pathology of devastating neurological diseases such as neurodegenerative disorders, stroke or trauma. However, little is known about the molecular mechanisms that bring about necrotic cell death. Proteases play crucial roles in neuron degeneration by exerting both regulatory and catabolic functions. Elevated intracellular calcium is the most ubiquitous feature of neuronal death with the concomitant activation of cysteine calcium-dependent proteases, calpains. Calpains and lysosomal, catabolic aspartyl proteases, play key roles in the necrotic death of neurons. In this review, we survey the recent literature on the role of cysteine and aspartyl proteases in necrosis and neurodegeneration, aiming to delineate common proteolytic mechanisms mediating cellular destruction.     

Cortical Cytochrome Oxidase Activity Is Reduced in Alzheimer's Disease

Abstract: A defect in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. In the present study, we examined the activities of the enzymes that catalyze oxidative phosphorylation in frontal, temporal, parietal, and occipital cortex from Alzheimer's disease patients and age-matched controls. Complex I and complex II–III activities showed a small decrease in occipital cortex, but were unaffected in the other cortical areas. The most consistent change was a significant decrease of cytochrome oxidase (complex IV) activity of 25–30% in the four cortical regions examined. These results provide further evidence of a cytochrome oxidase defect in Alzheimer's disease postmortem brain tissue. A deficiency in this key energy-metabolizing enzyme could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process.     

Entorhinal Cortex Lesions Transiently Alter Glucocorticoid but Not Mineralocorticoid Receptor Gene Expression in the Rat Hippocampus

Abstract: The mechanisms of delayed onset and cell death in Huntington's disease (HD) are unknown. One possibility is that a genetic defect in energy metabolism may result in slow excitotoxic neuronal death. Therefore, we examined the effects of age on striatal lesions produced by local administration of the mitochondrial toxin 3-nitropropionic acid in rats. In vivo chemical shift magnetic resonance imaging showed marked increases in striatal lactate concentrations that significantly correlated with increasing age. Histologic and neurochemical studies showed a striking age dependence of the lesions, with 4- and 12-month-old animals being much more susceptible than 1-month-old animals. Continuous systemic administration of low doses of 3-nitropropionic acid for 1 month resulted in striatal lesions showing growth-related changes in dendrites of striatal spiny neurons using the Golgi technique. These results show that a known mitochondrial toxin can produce selective axon-sparing striatal lesions showing both the age dependence and striatal spiny neuron dendritic changes that characterize HD.     

Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

The role of mitochondria in inherited neurodegenerative diseases

In the past decade, the genetic causes underlying familial forms of many neurodegenerative disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, hereditary spastic paraplegia, dominant optic atrophy, Charcot-Marie-Tooth type 2A, neuropathy ataxia and retinitis pigmentosa, and Leber's hereditary optic atrophy have been elucidated. However, the common pathogenic mechanisms of neuronal death are still largely unknown. Recently, mitochondrial dysfunction has emerged as a potential 'lowest common denominator' linking these disorders. In this review, we discuss the body of evidence supporting the role of mitochondria in the pathogenesis of hereditary neurodegenerative diseases. We summarize the principal features of genetic diseases caused by abnormalities of mitochondrial proteins encoded by the mitochondrial or the nuclear genomes. We then address genetic diseases where mutant proteins are localized in multiple cell compartments, including mitochondria and where mitochondrial defects are likely to be directly caused by the mutant proteins. Finally, we describe examples of neurodegenerative disorders where mitochondrial dysfunction may be 'secondary' and probably concomitant with degenerative events in other cell organelles, but may still play an important role in the neuronal decay. Understanding the contribution of mitochondrial dysfunction to neurodegeneration and its pathophysiological basis will significantly impact our ability to develop more effective therapies for neurodegenerative diseases.     

TRPC1 inhibits apoptotic cell degeneration induced by dopaminergic neurotoxin MPTP/MPP

Disturbances in Ca²⁺ homeostasis have been implicated in a variety of neuropathological conditions including Parkinson's disease (PD). However, the importance of store-operated Ca²⁺ entry (SOCE) channels in PD remains to be investigated. In the present study, we have scrutinized the significance of TRPC1 in 1-methyl-4-phenyl-1,2,3,6-tetrahyrdro-pyridine (MPTP)-induced PD using C57BL/6 animal model and PC12 cell culture model. Both sub-acute and sub-chronic treatments of MPTP significantly reduced TRPC1, and tyrosine hydroxylase levels, but not TRPC3, along with increased neuronal death. Furthermore, MPTP induces mitochondrial dysfunction, which was associated with reduced mitochondrial membrane potential, decreased level of Bcl₂, Bcl-xl, and an altered Bcl-xl/Bax ratio thereby initiating apoptosis. Importantly, TRPC1 overexpression in PC12 cells showed significant protection against MPP⁺ induced neuronal apoptosis, which was attributed to the restoration of cytosolic Ca²⁺ and preventing loss of mitochondrial membrane potential. Silencing of TRPC1 or addition of TRPC1 channel blockers decreased mitochondrial membrane potential, whereas activation of TRPC1 restored mitochondrial membrane potential in cells overexpressing TRPC1. TRPC1 overexpression also inhibited Bax translocation to the mitochondria and thereby prevented cytochrome c release and mitochondrial-mediated apoptosis. Overall, these results provide compelling evidence for the role of TRPC1 in either onset/progression of PD and restoration of TRPC1 levels could limit neuronal degeneration in MPTP mediated PD.     

Reduction of glyceraldehyde-3-phosphate dehydrogenase activity in Alzheimer's disease and in Huntington's disease fibroblasts

New functions have been identified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds specifically to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the beta-amyloid precursor protein and the huntingtin protein. However, the pathophysiological significance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole-cell sonicates of Alzheimer's and Huntington's disease fibroblasts. However, subcellular-specific GAPDH-protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole-cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH-protein distribution as a function of its subcellular localization in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age-matched controls. In the nuclear fraction, deficits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH:neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis.     

Antagonistic pleiotropy,mutation accumulation,and human genetic disease

The antagonistic pleiotropy theory of senescence is the most convincing theoretical explanation of the existence of aging. As yet, no locus or allele has been identified in a wild population with the features predicted by the pleiotropic theory. Human genetic diseases offer the opportunity to identify potentially pleiotropic alleles/loci. Four human genetic diseases—Huntington's disease, idiopathic hemochromatosis, myotonic dystrophy, and Alzheimer's disease—may exhibit pleiotropic effects and further study of these diseases might result in the identification of pleiotropic genes causing aging. Inability to find an early life selective benefit associated with these disease-causing alleles would favor the major alternative genetic explanation for aging, the mutation accumulation theory.     

Beneficial effects of dietary restriction on cerebral cortical synaptic terminals: preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid beta-peptide, iron, and 3-nitropropionic acid

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.     

研究细胞凋亡的新模式生物——酵母

细胞凋亡是受到严格调控的细胞自杀过程,凋亡机制从酵母到动物细胞高度保守.酵母细胞的凋亡过程虽发现较晚,但研究进展迅速.多个证据表明,酵母确实能发生细胞凋亡且细胞凋亡机制具有较高的保守性.酵母已成功用于发现新的细胞凋亡因子.近来,酵母还用作亨丁顿舞蹈症、帕金森氏病等凋亡相关疾病的细胞模型,为治愈这些疾病提供思路和指导·综述了酵母作为凋亡研究模式生物的可行性和独特的优势,其应用前景、存在的瓶颈问题及可能的解决方案.利用酵母为模式生物研究细胞凋亡和疾病发生,将大大加快发现新凋亡因子的过程,同时酵母作为凋亡相关疾病模式生物具有广阔的发展空间.     

Polycystin and calcium signaling in cell death and survival

Mutations in polycystin-1 (PC1) and polycystin-2 (PC2) result in a commonly occurring genetic disorder, called Autosomal Dominant Polycystic Kidney Disease (ADPKD), that is characterized by the formation and development of kidney cysts. Epithelial cells with loss-of-function of PC1 or PC2 show higher rates of proliferation and apoptosis and reduced autophagy. PC1 is a large multifunctional transmembrane protein that serves as a sensor that is usually found in complex with PC2, a calcium (Ca²⁺)-permeable cation channel. In addition to decreased Ca²⁺ signaling, several other cell fate-related pathways are de-regulated in ADPKD, including cAMP, MAPK, Wnt, JAK-STAT, Hippo, Src, and mTOR. In this review we discuss how polycystins regulate cell death and survival, highlighting the complexity of molecular cascades that are involved in ADPKD.