Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death

Mitochondria are critically involved in necrotic cell death induced by Ca(2+) overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore - a protein complex that spans both the outer and inner mitochondrial membranes - is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix. Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1-, Vdac3-, and Vdac1-Vdac3-null mice exhibited a Ca(2+)- and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca(2+)- and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1-Vdac3 and Vdac1-Vdac2-Vdac3. Wild-type and Vdac-deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.     

Full-length expanded ataxin-3 enhances mitochondrial-mediated cell death and decreases Bcl-2 expression in human neuroblastoma cells

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. An unstable CAG trinucleotide repeat expansion in MJD gene on long arm of chromosome 14 has been identified as the pathologic mutation of MJD and apoptosis was previously shown to be responsible for the neuronal cell death of the disease. In this study, we utilized human neuronal SK-N-SH cells stably transfected with HA-tagged full-length MJD with 78 polyglutamine repeats to examine the effects of polyglutamine expansion on neuronal cell survival in the early stage of disease. Various pro-apoptotic agents were used to assess the tolerance of the mutant cells and to compare the differences between cells with and without mutant ataxin-3. Concentration- and time-dependent experiments showed that the increase in staurosporine-induced cell death was more pronounced and accelerated in cells containing expanded ataxin-3 via MTS assays. Interestingly, under basal conditions, Western blot and immunocytochemical analyses showed a significant decrease of Bcl-2 protein expression and an increase of cytochrome c in cells containing expanded ataxin-3 when compared with those of the parental cells. The same reduction of Bcl-2 was further confirmed in fibroblast cells with mutant ataxin-3. In addition, exogenous expression of Bcl-2 desensitized SK-N-SH-MJD78 cells to poly-Q toxicity. These results indicated that mitochondrial-mediated cell death plays a role in the pathogenesis of MJD. In our cellular model, full-length expanded ataxin-3 that leads to neurodegenerative disorders significantly impaired the expression of Bcl-2 protein, which may be, at least in part, responsible for the weak tolerance to polyglutamine toxicity at the early stage of disease and ultimately resulted in an increase of stress-induced cell death upon apoptotic stress.     

Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 °C and the Q₁₀ value of 5.6. After 1 h exposure to acidic solution at 37 °C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 °C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.     

Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 °C and the Q₁₀ value of 5.6. After 1 h exposure to acidic solution at 37 °C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 °C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.     

Induced pluripotent stem cells from patients with Huntington's disease show CAG-repeat-expansion-associated phenotypes

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.     

From neuronal inclusions to neurodegeneration: neuropathological investigation of a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease caused by the expansion of a polyglutamine repeat sequence within a novel protein. Recent work has shown that abnormal intranuclear inclusions of aggregated mutant protein within neurons is a characteristic feature shared by HD and several other diseases involving glutamine repeat expansion. This suggests that in each of the these disorders the affected nerve cells degenerate as a result of these abnormal inclusions. A transgenic mouse model of HD has been generated by introducing exon 1 of the HD gene containing a highly expanded CAG sequence into the mouse germline. These mice develop widespread neuronal intranuclear inclusions and neurodegeneration specifically within those areas of the brain known to degenerate in HD. We have investigated the sequence of pathological changes that occur after the formation of nuclear inclusions and that precede neuronal cell death in these cells. Although the relation between inclusion formation and neurodegeneration has recently been questioned, a full characterization of the pathways linking protein aggregation and cell death will resolve some of these controversies and will additionally provide new targets for potential therapies.     

Does the voltage dependent anion channel modulate cardiac ischemia–reperfusion injury?

The voltage dependent anion channel (VDAC) provides exchange of metabolites, anions, and cations across the outer mitochondrial membrane. VDAC provides substrates and adenine nucleotides necessary for electron transport and therefore plays a key role in regulating mitochondrial bioenergetics. VDAC has also been suggested to regulate the response to cell death signaling. Emerging data show that VDAC is regulated by protein–protein interactions as well as by post-translational modifications. This review will focus on the regulation of VDAC and its potential role in regulating cell death in cardiac ischemia–reperfusion. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation is prevented by cycloheximide, 12-O-tetradecanoylphorbol-13-acetate, or vanadate

Serum-free mouse embryo cells cultured in medium supplemented with insulin, transferrin, high-density lipoprotein, and fibronectin are dependent on epidermal growth factor for survival. Cycloheximide or actinomycin D prevented cell death caused by growth factor deprivation, suggesting that cell death required the synthesis of RNA and protein, a phenomenon similar to that reported for neuronal cell death in the absence of nerve growth factor. Orthovanadate, an inhibitor of phosphotyrosine phosphatases, and 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, also prevented serum-free mouse embryo cell death in the absence of epidermal growth factor.     

Genetic strategies for dissecting mammalian and Drosophila voltage-dependent anion channel functions

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are a family of small pore-forming proteins of the mitochondrial outer membrane that are found in all eukaryotes. VDACs are thought to play important roles in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, in overall energy metabolism via interactions with cytosolic kinases, and a debated role in programmed cell death (apoptosis). The mammalian genome contains three VDAC loci termed Vdac1, Vdac2, and Vdac3, raising the question as to what function each isoform may be performing. Based upon expression studies of the mouse VDACs in yeast, biophysical differences can be identified but the physiologic significance of these differences remains unclear. Creation of “knockout” cell lines and mice that lack one or more VDAC isoforms has led to the characterization of distinct phenotypes that provide a different set of insights into function which must be interpreted in the context of complex physiologic systems. Functions in male reproduction, the central nervous system and glucose homeostasis have been identified and require a deeper and more mechanistic examination. Annotation of the genome sequence of Drosophila melanogaster has recently revealed three additional genes (CG17137, CG17139, CG17140) with homology to porin, the previously described gene that encodes the VDAC of D. melanogaster. Molecular analysis of these novel VDACs has revealed a complex pattern of gene organization and expression. Sequence comparisons with other insect VDAC homologs suggest that this gene family evolved through a mechanism of duplication and divergence from an ancestral VDAC gene during the radiation of the genus Drosophila. Striking similarities to mouse VDAC mutants can be found that emphasize the conservation of function over a long evolutionary time frame.     

Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis.

Most, but not all, cell types release intracellular organic solutes (e.g. taurine) in response to cell swelling to achieve cell volume regulation. Although this efflux is blocked by classical inhibitors of the electroneutral anion exchanger band 3 (AE1), it is thought to involve an anion channel. The role of band 3 in volume-dependent taurine transport was determined by expressing, in Xenopus oocytes, band 3 from erythrocytes which do (trout) or do not (mouse) release taurine when swollen. AE1 of both species elicited anion exchange activity, but only trout band 3 showed chloride channel activity and taurine transport. Chimeras constructed from trout and mouse band 3 allowed the identification of some protein domains critically associated with channel activity and taurine transport. The data provide evidence that swelling-induced taurine movements occur via an anion channel which is dependent on, or controlled by, band 3. They suggest the involvement of proteins of the band 3 (AE) family in cell volume regulation.