Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.     

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.     

Mitochondrial pathobiology in ALS

Amyotrophic lateral sclerosis (ALS) is the third most common human adult-onset neurodegenerative disease. Some forms of ALS are inherited, and disease-causing genes have been identified. Nevertheless, the mechanisms of neurodegeneration in ALS are unresolved. Genetic, biochemical, and morphological analyses of human ALS as well as cell and animal models of ALS reveal that mitochondria could have roles in this neurodegeneration. The varied functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations. Changes occur in mitochondrial respiratory chain enzymes and mitochondrial programmed cell death proteins in ALS. Transgenic mouse models of ALS reveal possible principles governing the biology of neurodegeneration that implicate mitochondria and the mitochondrial permeability transition pore. This paper reviews how mitochondrial pathobiology might contribute to the mechanisms of neurodegeneration in ALS.     

Lysyl-tRNA synthetase is a target for mutant SOD1 toxicity in mitochondria

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS.     

Polyglutamine tract-binding protein-1 dysfunction induces cell death of neurons through mitochondrial stress

Polyglutamine tract-binding protein-1 (PQBP-1) is a nuclear protein that interacts and colocalizes with mutant polyglutamine proteins. We previously reported that PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype and cell death of motor neurons analogous to human neurodegeneration. To investigate the molecular mechanisms underlying the motor neuron death, we performed microarray analyses using the anterior horn tissues of the spinal cord and compared gene expression profiles between pre-symptomatic transgenic and age-matched control mice. Surprisingly, half of the spots changed more than 1.5-fold turned out to be genes transcribed from the mitochondrial genome. Northern and western analyses confirmed up-regulation of representative mitochondrial genes, cytochrome c oxidase (COX) subunit 1 and 2. Immunohistochemistry revealed that COX1 and COX2 proteins are increased in spinal motor neurons. Electron microscopic analyses revealed morphological abnormalities of mitochondria in the motor neurons. PQBP-1 overexpression in primary neurons by adenovirus vector induced abnormalities of mitochondrial membrane potential from day 5, while cytochrome c release and caspase 3 activation were observed on day 9. An increase of cell death by PQBP-1 was also confirmed on day 9. Collectively, these results indicate that dysfunction of PQBP-1 induces mitochondrial stress, a key molecular pathomechanism that is shared among human neurodegenerative disorders.     

Mitochondrial permeability transition pore in Alzheimer's disease: Cyclophilin D and amyloid beta

Amyloid beta (Aβ) plays a critical role in the pathophysiology of Alzheimer's disease. Increasing evidence indicates mitochondria as an important target of Aβ toxicity; however, the effects of Aβ toxicity on mitochondria have not yet been fully elucidated. Recent biochemical studies in vivo and in vitro implicate mitochondrial permeability transition pore (mPTP) formation involvement in Aβ-mediated mitochondrial dysfunction. mPTP formation results in severe mitochondrial dysfunction such as reactive oxygen species (ROS) generation, mitochondrial membrane potential dissipation, intracellular calcium perturbation, decrease in mitochondrial respiration, release of pro-apoptotic factors and eventually cell death. Cyclophilin D (CypD) is one of the more well-known mPTP components and recent findings reveal that Aβ has significant impact on CypD-mediated mPTP formation. In this review, the role of Aβ in the formation of mPTP and the potential of mPTP inhibition as a therapeutic strategy in AD treatment are examined.     

Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration

A dinucleotide deletion in human ubiquitin (Ub) B messenger RNA leads to formation of polyubiquitin (UbB)+1, which has been implicated in neuronal cell death in Alzheimer's and other neurodegenerative diseases. Previous studies demonstrate that UbB+1 protein causes proteasome dysfunction. However, the molecular mechanism of UbB+1-mediated neuronal degeneration remains unknown. We now report that UbB+1 causes neuritic beading, impairment of mitochondrial movements, mitochondrial stress and neuronal degeneration in primary neurons. Transfection of UbB+1 induced a buildup of mitochondria in neurites and dysregulation of mitochondrial motor proteins, in particular, through detachment of P74, the dynein intermediate chain, from mitochondria and decreased mitochondria-microtubule interactions. Altered distribution of mitochondria was associated with activation of both the mitochondrial stress and p53 cell death pathways. These results support the hypothesis that neuritic clogging of mitochondria by UbB+1 triggers a cascade of events characterized by local activation of mitochondrial stress followed by global cell death. Furthermore, UbB+1 small interfering RNA efficiently blocked expression of UbB+1 protein, attenuated neuritic beading and preserved cellular morphology, suggesting a potential neuroprotective strategy for certain neurodegenerative disorders.     

Mitochondrial dysfunction and its role in motor neuron degeneration in ALS

Mitochondria play a pivotal role in many metabolic and apoptotic pathways that regulate the life and death of cells. Accumulating evidence suggests that mitochondrial dysfunction is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mitochondrial dysfunction may cause motor neuron death by predisposing them to calcium-mediated excitotoxicity, by increasing generation of reactive oxygen species, and by initiating the intrinsic apoptotic pathway. Morphological and biochemical mitochondrial abnormalities have been described in sporadic human ALS cases, but the implications of these findings in terminally ill individuals or in post-mortem tissues are difficult to decipher. However, remarkable mitochondrial abnormalities have also been identified in transgenic mouse models of familial ALS expressing mutant Cu, Zn superoxide dismutase (SOD1). Detailed studies in these mouse models indicate that mitochondrial abnormalities begin prior to the clinical and pathological onset of the disease, suggesting that mitochondrial dysfunction may be causally involved in the pathogenesis of ALS. Although the mechanisms whereby mutant SOD1 damages mitochondria remain to be fully understood, the finding that a portion of mutant SOD1 is localized in mitochondria, where it forms aberrant aggregates and protein interactions, has opened a number of avenues of investigation. The future challenges are to devise models to better understand the effects of mutant SOD1 in mitochondria and the relative contribution of mitochondrial dysfunction to the pathogenesis of ALS, as well as to identify therapeutic approaches that target mitochondrial dysfunction and its consequences.     

Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.     

Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset

Mutations in superoxide dismutase (SOD1) are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.     

Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis

Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial swelling and vacuolization are among the earliest pathologic features found in two strains of transgenic amyotrophic lateral sclerosis (ALS) mice with SOD1 mutations. Mice with the G93A human SOD1 mutation have altered electron transport enzymes, and expression of the mutant enzyme in vitro results in a loss of mitochondrial membrane potential and elevated cytosolic calcium concentration. Mitochondrial dysfunction may lead to ATP depletion, which may contribute to cell death. If this is true, then buffering intracellular energy levels could exert neuroprotective effects. Creatine kinase and its substrates creatine and phosphocreatine constitute an intricate cellular energy buffering and transport system connecting sites of energy production (mitochondria) with sites of energy consumption, and creatine administration stabilizes the mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore. We found that oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival in G93A transgenic mice, and it protected mice from loss of both motor neurons and substantia nigra neurons at 120 days of age. Creatine administration protected G93A transgenic mice from increases in biochemical indices of oxidative damage. Therefore, creatine administration may be a new therapeutic strategy for ALS.     

Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn-superoxide dismutase

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor function and eventual death as a result of degeneration of motor neurons in the spinal cord and brain. The discovery of mutations in SOD1, the gene encoding the antioxidant enzyme Cu/Zn-superoxide dismutase (CuZnSOD), in a subset of ALS patients has led to new insight into the pathophysiology of ALS. Utilizing a novel adenovirus gene delivery system, our laboratory has developed a human cell culture model using chemically differentiated neuroblastoma cells to investigate how mutations in SOD1 lead to neuronal death. Expression of mutant SOD1 (G37R) resulted in a time and dose-related death of differentiated neuroblastoma cells. This cell death was inhibited by overexpression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). These observations support the hypothesis that mutant SOD1-associated neuronal death is associated with alterations in oxidative stress, and since MnSOD is a mitochondrial enzyme, suggest that mitochondria play a key role in disease pathogenesis. Our findings in this model of inhibition of mutant SOD1-associated death by MnSOD represent an unique approach to explore the underlying mechanisms of mutant SOD1 cytotoxicity and can be used to identify potential therapeutic agents for further testing.     

Protein misfolding, mitochondrial dysfunction and muscle loss are not directly dependent on soluble and aggregation state of mSOD1 protein in skeletal muscle of ALS

Mutant superoxide dismutase 1 (mSOD1) is often found as aggregates at the outer-membrane of mitochondria in motor neurons of various mouse models and familial amyotrophic lateral sclerosis (f-ALS) patients. It has been postulated that disruption of mitochondrial function by physical association of misfolded mSOD1 aggregates may actually be the trigger for initiation of degeneration of motor neurons in ALS. However, it was not clear if the same mechanism is involved in muscle degeneration and mitochondrial dysfunction in skeletal muscles of ALS. Recent study from our laboratory show that two skeletal muscle proteins, namely creatine kinase (CK) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) undergo major conformational and functional changes in the f-ALS mouse model of ALS (G93A). In this paper, we report two intriguing observations which are as follows:(i) G93A protein does not form aggregates in skeletal muscle at any stages of disease process probably due to high chymotrypsin-like activity of proteasome and thus G93A protein aggregates have no direct effects on progressive loss of muscle mass and global changes in protein conformation in ALS, and (ii) the soluble G93A protein does not have direct effects on mitochondrial dysfunction as determined by quantifying the release of reactive oxygen species (ROS) in skeletal muscle mitochondria; instead, the proteins affected by G93A possibly affect mitochondrial ROS release. These data strongly suggest for the first time that unlike in motor neurons, the soluble and aggregation states of the G93A protein do not have direct effects on protein misfolding and mitochondrial dysfunction in skeletal muscle during ALS.     

NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.     

Mitochondrial involvement in amyotrophic lateral sclerosis

Despite numerous reports demonstrating mitochondrial abnormalities associated with amyotrophic lateral sclerosis (ALS), the role of mitochondrial dysfunction in the disease onset and progression remains unknown. The intrinsic mitochondrial apoptotic program is activated in the central nervous system of mouse models of ALS harboring mutant superoxide dismutase 1 protein. This is associated with the release of cytochrome-c from the mitochondrial intermembrane space and mitochondrial swelling. However, it is unclear if the observed mitochondrial changes are caused by the decreasing cellular viability or if these changes precede and actually trigger apoptosis. This article discusses the current evidence for mitochondrial involvement in familial and sporadic ALS and concludes that mitochondria is likely to be both a trigger and a target in ALS and that their demise is a critical step in the motor neuron death.     

Late onset of motor neurons in mice overexpressing wild-type peripherin

Peripherin, a type III intermediate filament (IF) protein, upregulated by injury and inflammatory cytokines, is a component of IF inclusion bodies associated with degenerating motor neurons in sporadic amyotrophic lateral sclerosis (ALS). We report here that sustained overexpression of wild-type peripherin in mice provokes massive and selective degeneration of motor axons during aging. Remarkably, the onset of peripherin-mediated disease was precipitated by a deficiency of neurofilament light (NF-L) protein, a phenomenon associated with sporadic ALS. In NF-L null mice, the overexpression of peripherin led to early- onset formation of IF inclusions and to the selective death of spinal motor neurons at 6 mo of age. We also report the formation of similar peripherin inclusions in presymptomatic transgenic mice expressing a mutant form of superoxide dismutase linked to ALS. Taken together, these results suggest that IF inclusions containing peripherin may play a contributory role in motor neuron disease.     

MFN2 Couples Glutamate Excitotoxicity and Mitochondrial Dysfunction in Motor Neurons

Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca²⁺-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction.     

Removing dysfunctional mitochondria from axons independent of mitophagy under pathophysiological conditions

Chronic mitochondrial dysfunction has been implicated in major neurodegenerative diseases. Long-term cumulative pathological stress leads to axonal accumulation of damaged mitochondria. Therefore, the early removal of defective mitochondria from axons constitutes a critical step of mitochondrial quality control. We recently investigated the axonal mitochondrial response to mild stress in wild-type neurons and chronic mitochondrial defects in amyotrophic lateral sclerosis (ALS)- and Alzheimer disease (AD)-linked neurons. We demonstrated that remobilizing stressed mitochondria is critical for maintaining axonal mitochondrial integrity. The selective release of the mitochondrial anchoring protein SNPH (syntaphilin) from stressed mitochondria enhances their retrograde transport toward the soma before PARK2/Parkin-mediated mitophagy is activated. This SNPH-mediated response is robustly activated during the early disease stages of ALS-linked motor neurons and AD-related cortical neurons. Our study thus reveals a new mechanism for the maintenance of axonal mitochondrial integrity through SNPH-mediated coordination of mitochondrial stress and motility that is independent of mitophagy.