Dr Lee J Martin
email: martinl@jhmi.edu

The Mitochondrial Permeability Transition Pore: A Molecular Target for Amyotrophic Lateral Sclerosis Therapy

Keywords: adenine nucleotide translocator, apoptosis, cell death, cyclophilin D, excitotoxicity, mitochondria, motor neuron, ppif, voltage-dependent anion channel

Effective therapies are needed for the treatment of amyotrophic lateral sclerosis (ALS), a fatal type of motor neuron disease. Morphological, biochemical, molecular genetic, and cell/animal model studies suggest that mitochondria have potentially diverse roles in neurodegenerative disease mechanisms and neuronal cell death. In human ALS, abnormalities have been found in mitochondrial structure, mitochondrial respiratory chain enzymes, and mitochondrial cell death proteins indicative of some non-classical form of programmed cell death. Mouse models of ALS are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria. This minireview summarizes work on the how malfunctioning mitochondria might contribute to neuronal death in ALS through the biophysical entity called the mitochondrial permeability pore (mPTP). The major protein components of the mPTP are enriched in mouse motor neurons. Early in the course of disease in ALS mice expressing human mutant superoxide dismutase-1, mitochondria in motor neurons undergo trafficking abnormalities and dramatic remodeling resulting in the formation of mega-mitochondria and coinciding with increased protein carbonyl formation and nitration of mPTP components. The genetic deletion of a major mPTP component, cyclophilin D, has robust effects in ALS mice by delaying disease onset and extending survival. Thus, attention should be directed to the mPTP as rational target for the development of drugs designed to treat ALS.

Introduction

ALS is a progressive and severely disabling fatal neurological disease in humans characterized by initial muscle weakness, and then muscle atrophy, spasticity, and eventual paralysis and death typically within 3 to 5 years after symptoms begin [START_REF] Rowland | Amyotrophic lateral sclerosis[END_REF]. The cause of the spasticity, paralysis and death is progressive degeneration and elimination of upper motor neurons (MNs) in cerebral cortex and lower MNs in brainstem and spinal cord [START_REF] Rowland | Amyotrophic lateral sclerosis[END_REF][START_REF] Sathasivam | Apoptosis in amyotrophic lateral sclerosis: a review of the evidence[END_REF]. Degeneration and loss of spinal and neocortical interneurons has also been found in human ALS [START_REF] Stephens | Widespread loss of neuronal populations in spinal ventral horn in sporadic motor neuron disease. A morphometric study[END_REF][START_REF] Maekawa | Cortical selective vulnerability in motor neurons disease: a morphometric study[END_REF]. More than 5000 people in the United States are diagnosed with ALS each year (ALS Association, www.alsa.org), and, in parts of the United Kingdom, three people die everyday from some form of MN disease (www.uk-mnd-professionalnetwork.com). Other than life support management, no effective treatments exist for ALS [START_REF] Cozzolino | Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications[END_REF].

It is still not understood why specific neuronal populations are selectively vulnerable in ALS, such as certain somatic MNs and interneurons [START_REF] Stephens | Widespread loss of neuronal populations in spinal ventral horn in sporadic motor neuron disease. A morphometric study[END_REF][START_REF] Maekawa | Cortical selective vulnerability in motor neurons disease: a morphometric study[END_REF]. The molecular pathogenesis of ALS is understood poorly, contributing to the lack of appropriate target identification and effective mechanism-based therapies to treat this disease. Two forms of ALS exist: idiopathic (sporadic) and heritable (familial).

The majority of ALS cases are sporadic with few known genetic contributions, except for missense mutations in TAR-DNA binding protein [START_REF] Kabashi | TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis[END_REF]. Aging is a strong risk factor for ALS because the average age of onset is 55 (ALS Association, www.alsa.org). Familial forms of ALS (fALS) have autosomal dominant or autosomal recessive inheritance patterns and make up ~10% or less of all ALS cases. ALS-linked mutations occur in the genes encoding SOD1 (ALS1), Alsin (ALS2), senataxin (ALS4), vesicle associated membrane protein (VAMP/synaptobrevin)-associated protein B (ALS8), dynactin, TAR-DNA binding protein, and fused in sarcoma (FUS, ALS6) [START_REF] Martin | Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis[END_REF][START_REF] Schymick | Genetics of amyotrophic lateral sclerosis[END_REF].

Mitochondrial perturbations have been known for a long time to participate in the mechanisms of neuropathology, particularly disorders involving acute interruptions in O 2 and substrate delivery to the brain and bioenergetic failure as seen in tissue ischemia [START_REF] Hou | Molecular mechanisms of cerebral ischemia-induced neuronal death[END_REF][START_REF] Zorov | The mitochondrion as Janus Bifrons[END_REF]. An exciting new understanding of

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ACCEPTED MANUSCRIPT Martin 4 mitochondrial biology has emerged over the past decade that is likely to be very relevant to agerelated neurodegenerative disorders [START_REF] Martin | Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis[END_REF]. Mitochondria are multi-functional organelles [START_REF] Zorov | The mitochondrion as Janus Bifrons[END_REF]. In addition to their critical role in the production of ATP through the electron transport chain (Fig. 1), these organelles function in intracellular Ca 2+ homeostasis, steroid, heme and iron-sulfur cluster synthesis, and programmed cell death [START_REF] Zorov | The mitochondrion as Janus Bifrons[END_REF][START_REF] Nicholls | Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease[END_REF]. They are also sites of formation of reactive oxygen species (ROS), including superoxide anion (O 2 •-) and the highly reactive hydroxyl radical ( • OH) or its intermediates [START_REF] Zorov | The mitochondrion as Janus Bifrons[END_REF], and reactive nitrogen species such as nitric oxide ( • NO) [START_REF] Zorov | The mitochondrion as Janus Bifrons[END_REF]. Thus, mitochondria have functions and properties that might confer an intrinsic susceptibility of subsets of long-lived postmitotic cells such as neurons to aging and stress. In this regard varying degrees of mitochondrial dysfunction and aberrant mitochondrial maintance and repair could be critical determinants in the regulation of disease and neuronal cell death ranging from necrosis and apoptosis to autophagy [START_REF] Cozzolino | Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications[END_REF][START_REF] Martin | Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis[END_REF][START_REF] Northington | Apoptosis in perinatal hypoxic-ischemic brain injury: how important is it and should it be inhibited?[END_REF][START_REF] Northington | Failure to complete apoptosis following neonatal hypoxia-ischemia manifests as "continuum" phenotype of cell death and occurs with multiple manifestations of mitochondrial dysfunction in rodent forebrain[END_REF].

Mitochondrial abnormalities in human ALS

Evidence for mitochondrial abnormalities in human ALS pathogenesis lacks definite causal relationships

Mitochondrial dysfunction has been implicated in the pathogenesis of ALS in humans. Electron microscopy studies have shown mitochondrial morphology abnormalities in skeletal muscle, liver, spinal MNs and cortical upper MN regions of ALS patients [START_REF] Sasaki | Ultrastructural changes of synapses of Betz cell in patients with amyotrophic lateral sclerosis[END_REF][START_REF] Menzies | Mitochondrial involvement in amyotrophic lateral sclerosis[END_REF]. A mutation in cytochrome c oxidase subunit I was found in a patient with a MN disease phenotype [START_REF] Comi | Cytochrome c oxidase subunit I microdeletion in a paitent with motor neuron disease[END_REF]. Another patient with MN disease had a mutation in a mitochondrial tRNA gene [START_REF] Borthwick | Motor neuron disease in a patient with a mitochondrial tRNAIle mutation[END_REF]. One type of mitochondrial DNA (mtDNA) mutation, called the common mtDNA deletion (mtDNA4977), is found non-uniformly within different human brain areas; the highest levels are detected in the striatum and substantia nigra [START_REF] Soong | Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain[END_REF][START_REF] Corral-Debrinski | Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age[END_REF].

However, no significant accumulation of the 5kb common deletion in mtDNA has been found by single-cell analysis of MNs from sporadic ALS cases [START_REF] Mawrin | Single-cell analysis of mtDNA levels in sporadic amyotrophic lateral sclerosis[END_REF]. Some ALS patients with defects in mitochondrial oxidative phosphorylation in skeletal muscle have a novel SOD1 mutation [START_REF] Corti | Amyotrophic lateral sclerosis linked to a novel SOD1 mutation with muscle mitochondrial dysfunction[END_REF].

Intracellular Ca 2+ abnormalities and excitotoxicity in human ALS pathogenesis: links to mitochondrial dysfunction and oxidative stress

Mitochondria function in the regulation of intracellular Ca 2+ levels [START_REF] Nicholls | Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease[END_REF][START_REF] Babcock | Mitochondrial oversight of cellular Ca 2+ signaling[END_REF]. Regarding ALS, skeletal muscle biopsies of patients with sporadic disease show ultrastructural changes indicative of elevated Ca 2+ in MN terminals, with some mitochondria showing an augmented Ca 2+ signal [START_REF] Siklos | Ultrastructural evidence for altered calcium in motor nerve terminals in amyotrophic lateral sclerosis[END_REF]. Utilizing specific transport systems mitochondria can move Ca 2+ from the cytosol into the matrix by the Ca 2+

uniporter and eject Ca 2+ via the Na + /Ca 2+ exchanger [START_REF] Nicholls | Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease[END_REF] and more catastrophically through the mitochondrial permeability transition pore (mPTP) [START_REF] Zoratti | The mitochondrial permeability transition[END_REF]. Under conditions of elevated cytoplasmic Ca 2+ , whenever the local free Ca 2+ concentration rises above a set-point of ~0.5 μM, mitochondria avidly accumulate Ca 2+ to a fixed capacity [START_REF] Nicholls | Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease[END_REF]. The electrical gradient across the mitochondrial inner membrane, the ΔΨ m , established by electron transport chain activity (Fig. 1), provides the driving force for the accumulation of Ca 2+ into the mitochondrial matrix [START_REF] Babcock | Mitochondrial oversight of cellular Ca 2+ signaling[END_REF]. Cytosolic Ca 2+ concentrations above set-point levels are believed to be achieved during tetanic stimulation and by activation of glutamate receptors on the plasma membrane [START_REF] Nicholls | Mitochondrial function and dysfunction in the cell: its relevance to aging and aging-related disease[END_REF]. In settings of the pathological process called excitotoxicity, resulting from excessive overstimulation of glutamate receptors [START_REF] Olney | Brain lesion, obesity, and other disturbances in mice treated with monosodium glutamate[END_REF], Ca 2+ overload in neurons is significant and cause cell death [START_REF] Choi | Cellular defences destroyed[END_REF]. When mitochondria become overloaded with Ca 2+ , they undergo mitochondrial permeability transition (see below) resulting in osmotic swelling and rupture of the outer mitochondrial membrane (Fig. 1). Interestingly, mitochondria within synapses appear to be more susceptible than non-synaptic mitochondria to Ca 2+ overload [START_REF] Brown | Synaptic mitochondria are more susceptible to Ca 2+ overload than nonsynaptic mitochondria[END_REF].

Exitotoxicity has been implicated in the pathogenesis of ALS for a long time [START_REF] Rothstein | Decreased glutamate transport by brain and spinal cord in amyotrophic lateral sclerosis[END_REF] and is another possible mechanism by which MNs can be damaged in ALS [START_REF] Choi | Cellular defences destroyed[END_REF]. Sporadic ALS patients have Martin 6 reduced levels of synaptosomal high-affinity glutamate uptake [START_REF] Rothstein | Decreased glutamate transport by brain and spinal cord in amyotrophic lateral sclerosis[END_REF] and astroglial glutamate transporter EAAT2 (excitatory amino acid transporter 2 or GLT1) in motor cortex and spinal cord [START_REF] Rothstein | Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis[END_REF].

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Reductions in levels of activity of EAAT2 in spinal cord could increase the extracellular concentrations of glutamate at synapses on MNs. MNs are sensitive to glutamate excitotoxicity because they have a low proportion of GluR2-edited or under-edited AMPA subtype glutamate receptor on their surfaces, predisposing MNs to risk of excess Ca 2+ entry and mitochondrial perturbations [START_REF] Heath | Quantitative assessment of AMPA receptor mRNA in human spinal motor neurons isolated by laser capture microdissection[END_REF][START_REF] Kwak | Deficient RNA editing of GluR2 and neuronal death in amyotrophic lateral sclerosis[END_REF]. Excess glutamate receptor activation in neurons can cause increased intracellular Ca 2+ , mitochondrial ROS production, bioenergetic failure, and mitochondrial trafficking abnormalities [START_REF] Chang | Mitochondrial trafficking and motphology in healthy and injured neurons[END_REF]. Ca 2+ -induced generation of ROS in brain mitochondria is mediated by mitochondrial permeability transition [START_REF] Hansson | Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition[END_REF].

MNs are particularly affected by inhibition of mitochondrial metabolism which causes elevated cytosolic Ca 2+ levels and increased excitability [START_REF] Bergmann | Impact of mitochondrial inhinition on excitability and cytosolic Ca 2+ levels in brainstem motoneurones[END_REF].

Mitochondria generate endogenous ROS as by-products of oxidative phosphorylation (Fig. 1) [START_REF] Wallace | A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn of evolutionary medicine[END_REF].

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nucleophile and oxidant and nitrating agent peroxynitrite (ONOO -) (Fig. 1) [START_REF] Beckman | ALS, SOD and peroxynitrite[END_REF]. ONOO -or products of ONOO -can damage proteins by nitration [START_REF] Beckman | ALS, SOD and peroxynitrite[END_REF]. ONOO -is genotoxic directly to neurons by causing single-and double-strand breaks in DNA [START_REF] Martin | DNA damage profiling in motor neurons: a single-cell analysis by comet assay[END_REF]. NO can be produced in mitochondria [START_REF] Giulini | Characterization and function of mitochondrial nitric-oxide synthase, Free Radic[END_REF] and has direct effects in mitochondria. NO at nanomolar concentrations can inhibit rapidly and reversibly respiration [START_REF] Brown | Nitric oxide, cytochrome c and mitochondriam[END_REF].

Markers of oxidative stress and ROS damage are elevated in ALS tissues [START_REF] Beal | Oxidatively modified protein in aging and disease[END_REF]. In human sporadic ALS, protein carbonyls are elevated in motor cortex [START_REF] Ferrante | Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis[END_REF]. Tyrosine nitration is elevated in human ALS nervous tissues [START_REF] Abe | Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis[END_REF][START_REF] Beal | Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis[END_REF][START_REF] Sasaki | iNOS and nitrotyrosine immunoreactivity in amyotrophic lateral sclerosis[END_REF]. Studies of respiratory chain enzyme activities are discrepant. Studies have reported increases in complex I, II, and III activities in vulnerable and non-vulnerable brain regions in patients with mutant SOD1-fALS [START_REF] Browne | Metabolic dysfunction in familial, but not sporadic, amyotrophic lateral sclerosis[END_REF], but other studies have found decreased complex IV activity in spinal cord ventral horn [START_REF] Borthwick | Mitochondrial enzyme activity in amyotrophic lateral sclerosis: implications for the role of mitochondria in neuronal cell death[END_REF] and skeletal muscle [START_REF] Vielhaber | Mitochondrial DNA abnormalities in skeletal muscle of patients with sporadic amyotrophic lateral sclerosis[END_REF] of sporadic ALS cases. In sporadic ALS skeletal muscle, reductions in activity of respiratory chain complexes with subunits encoded by the mitochondrial genome are associated with decreased neuronal NO synthase levels [START_REF] Soraru | Activities of mitochondrial complexes correlate with nNOS amount in muscle from ALS patients[END_REF]. Alterations in skeletal muscle mitochondria are progressive [START_REF] Echaniz-Laguna | Muscular mitochondrial function in amyotrophic lateral sclerosis is progressively altered as the desiese develops; a temporal study in man[END_REF] and could be intrinsic to skeletal muscle [START_REF] Vielhaber | Visualization of defective mitochondrial function in skeletal muscle fibers of patients with sporadic amyotrophic lateral sclerosis[END_REF] and not due merely to neurogenic atrophy as assumed commonly.

Human ALS and mitochondrial orchestrated programmed cell death (PCD) involving p53

Degeneration of motor neurons in human ALS has features of PCD

PCD is physiologically regulated cell death [START_REF] Martin | Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis[END_REF]. Apoptosis, a form of PCD, is a structurally and biochemically organized, transcriptionally-dependent or -independent, form of cell death. The basic machinery of apoptosis is conserved in yeast, hydra, nematode, fruit fly, zebrafish, mouse, and human [START_REF] Ameisen | On the origin, evolution, and nature of programmed cell death: a timeline of four billion years[END_REF]. Mitochondria regulate cell death processes (Figs. [START_REF] Rowland | Amyotrophic lateral sclerosis[END_REF][START_REF] Sathasivam | Apoptosis in amyotrophic lateral sclerosis: a review of the evidence[END_REF]. A variety of mitochondrial proteins function in apoptosis (Table 1) including Bcl-2 family members, cytochrome c, apoptosis inducing factor (AIF), endonuclease G, second mitochondrial activator of caspases (Smac/DIABLO),

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and Omi/high-temperature requirement protein A2 (HtrA2) inhibitor of the inhibitors of apoptosis proteins (Fig. 1) [START_REF] Merry | Bcl-2 gene family in the nervous system[END_REF][START_REF] Cory | The bcl-2 family: regulators of the cellular life-or-death switch[END_REF][START_REF] Lorenzo | Mitochondrial effectors in caspase-independent cell death[END_REF]. Other proteins (e.g., humanin, Ku70, 14-3-3 proteins) that are not mitochondrial can modulate mitochondrially translocated cell death proteins by binding and sequestration. For example, Ku70 blocks the translocation of Bax to mitochondria to restrain cell death [START_REF] Cohen | Acetylation of the C terminus of Ku70 by CBP and PCAF controls Bax-mediated apoptosis[END_REF].

PCD appears to contribute to the selective degeneration of MNs in human sporadic ALS and fALS, albeit seemingly as a non-classical form differing from apoptosis (Fig. 2) [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF]. MNs appear to pass through sequential stages of chromatolysis (suggestive of initial axonal injury), somatodendritic attrition without extensive cytoplasmic vacuolation, and then nuclear DNA fragmentation, nuclear condensation, and cell death (Fig. 3) [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF]. MNs in individuals dying from sporadic ALS and fALS show the same patterns of degeneration [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF]. This cell death in human MNs is defined clearly by genomic DNA fragmentation (determined by DNA agarose gel electrophoresis and in situ DNA nick-end labeling) and cell loss and is associated with accumulation of mitochondria, cytochrome c, and cleaved caspase-3 (Figs. 2,3) [START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF]. However, the morphology of this cell death is distinct from classical apoptosis, despite the nuclear condensation [START_REF] Martin | Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis[END_REF][START_REF] Martin | Neuronal cell death in nervous system development, disease, and injury[END_REF]. Nevertheless, Bax and Bak1 protein levels are increased in mitochondria-enriched fractions of selectively vulnerable motor regions (spinal cord anterior horn and motor cortex gray matter), but not in regions unaffected by the disease (somatosensory cortex gray matter). In marked contrast, Bcl-2 protein is severely depleted in mitochondria-enriched fractions of affected regions and is sequestered in the cytosol (Fig. 2) [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF].

Although these western blot observations lacked direct specificity for MN events [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF], subsequent immunohistochemistry (Figs. 2,3) [START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF] and laser capture microdissection of MNs combined with mass spectroscopy-protein profiling have confirmed the presence of intact active caspase-3 in human ALS MNs [START_REF] Ginsberg | Cell and tissue microdissection in combination with genomic and proteomic profiling[END_REF].

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Degeneration of motor neurons in human ALS is associated with p53 activation

Neuronal apoptosis can be driven by the tumor suppressor p53 [START_REF] Martin | Molecular regulation of DNA damageinduced apoptosis in neurons of cerebral cortex[END_REF]. This cell death can be transcriptionally-dependent and transcriptionally-independent [START_REF] Martin | Molecular regulation of DNA damageinduced apoptosis in neurons of cerebral cortex[END_REF]. p53 can mediate mitochondrial permeabilization through direct physical interaction with Bcl-2 family members [START_REF] Chipuk | Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis[END_REF]. p53 is activated in MNs in human ALS [START_REF] Martin | p53 is abnormally elevated and active in the CNS of patients with amyotrophic lateral sclerosis[END_REF]. p53 levels increase in vulnerable regions in individuals ALS, and p53

accumulates specifically in ALS MN [START_REF] Martin | p53 is abnormally elevated and active in the CNS of patients with amyotrophic lateral sclerosis[END_REF]. This p53 is active functionally because it is phosphorylated at serine 392 and has increased DNA binding activity [START_REF] Martin | Neuronal cell death in nervous system development, disease, and injury[END_REF][START_REF] Martin | p53 is abnormally elevated and active in the CNS of patients with amyotrophic lateral sclerosis[END_REF].

These data support the concept of an aberrant re-emergence of a PCD mechanism, involving p53 activation and cytosol-to-mitochondria redistributions of cell death proteins, participating in the pathogenesis of MN degeneration in human ALS [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF][START_REF] Pacher | Nitric oxide and peroxynitrite in health and disease[END_REF]. The morphological and biochemical changes seen in human ALS are modeled robustly and faithfully at structural and molecular levels in axotomy models of MN degeneration in adult mouse [START_REF] Martin | Injury-induced spinal motor neuron apoptosis is preceded by DNA singlestrand breaks and is p53-and Bax-dependent[END_REF] but not in the current commonly used human mutant SOD1 transgenic mouse models (Fig. 3) [START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF].

Mitochondrial pathobiology in cell and mouse models of ALS

A common mutation in human SOD1 that is linked to fALS is the substitution of glycine by alanine at position 93 (G93A) [START_REF] Bendotti | Lessons from models of SOD1-linked familial ALS[END_REF][START_REF] Turner | Transgenics, toxicity and therapeutics in rodent models of mutant SOD1mediated familial ALS[END_REF]. SOD1 is a metalloenzyme of 153 amino acids (~16 kDa) that binds one copper ion and one zinc ion per subunit [START_REF] Fridovich | Superoxide radical and superoxide dismutases[END_REF]. This enzyme, functioning as a ~32 kDa non-covalently linked homodimer, is responsible for the detoxification and maintenance of intracellular O 2

•concentration in the low femtomolar range by catalyzing its dismutation [START_REF] Fridovich | Superoxide radical and superoxide dismutases[END_REF][START_REF] Mccord | Superoxide dismutase, an enzymic function for erythrocuprein (hemocuprein)[END_REF]. SOD1 is ubiquitous (intracellular SOD concentrations are typically ~10-40 μM) in most tissues and possibly greater in neurons [START_REF] Rakhit | Monomeric Cu, Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic sclerosis[END_REF].

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Evidence for mitochondrial abnormalities in cell models of ALS

Data from cell culture studies argue strongly for mitochondria dysfunction, possibly MN selective, in the presence of mSOD1 [START_REF] Bendotti | Lessons from models of SOD1-linked familial ALS[END_REF][START_REF] Ferri | Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials[END_REF]. Expression of several human SOD1 mutants increases mitochondrial O 2

•-levels and causes toxicity in primary MNs [START_REF] Estévez | Induction of nitric oxide-dependent apoptosis in motor neurons by zinc-deficient superoxide dismutase[END_REF], neuroblastoma cells [START_REF] Flanagan | Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn-superoxide dismutase[END_REF], and NSC-34 cells (a hybrid cell line with some MN-like characteristics, produced by fusion of MN-enriched embryonic mouse spinal cord cells with mouse neuroblastoma cells) [START_REF] Bilsland | Expression of mutant SOD1G93A in astrocytes induces functional deficits in motoneuron mitochondria[END_REF]. These responses can be attenuated by over-expression of MnSOD [START_REF] Flanagan | Overexpression of manganese superoxide dismutase attenuates neuronal death in human cells expressing mutant (G37R) Cu/Zn-superoxide dismutase[END_REF]. ALS-mSOD1 variants, compared to wild-type SOD1, associate more with mitochondria in NSC-34 cells and appear to form cross-linked oligomers that shift the mitochondrial GSH/GSSH ratio toward oxidation [START_REF] Ferri | Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials[END_REF].

Most evidence for mitochondrial abnormalities in mouse ALS pathogenesis lacks definite causal relationships

Gurney et al were the first to develop transgenic (tg) mice that express the G93A mutant form of human SOD1 [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF][START_REF] Dal Canto | Development of central nervous system pathology in a murine transgenic model of human amyotrophic lateral sclerosis[END_REF]. Now, these mice are used widely as an animal model of ALS [START_REF] Cozzolino | Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications[END_REF][START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF][START_REF] Bendotti | Lessons from models of SOD1-linked familial ALS[END_REF][START_REF] Turner | Transgenics, toxicity and therapeutics in rodent models of mutant SOD1mediated familial ALS[END_REF].

Human mutant SOD1 (mSOD1) is expressed ubiquitously in these mice by its endogenous promoter in a tissue/cell non-selective pattern against a background of normal mouse SOD1 [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF]. Effects of this human mutant gene in mice are profound. Hemizygous tg mice expressing high copy number of the G93A variant of mSOD1 become completely paralyzed and die at ~16-18 weeks of age [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF]. G93A-mSOD1 mice with reduced transgene copy number have a much slower disease progression and die at ~8-9 months of age [START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF][START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Spinal MNs and interneurons in mice expressing G93A high -mSOD1 undergo prominent degeneration; about 70% of lumbar MNs are eliminated by end-stage disease [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF][START_REF] Chang | Glycinergic Innervation of motoneurons is deficient in amyotrophic lateral sclerosis mice: a confocal quantitative analysis[END_REF]. More work is needed on the cell death and its mechanisms in G93A low -expressing mice.

Subsets of spinal interneurons are lost before MNs G93A high -mSOD1 [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF], some of which are the glycinergic Renshaw cells [START_REF] Chang | Glycinergic Innervation of motoneurons is deficient in amyotrophic lateral sclerosis mice: a confocal quantitative analysis[END_REF]. Unlike the degeneration of MN in human ALS, MNs in these mice do not degenerate with a morphology resembling any form of apoptosis (Fig. 3) [START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF][START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF][START_REF] Bendotti | Early vacuolization and mitochondrial damage in motor neurons of FALS mice are not associated with apoptosis or with changes in cytochrome oxidase histochemical reactivity[END_REF]. The MN degeneration seen in G93A high -mSOD1 mice more closely resembles a prolonged necrotic-like cell death process (Fig. 3) [START_REF] Martin | Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis[END_REF] involving early-occurring mitochondrial damage, cellular swelling, and dissolution [START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF][START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF][START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF][START_REF] Chang | Glycinergic Innervation of motoneurons is deficient in amyotrophic lateral sclerosis mice: a confocal quantitative analysis[END_REF]. Biochemically, the death of MNs is characterized by somal and mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in nuclear DNA and mitochondrial DNA [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. The MN death is independent of activation of caspases-1 and 3, and also appears to be independent of capsase-8 and apoptosis-inducing factor activation within MNs [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. Indeed, caspase-dependent and p53-mediated apoptosis mechanisms might be blocked actively in G93A high -mSOD1 mouse MNs, possibly by upregulation of inhibitors of apoptosis and changes nuclear import of proteins [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF].

Mitochondrial pathology has been implicated in the mechanisms of human and mouse ALS [START_REF] Martin | Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis[END_REF], but most evidence is circumstantial. In different mSOD1 mouse models of ALS, mitochondria in spinal cord neurons exhibit structural pathology [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF][START_REF] Wong | An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria[END_REF][START_REF] Kong | Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1[END_REF][START_REF] Jaarsma | CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations[END_REF][START_REF] Higgins | Mutant Cu, Zn Superoxide dismutase that causes motoneuron degeneration is present in mitochondria in the CNS[END_REF][START_REF] Higgins | ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes[END_REF] and some of the mitochondrial degeneration occurs very early in the course of the disease [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF][START_REF] Bendotti | Early vacuolization and mitochondrial damage in motor neurons of FALS mice are not associated with apoptosis or with changes in cytochrome oxidase histochemical reactivity[END_REF]. Mitochondrial microvacuolar damage in MNs emerges by 4 weeks of age in G93A mice with high expression [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF][START_REF] Bendotti | Early vacuolization and mitochondrial damage in motor neurons of FALS mice are not associated with apoptosis or with changes in cytochrome oxidase histochemical reactivity[END_REF]. It has been argued that mitochondrial damage in G93A high -mSOD1 mice is related to supra-normal levels of SOD1 and might not be related causally to the disease process because transgenic mice expressing high levels of human wildtype SOD1 show some mitochondrial pathology [START_REF] Jaarsma | Swelling and vacuolation or mitochondria in transgenic SOD1-ALS mice: a consequence of supranormal SOD1 expression?[END_REF], but mitochondrial abnormalities have been found histologically also in G93A low -mSOD1 mice [START_REF] Sasaki | Ultrastructural study of mitochondria in the spinal cord of transgenic mice with a G93A mutant SOD1 gene[END_REF](Martin LJ et al, unpublished observations). Thus, mitochondria could be primary sites of human SOD1 toxicity in transgenic mice irrespective of transgene copy number and expression level of human SOD1, but direct, unequivocal causal relationships have been lacking.

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ACCEPTED MANUSCRIPT Martin 12 Human SOD1 mutant proteins appear to gain a toxic property or function, rather than having diminished O 2 -scavenging activity [START_REF] Deng | Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase[END_REF][START_REF] Borchelt | Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity[END_REF][START_REF] Yim | A gain-offunction of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: an enhancement of free radical formation due to a decrease in K m for hydrogen peroxide[END_REF], and wild-type SOD1 can gain toxic properties through oxidative modification [START_REF] Kabashi | Oxidized/misfolded superoxide dismutase-1: the cause of all amyotrophic lateral sclerosis?[END_REF][START_REF] Ezzi | Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation[END_REF]. A gain in aberrant oxidative chemistry could contribute to the mechanisms of mitochondriopathy in G93A high mice [START_REF] Beckman | ALS, SOD and peroxynitrite[END_REF][START_REF] Liochev | Mutant Cu,Zn superoxide dismutases and familial amyotrophic lateral sclerosis: evaluation of oxidative hypotheses[END_REF]. G93A-mSOD1 has enhanced free radical-generating capacity compared to wild-type enzyme [START_REF] Yim | A gain-offunction of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: an enhancement of free radical formation due to a decrease in K m for hydrogen peroxide[END_REF] and can catalyze protein oxidation by hydroxyl-like intermediates and carbonate radical [START_REF] Pacher | Nitric oxide and peroxynitrite in health and disease[END_REF]. G93A high mice have increased protein carbonyl formation in total spinal cord tissue extracts at pre-symptomatic disease [START_REF] Andrus | Protein oxidative damage in a transgenic mouse medel of familial amyotrophic lateral sclerosis[END_REF]. Protein carbonyl formation in mitochondrial membrane-enriched fractions of spinal cord is a robust signature of incipient disease [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. A mass spectroscopy study of G93A high mice identified proteins in total spinal cord tissue extracts with greater than baseline carbonyl modification, including SOD1, translationally controlled tumor protein, and ubiquitin carboxyl-terminal hydrolase-L1 [START_REF] Poon | Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice-a model of familial amyotrophic lateral sclerosis[END_REF]. Nitrated and aggregated cytochrome c oxidase subunit-I and α-synuclein accumulate in G93A high mouse spinal cord [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF].

Nitrated MnSOD accumulates also in G93A high mouse spinal cord [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. Toxic properties of mSOD1 could also be mediated through protein binding or aggregation. Wild-type SOD1 and human mSOD1 associate with mitochondria [START_REF] Higgins | Mutant Cu, Zn Superoxide dismutase that causes motoneuron degeneration is present in mitochondria in the CNS[END_REF][START_REF] Okado-Matsumoto | Subcellular distribution of superoxide (SOD) in rat liver[END_REF]. Human SOD1 mutants associate with spinal cord mitochondria in mSOD1 mice and can bind Bcl-2 [99,100], thus potentially being decoys or dominant negative regulators of cell survival molecules (Fig. 2), but it is not known if this process is occurring specifically in mouse MNs. Bcl-2 is depleted from mitochondria isolated from micropunches of human ALS anterior horn gray matter and accumulates in the cytosol (Fig. 2) [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF]. Binding of mSOD1 (and perhaps its low-mobility species) to mitochondria has been reported to be spinal cord selective and ACCEPTED MANUSCRIPT Martin 13 mitochondrial redox potential when expressed in cultured cells [START_REF] Ferri | Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials[END_REF]. Nevertheless, the direct links between the physicochemical changes in mSOD1 and wild-type SOD1 and the mitochondrial functional and structural changes associated with ALS and MN degeneration remain uncertain.

EM studies have shown that the outer mitochondrial membrane (OMM) remains relatively intact to permit formation of mega-mitochondria in MNs cell bodies in G93A high mice [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF][START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. Moreover, early in the disease of these mice, mitochondria in dendrites in spinal cord ventral horn undergo extensive cristae and matrix remodeling, while few mitochondria in MN cell bodies show major structural changes [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Another interpretation of ultrastructural findings is that the mSOD1 causes mitochondrial degeneration by inducing OMM extension and leakage and intermembrane space expansion [START_REF] Higgins | ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes[END_REF]. Mechanisms for this damage could be related to mSOD1 gaining access to the mitochondrial intermembrane space [START_REF] Kong | Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1[END_REF]102] and the matrix [103] and inducing disturbances in oxidative phosphorylation [104] and antioxidant activity. This mitochondrial conformation seen by EM might favor the formation of the mPTP (Fig. 1); indeed, we found evidence for increased contact sites between the OMM and inner mitochondrial membrane (IMM) in dendritic mitochondria in G93A high mice [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Another feature of MNs of young G93A high mice before symptoms is apparent fission of ultrastructurally normal mitochondria in cell bodies and fragmentation of abnormal mitochondria [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF].

It is not clear if the cristae and matrix remodeling and the apparent fragmentation and fission mitochondria are related or independent events and if these abnormalities interfere with mitochondrial trafficking; nevertheless, morphological observations enforce the idea that mitochondria are critical to the pathobiology of mSOD1 toxicity to MNs in G93A high mice.

The possibility of changes in mitochondrial trafficking in MNs of mSOD1 mice is mostly unexplored.

Some data support the novel idea that mitochondria might act as messengers from distal regions of MNs in mSOD1 mice [START_REF] Martin | Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis[END_REF]. G93A high -mSOD1 mouse MNs accumulate mitochondria from the axon terminals and generate higher levels of O 2 •-, • NO, and ONOO -than MNs in transgenic mice expressing human wild-type SOD1 [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. This mitochondrial accumulation occurs at a time when MN cell body volume is increasing, suggestive of ongoing problems with ATP production or plasma membrane Na,K ATPase [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. G93A-mSOD1 perturbs anterograde axonal transport of mitochondria in cultured primary embryonic MNs [105] making it possible that retrogradely transported mitochondria with toxic properties from the neuromuscular junction fail to be returned to distal processes [START_REF] Martin | Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis[END_REF][START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF].

Mitochondria with enhanced toxic potential from distal axons and terminals could therefore have a "Trojan horse" role in triggering degeneration of MNs in ALS via retrograde transport from diseased skeletal muscle.

MNs in G93A high -mSOD1 mice also accumulate higher levels of intracellular Ca 2+ than MNs in transgenic human wild-type SOD1 mice [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. The intracellular Ca 2+ signal in MNs is very compartmental and mitochondrial-like in its appearance [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]106] NO signaling mechanisms in mitochondria of ALS mice have also been implicated in the pathogenesis (Fig. 1). MNs seem to be unique regarding NO production because the express constitutively low levels of inducible NO synthase (iNOS) [106]. G93A high -mSOD1 mouse MNs accumulate nicotinamide adenine dinucleotide phosphate diaphorase and iNOS-like immunoreactivity [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. iNOS is also up-regulated aberrantly in human sporadic ALS MNs [111]. iNOS gene deletion extends significantly the lifespan of G93A high -mSOD1 mice [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. Thus, mitochondrial oxidative stress, Ca 2+ dysregulation, iNOS activation, protein nitration, and protein aggregation (not necessarily SOD1

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though) are all likely intrinsic, cell automonous mechanisms in the process of MN degeneration caused by mSOD1 in mice [START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. The mechanistic basis for the differences between human ALS and mSOD1 mice, regarding cell death phenotype (Fig. 3), is not yet clear but could be related to the supra-normal expression of toxic mSOD1 or to fundamental differences in cell death mechanisms [START_REF] Martin | Opportunities for neuroprotection in ALS using cell dealth mechanism rationales[END_REF] or the tissue inflammatory milieu (Martin LJ, unpublished observations) that drive MNs in mSOD1 transgenic mice to necrotic-like death along the apoptosis-necrosis cell death continuum [START_REF] Martin | Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: a perspective on the contributions of apoptosis and necrosis[END_REF][START_REF] Northington | Apoptosis in perinatal hypoxic-ischemic brain injury: how important is it and should it be inhibited?[END_REF]106].

Another contributing factor for this difference between human and mouse MNs is that mitochondria are functionally diverse and have species-specific activities and molecular compositions, including the makeup of the mitochondrial permeability transition pore [112]. These possiblities allow for skepticism regarding the suitability of existing transgenic mSOD1 mouse lines to model human ALS.

The mitochondrial permeability transition pore contributes to the causal mechanisms of

ALS in mice

Despite the implication of toxic effects of mSOD1 on mitochondria in mouse ALS, cause-effect relationships between abnormal functioning of mitochondria and initiation and progression of disease have been uncertain. These relationships need to be known because this knowledge could provide a rationale for new mechanism-based treatments for ALS. One venue of investigation for mitochondrial damage causality is the mPTP and its possible involvement in ALS.

The mPTP: definition and molecular composition

Mitochondrial permeability transition is a mitochondrial state in which the proton-motive force is disrupted reversibly or irreversibly [START_REF] Zoratti | The mitochondrial permeability transition[END_REF][START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF][113][114][115][116][117]. Conditions of mitochondrial Ca 2+ overload, excessive oxidative stress, and decreased electrochemical gradient (ΔP), ADP, and ATP can favor mitochondrial permeability transition. This alter state of mitochondria involves the mPTP that functions

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ACCEPTED MANUSCRIPT Martin 16 as a voltage, thiol, and Ca 2+ sensor [START_REF] Zoratti | The mitochondrial permeability transition[END_REF][START_REF] Gurney | Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation[END_REF][113][114][115][116][117]. The mPTP is believed to be a poly-protein transmembrane channel formed at the contact sites between the IMM and the OMM. The collective components of the mPTP are still controversial, but the voltage-gated anion channel (VDAC, or porin) in the OMM, the adenine nucleotide translocator (ANT, or solute carrier family 25) in the IMM, and cyclophilin D (CyPD) in the matrix are believed to be the core components (

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Expression and localization of mPTP components in the CNS

Very few studies have been published on the localizations of mPTP components in the mammalian CNS; thus, details about the cellular expressions in different nervous system cell types are lacking. In mouse spinal cord, the core components of the mPTP (VDAC, ANT, and CyPD) are enriched in MNs as determined by immunohistochemistry [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. The specific isoforms of ANT and VDAC in MNs have not been determined. CyPD, ANT, and VDAC have mitochondrial and non-mitochondrial localizations in MNs [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. They are all nuclear-encoded mitochondrial-targeted proteins, thus a possible explanation for their non-mitochondrial localizations is that they are pre-mitochondrial forms.

Some cyclophilins are located in the cytoplasm [146], but CyPD immunoreactivity is annulled in ppif -/- mice, demonstrating that the antibody is detecting only CyPD [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Spinal cord, brainstem, and forebrain had similar levels of CyPD, as well as similar levels of ANT and VDAC [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Thus, differences in the levels of individual mPTP components cannot explain the intrinsic differences in the sensitivity to Ca 2+ -induced permeability transition seen in spinal cord and brain isolated mitochondria [147,148]. Not all mitochondria within individual MNs contained CyPD, ANT, and VDAC [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]; this observation supports that idea of that mitochondria in individual cells are not only heterogeneous in shape [149,150] but also in biochemical composition, notably metabolism [151] and genetics [START_REF] Wallace | A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn of evolutionary medicine[END_REF].

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The mPTP contributes to the pathogenesis of mouse ALS

The mPTP was first implicated in ALS pathogenesis using pharmacological approaches.

Cyclopsorine A treatment of G93A high mice, delivered intracerebroventricularly or systemically to mice on a multiple drug resistance type 1a/b background, modestly improved outcome [152-154], but these studies are confounded by the immunosuppressant actions of cyclopsorine A through calcineurin inhibition. Pharmacological studies using CyPD inhibitors devoid of effects on calcineurin need to be done on ALS mice. Another study showed that treatment with cholest-4-en-3-one oxime (TRO19622), a drug that binds VDAC and the 18 kDa translocator protein (TSPO, or peripheral benzodiazepine receptor), improved motor performance, delayed disease onset, and extended survival of G93A high mice [155]. However, another study using a different TSPO ligand (Ro-4864) did not show positive effects with G93A high mice [156].

CyPD and ANT have been identified as targets of nitration in ALS mice [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. CyPD nitration is elevated in early-to mid-symptomatic stages, but declines to baseline at end-stage disease [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. ANT nitration is notable particularly because it is found in pre-symptomatic and symptomatic stages but not at end-stage disease or in transgenic mice expressing human wild-type SOD1 [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. The ANT is Together these data and future work could reveal that oxidative and nitrative damage to proteins,

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ACCEPTED MANUSCRIPT Martin 21 some of which are core components of the mPTP, in G93A high mice is targeted rather than stochastic and could impinge on the functioning of the mPTP.

The role of CyPD in the process of MN disease has been examined in ALS mice through geneablation [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. G93A high -mSOD1 mice without CyPD show markedly delayed disease onset and lived significantly longer than transgenic mice with CyPD. The effect of CyPD deletion was much more prominent in females than in males [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Female mice even showed positive effects with haplodeletion of CyPD. Ppif gene ablation in transgenic mice with much lower levels of human mSOD1 expression and a slower disease progression (G93A low -mSOD1 mice) also show significantly delayed disease onset and lived significantly longer than transgenic mice with CyPD [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF]. Thus, some form of mitochondrial pathobiology is occurring regardless of whether transgene expression of G93A is high

Summary and outlook

ALS is the 3 rd most common human neurodegenerative disease with an adult onset [START_REF] Rowland | Amyotrophic lateral sclerosis[END_REF][START_REF] Sathasivam | Apoptosis in amyotrophic lateral sclerosis: a review of the evidence[END_REF]. It is a paralytic disease that destroys MNs and skeletal muscle and cannot yet be cured or treated effectively; thus contracting the disease is fatal [START_REF] Cozzolino | Amyotrophic lateral sclerosis: from current developments in the laboratory to clinical implications[END_REF]. Mitochondria have diverse functions and properties and could be critically important for the development of human ALS [START_REF] Martin | Mitochondriopathy in Parkinson disease and amyotrophic lateral sclerosis[END_REF]. Structural and biochemical data from studies of human ALS and cell/animal models of ALS suggest that mitochondrial dysfunction is a trigger or propagator of neurodegeneration. Mitochondria in subsets of and apoptosome-mediated activation of caspase-3 in ALS motor neurons. These changes appear to be occurring slowly in subsets of motor neurons and could lead to cell death that is a variant phenotype of apoptosis (see Figure 3). In human ALS motor neurons, it is not known yet if alterations

Modulator of mPTP

Hexokinase

Modulator of VDAC *Known to be changed in human ALS [START_REF] Martin | Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism[END_REF][START_REF] Martin | p53 is abnormally elevated and active in the CNS of patients with amyotrophic lateral sclerosis[END_REF] + Oxidatively modified in mouse ALS [START_REF] Martin | The mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology of ALS mice[END_REF] A C C E P T E D M A N U S C R I P T

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  age-dependent [101], but this work lacks cellular resolution. A recent biochemical in vitro study has shown that endogenous SOD1 in the mitochondrial intermembrane space controls cytochrome ccatalyzed peroxidation and that G93A-mSOD1 mediates greater ROS production in the intermembrane space compared to wild-type SOD1 [102]. Human SOD1 mutants can also shift A C C E P T E D M A N U S C R I P T

  VDAC expression patterns are complicated by alternative splicing that generates two different VDAC1 mRNAs, three different VDAC2 mRNAs, and two different VDAC3 mRNAs [118]. Studies of nervous tissue have found VDAC in neurons and glial cells [139] and associated with mitochondria, the endoplasmic reticulum, and the plasma membrane [140,141]. Non-mitochondrial localizations of VDAC have been disputed [142]. Information on ANT localizations in nervous tissue is particularly scarce. ANT appears to be expressed in reactive astrocytes [143]. The few existing studies on CyPD localization in mammalian CNS have found it enriched in subsets of neurons in adult rat brain, with some interneurons being positive [144], and relative low levels in astrocytes [145,146].

  important in the context of age-related neurodegenerative disease because it undergoes carbonyl modification during aging in housefly flight muscle [157] and rat brain [158]. In vitro cell-free and cell experiments have shown that NO and ONOO -can act directly on the ANT to induce mitochondrial permeabilization in a cyclosporine A-sensitive manner [159]. Oxidative stress enhances the binding of CyPD to ANT [160]. Some SOD1 mutants are unstable and loose copper [67], and interestingly, copper interactions with ANT and thiol modification of ANT can cause mPTP opening [161-163].

  -mSOD1 mice without CyPD develop eventually MN disease and die. Other work on CypD null mice has shown that high concentrations of Ca 2+ (2 mM) can still lead to mPTP activation without CyPD and that cell deaths caused by Bid, Bax, DNA damage and TNF-α are not affected [164]. The effects of CyPD deficiency on MN cell mechanisms thus need to be examined in more detail, but the cell death phenotype might be switched or converted to another form with the attenuation of mitochondrial swelling. A switch in the cell death morphology and molecular mechanisms in MNs of mSOD1 mice without CyPD is an outcome consistent with the cell death continuum concept [12].

  neurons and skeletal muscle cells could be compromised in ALS, rendering these cells intrinsically susceptible to cellular aging and stress. Novel mechanisms for mitochondriopathy and MN degeneration in human and mouse ALS could involve apoptosis, accumulation of intracellular Ca 2+ , abnormal trafficking of mitochondria with enhanced ROS toxic potential from distal dendrites or terminals at the neuromuscular junction, distal axonopathy and target deprivation, NOS trafficking and localization to MN mitochondria, and ONOO -damage[START_REF] Martin | Motor neuron degeneration in amyotrophoc lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death[END_REF]. The mPTP actively participates in the mechanisms of MN death in ALS mice in a gender-dependent pattern. Thus, mPTP activation is a possible triggering event for MN degeneration and MN selective vulnerability in ALS could be related to amount, composition, and trafficking of mitochondria in MNs and the association of MN terminals with skeletal muscle cells. There is precedence for this logic in mouse models of Alzheimer's disease [165], multiple sclerosis [166], and stroke [167]. Further study of mitochondria in neurons and skeletal myocytes can define new mechanisms of disease in ALS and can lead to the identification of molecular mechanism-based therapies for treating this fatal disease.

Figure 1 .

 1 Figure 1. Mitochrondrial regulation of cell life and death in schematic representation. The

Figure 2 .

 2 Figure 2. PCD mechanisms regulate the degeneration of motor neurons in human amyotrohpic

  components of the mPTP (top schematic drawing), such as cyclophilin D (CypD), adenine nucleotide translocator (ANT), and voltage-dependent anion channel (VDAC). Scale bars = 6 µm (A); 3 µm (B).

Figure 3 .

 3 Figure 3. Comparison of motor neuron death phenotypes in human and transgenic mouse

Table 1

 1 The ANT and CyPD interact directly[137]. The molar concentration of CyPD (in heart mitochondria) is much less (>5%) than ANT; thus, under normal conditions only a minor fraction of the ANT can be in a complex withCyPD [117,138]. When this occurs, small ions and metabolites permeate freely across the IMM and oxidation of metabolites by O 2 proceeds with electron flux not coupled to proton pumping, resulting in collapse of ΔP,

	) [114-116]. Other

The VDAC family in human and mouse consists of three proteins of ~31 kDa (VDAC1-3) encoded by three different genes

[118]

. VDACs are the major transport proteins in the OMM, functioning in ATP rationing, Ca

2+

homeostasis, oxidative stress response, and apoptosis [118]. Monomeric VDAC serves as the functional channel, although oligomerization of VDAC into dimers and tetramers can occur and might function in cell death [118]. The VDAC adopts an open conformation at low or zero membrane potentials and a closed conformation at potentials above 30-40 mV making the OMM permeable to most small hydrophilic molecules up to 1.3 kDa for free exchange of respiratory chain substrates [119]. Most data implicating VDAC opening or closing as an important regulator of cell death are based on in vitro conditions, while limited in vivo evidence is available [120]. are not equivalent, implying different functions. Mice deficient in either VDAC1 or VDAC3 are viable [122-124], but VDAC2 deficiency causes embryonic lethality [125]. Lack of both VDAC1 and VDAC3 causes growth retardation [124]. VDAC null mouse tissues exhibit deficits in mitochondrial respiration The mitochondrial ANT family in human consists of 3 members (ANT1-3, or solute carrier family 25, members 4, 5, and 6) encoded by three different genes, but in mouse only two isoforms of the ANT are present [128]. The proteins are ~33 kDa and function as homodimers [128]. They are multipass membrane proteins, with odd-numbered transmembrane helices having kinks because of proline residues, which mediate exchange of cytosolic ADP for mitochondrial ATP across the inner the mPTP at contact sites between the inner and outer mitochondrial membranes [131]. It has been proposed that, in the presence of high mitochondrial Ca 2+ , the binding of CyPD to proline residue 61 (Pro 61 ) in loop 1 of ANT1 results in a conformation that converts the ANT into a non-specific pore CyPD (also named cyclophilin F, peptidyl prolyl iosmerase F) is encoded by a single gene [114,116,134]. Despite confusing nomenclature, there is only one isoform of CyPD (EC 5.2.1.8, ppif gene product) in mouse and human. The ~20 kDa protein encoded by this gene is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. PPIases catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. CyPD binds ANT1 [129]. During normal mitochondrial function the OMM and the IMM are separated by the intermembrane space, and the VDAC and the ANT do not interact [114]. Permeability transition is activated by the formation of the mPTP; the IMM looses its integrity and the ANT changes its conformation from its native state into a non-selective pore [135]. This process is catalyzed by CyPD that functions in protein cis-trans isomerization and chaperoning [136]. dissipation of ATP production, production of ROS, equilibration of ions between the matrix and cytosol, matrix volume increases, and mitochondrial swelling [115,119].

Table 1 . Some Mitochondrial Associated Proteins That Function in Cell Death

 1 for Bcl-2/Bcl-X L promoting Bax/Bak pore formation Bid Proapoptotic, decoy for Bcl-2/Bcl-X L promoting Bax/Bak pore formation Noxa Proapoptotic, decoy for Bcl-2/Bcl-X L promoting Bax/Bak pore formation Puma Proapoptotic, decoy for Bcl-2/Bcl-X L promoting Bax/Bak pore formation p53* Antagonizes activity of Bcl-2/Bcl-X L , promotes Bax/Bak oligomerization

	Protein	Function
	Bcl-2*	Antiapoptotic, blocks Bax/Bak channel formation
	Bcl-X L	Antiapoptotic, blocks Bax/Bak channel formation
	Bax*	Proapoptotic, forms pores for cytochrome release
	Bak*	Proapoptotic, forms pores for cytochrome release
	Bad Proapoptotic, decoy Cytochrome c Activator of apoptosome
	Smac/DIABLO	IAP inhibitor
	AIF	Antioxidant flavoprotein/released from mitochondria to promote
		nuclear DNA fragmentation
	Endonuclease G	Released from mitochondria to promote nuclear DNA fragmentation
	HtrA2/Omi	IAP inhibitor
	VDAC	mPTP component in outer mitochondrial membrane
	ANT +	mPTP component in inner mitochondrial membrane
	Cyclophilin D +	mPTP component in mitochondrial matrix
	TSPO (peripheral
	benzodiazepine
	receptor)

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ACKNOWLEDGMENTS

The author thanks all of the individuals in his lab, particularly Yan Pan, Ann Price, and Barry Gertz for data generated on human ALS and G93A-mSOD1 mice. This work was supported by grants from the U.S. Public Health Service, NIH-NINDS (NS065895, NS052098) and NIH-NIA (AG016282).

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