cytochrome-c-t and 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.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Central Nervous System; Cytochromes c; Humans; Mice; Mitochondria; Motor Neurons; Mutation; Oxidative Stress; Superoxide Dismutase; Superoxide Dismutase-1

Apoptosis of motor neurons is an important feature in amyotrophic lateral sclerosis (ALS). A vital role of mitochondria in apoptosis and cell survival is well documented. Eventually mitochondria have shown to be an early target in the pathogenesis of ALS. On account of these facts, we investigated the involvement of mitochondrial-dependent apoptosis in ALS and control (CTR) cybrids, generated fusing human platelets with mitochondrial DNA-depleted NT2-neuroteratocarcinoma cells. After a 6 week selection process during which transferred subject mtDNA repopulated the NT2 cells and restored mitochondrial oxygen consumption, we assessed cell viability and two programmed cell death parameters, caspase 3 activity and cytosolic cytochrome c levels. Compared to the control cybrid lines (n = 5), the ALS cybrid lines (n = 10) showed 45% less XTT reduction and higher caspase 3 activity ( p < 0.05, two-way Student's t test) exhibiting lesser cell viability and execution of apoptosis. Elevated cytosolic cytochrome c levels in ALS cybrid lines (n = 8) than in CTR (n = 4) ( p < 0.05, two-way Student's t-test) indicating its mitochondrial release and initiation of apoptosis. This indicates apoptosis as one of the possible mechanisms of cell death in ALS. Our findings support the view that in ALS, subject's mitochondria are altered in non-degenerating tissues in such a way that intrinsic apoptotic pathway activity is relatively increased.

Topics: Amyotrophic Lateral Sclerosis; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; DNA, Mitochondrial; Humans; Hybrid Cells

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss. Evidence suggests that mitochondrial dysfunction, apoptosis, oxidative stress, inflammation, glutamate excitotoxicity, and proteasomal dysfunction are all responsible for ALS pathogenesis. N-acetyl-tryptophan has been identified as an inhibitor of mitochondrial cytochrome c release and therefore is a potential neuroprotective agent. By quantifying cell death, we demonstrate that N-acetyl-l-tryptophan (L-NAT) and N-acetyl-DL-tryptophan are neuroprotective in NSC-34 motor neuron-like cells and/or primary motor neurons, while their isomer N-acetyl-d-tryptophan has no protective effect. These findings are consistent with energy minimization and molecular modeling analysis, confirming that L-NAT generates the most stable complex with the neurokinin-1 receptor (NK-1R). L-NAT inhibits the secretion of Substance P and IL-1β (Enzyme-Linked Immunosorbent Assay and/or dot blots) and mitochondrial dysfunction by effectively inhibiting the release of cytochrome c/Smac/AIF from mitochondria into the cytoplasm and activation of apoptotic pathways, including the activation of caspase-1, -9, and -3, as well as proteasomal dysfunction through restoring chymotrypsin-like, trypsin-like, and caspase-like proteasome activity. These data provide insight into the molecular mechanisms by which L-NAT offers neuroprotection in models of ALS and suggest its potential as a novel therapeutic strategy for ALS. We demonstrate that L-NAT (N-acetyl-l-tryptophan), but not D-NAT, rescues NSC-34 cells and primary motor neurons from cell death. L-NAT inhibits the secretion of Substance P and IL-1β, and caspase-1 activation, the release of cytochrome c/Smac/AIF, and the activation of caspase -9, and -3, as well as proteasomal dysfunction. The data suggest the potential of L-NAT as a novel therapeutic strategy for amyotrophic lateral sclerosis (ALS). AIF, apoptosis-inducing factor.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Caspases; Cell Line; Cytochromes c; Drug Evaluation, Preclinical; Hybrid Cells; Interleukin-1beta; Mice; Mitochondria; Motor Neurons; Neurokinin-1 Receptor Antagonists; Neuroprotective Agents; Proteasome Endopeptidase Complex; Receptors, Neurokinin-1; Stereoisomerism; Substance P; Tryptophan

Glycogen synthase kinase-3β (GSK-3β) activity plays a central role in motor neuron degeneration. GSK-3β inhibitors have been shown to prolong motor neuron survival and suppress disease progression in amyotrophic lateral sclerosis (ALS). In this study, we evaluated the therapeutic effects of a new GSK-3b inhibitor, JGK-263, on ALS in G93A SOD1 transgenic mice.. Previously, biochemical efficacy of JGK-263 was observed in normal and mutant (G93A) hSOD1-transfected motor neuronal cell lines (NSC34). Based on these previous results, we administered JGK-263 orally to 93 transgenic mice with the human G93A-mutated SOD1 gene. The mice were divided into three groups: a group administered 20mg/kg JGK-263, a group administered 50mg/kg JGK-263, and a control group not administered with JGK-263. Clinical status, rotarod test, and survival rates of transgenic mice with ALS were evaluated. Sixteen mice from each group were selected for further biochemical study that involved examination of motor neuron count, apoptosis, and cell survival signals.. JGK-263 administration remarkably improved motor function and prolonged the time until symptom onset, rotarod failure, and death in transgenic mice with ALS compared to control mice. In JGK-263 groups, choline acetyltransferase (ChAT) staining in the ventral horn of the lower lumbar spinal cord showed a large number of motor neurons, suggesting normal morphology. The neuroprotective effects of JGK-263 in ALS mice were also suggested by western blot analysis of spinal cord tissues in transgenic mice.. These results suggest that JGK-263, an oral GSK-3β inhibitor, is promising as a novel therapeutic agent for ALS. Still, further biochemical studies on the underlying mechanisms and safety of JGK-263 are necessary.

Topics: Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Caspase 3; Choline O-Acetyltransferase; Cytochromes c; Disease Models, Animal; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mice, Transgenic; Motor Activity; Motor Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Single-Blind Method; Spinal Cord; Superoxide Dismutase; Time Factors

Caspase-mediated cell death contributes to the pathogenesis of motor neuron degeneration in the mutant SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS), along with other factors such as inflammation and oxidative damage. By screening a drug library, we found that melatonin, a pineal hormone, inhibited cytochrome c release in purified mitochondria and prevented cell death in cultured neurons. In this study, we evaluated whether melatonin would slow disease progression in SOD1(G93A) mice. We demonstrate that melatonin significantly delayed disease onset, neurological deterioration and mortality in ALS mice. ALS-associated ventral horn atrophy and motor neuron death were also inhibited by melatonin treatment. Melatonin inhibited Rip2/caspase-1 pathway activation, blocked the release of mitochondrial cytochrome c, and reduced the overexpression and activation of caspase-3. Moreover, for the first time, we determined that disease progression was associated with the loss of both melatonin and the melatonin receptor 1A (MT1) in the spinal cord of ALS mice. These results demonstrate that melatonin is neuroprotective in transgenic ALS mice, and this protective effect is mediated through its effects on the caspase-mediated cell death pathway. Furthermore, our data suggest that melatonin and MT1 receptor loss may play a role in the pathological phenotype observed in ALS. The above observations indicate that melatonin and modulation of Rip2/caspase-1/cytochrome c or MT1 pathways may be promising therapeutic approaches for ALS.

Topics: Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Antioxidants; Caspase 3; Cell Death; Cytochromes c; Disease Models, Animal; Disease Progression; Enzyme-Linked Immunosorbent Assay; Melatonin; Mice; Mice, Transgenic; Receptor, Melatonin, MT1; Signal Transduction; Superoxide Dismutase

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.

Topics: Activating Transcription Factor 6; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Cell Line, Tumor; Cytochromes c; Down-Regulation; Endoplasmic Reticulum Stress; Humans; Membrane Proteins; Mice; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Transcription Factor CHOP; Up-Regulation

Amyotrophic lateral sclerosis (ALS) is a devastating disorder involving loss of movement due to degeneration of motor neurons. Studies suggest that in ALS axonal dysfunction precedes the death of motor neurons. Pathologically, ALS is characterized by neurofilamentous swellings (spheroids) within the axons of motor neurons. However, the causes of this axonopathy and possible resulting axonal dysfunction are not known. Using a novel model of cultured mouse motor neurons, we have determined that these neurons are susceptible to proximal axonopathy, which is related to the glial environment. This axonopathy showed remarkable similarity, both morphologically and neurochemically, to spheroids that develop over months in SOD1(G93A) transgenic mice. Focal ubiquitination, as well as perturbations of neurofilaments and microtubules, occurred in the axonal spheroid-like swellings in vitro, and visualization of mitochondrial dynamics demonstrated that axonopathy resulted in impaired axonal transport. These data provide strong evidence for the involvement of non-neuronal cells in axonal dysfunction in ALS. This cell culture model may be of benefit for the development of therapeutic interventions directed at axonal preservation.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Axons; Bacterial Proteins; Cell Death; Cells, Cultured; Cytochromes c; Cytoskeleton; Disease Models, Animal; Edema; Glial Fibrillary Acidic Protein; Humans; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Neurons; Neurofilament Proteins; Neuroglia; Spinal Cord; Superoxide Dismutase; Synaptophysin; Time Factors; Transfection

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by a progressive, selective loss of motor neurons (MN) in brain and spinal cord. The mechanisms of selective and age-dependent MN degeneration in ALS have not been defined. Recent studies suggest that the elevation of intracellular oxidative toxicity contributes to death of MN, but the molecular pathways remain largely unknown. In order to study the possible molecular pathways that the oxidative toxicity induced MN death in ALS, a MN-like cell NSC34, a primary neuronal cell (PNC) of mouse prontal cortex, and a G93A-SOD1 transgenic mouse model were used. Exposure of NSC34 and PNC to cobalt chloride or chronic sustained hypoxic conditions showed a dramatic increase of cellular Hif-1α (hypoxia inducing factor-1α), HO-1 (heme oxygenases-1), and UCP4 (uncoupling protein 4) expression by Western blot analysis, accompanied with increasing cellular apoptosis by histone protein release assay. In an ALS mouse model, the caspase 3 activation, Aif (apoptosis inducing factor), cytochrome c redistribution in MN of spinal cord significantly increased at 70days of disease progression, and Hif-1α expression significantly increased at whole disease stages by an immunohistochemical positive cell counting and Western blot analysis, respectively. The data on this in vitro and in vivo study suggested that oxidative toxicity promoted multiple molecular pathways associated with MN death in ALS and at least were partially associated with the changes of Hif-1α, HO-1, UCP4 expressive increment, caspase 3 activation and Aif, cytochrome c redistribution.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Caspase 3; Cells, Cultured; Cytochromes c; Disease Models, Animal; Embryo, Mammalian; Gene Expression Regulation; Heme Oxygenase-1; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Transport Proteins; Mice; Mice, Transgenic; Mitochondrial Uncoupling Proteins; Motor Neurons; Prefrontal Cortex; Superoxide Dismutase; Time Factors

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disorder. Mutations in Cu,Zn superoxide dismutase (SOD1) cause approximately 20% of familial ALS. One of the possible mechanisms whereby they induce disease is mitochondrial dysfunction in motor neurons. Here we describe a patient with ALS and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation. Direct sequencing of SOD1 gene revealed a heterozygous mutation in codon 22 substituting a highly conserved amino acid, from glutamine to arginine (Q22R). Muscle biopsy showed a neurogenic pattern associated with cytochrome c oxidase (COX) deficiency in several muscle fibers. Western blot analysis demonstrated a reduction in SOD1 content in the cytoplasmic and mitochondrial fractions. These results suggest that a minute quantity of mutant SOD1 protein contributes to a mitochondrial toxicity also in muscle tissue.

Topics: Adult; Amyotrophic Lateral Sclerosis; Arginine; Cytochromes c; DNA Mutational Analysis; Family Health; Genetic Linkage; Genetic Predisposition to Disease; Glutamic Acid; Humans; Male; Mitochondria, Muscle; Mitochondrial Diseases; Mutation; Superoxide Dismutase; Superoxide Dismutase-1

Mutations in Cu, Zn-superoxide dismutase 1 (SOD1) are associated with degeneration of motor neurons in the disease, familial amyotrophic lateral sclerosis. Intracellular protein inclusions containing mutant SOD1 (mSOD1) are associated with disease but it is unclear whether they are neuroprotective or cytotoxic. We report here that the formation of mSOD1 inclusions in a motor neuron-like cell line (NSC-34) strongly correlates with apoptosis via the mitochondrial death pathway. Applying confocal microscopic analyses, we observed changes in nuclear morphology and activation of caspase 3 specifically in cells expressing mSOD1 A4V or G85R inclusions. Furthermore, markers of mitochondrial apoptosis (activation and recruitment of Bax, and cytochrome c redistribution) were observed in 30% of cells bearing mSOD1 inclusions but not in cells expressing dispersed SOD1. In the presence of additional apoptotic challenges (staurosporine, etoposide, and hydrogen peroxide), cells bearing mSOD1 inclusions were susceptible to further apoptosis suggesting they were in a pro-apoptotic state, thus confirming that inclusions are linked to toxicity. Surprisingly, cells displaying dispersed SOD1 [both wildtype (WT) and mutant] were protected against apoptosis upstream of mitochondrial apoptotic signaling, induced by all agents tested. This protection against apoptosis was unrelated to SOD1 enzymatic activity because the G85R that lacks enzymatic function protected cells similarly to both WT SOD1 and A4V that possesses WT-like activity. These findings demonstrate new aspects of SOD1 in relation to cellular viability; specifically, mSOD1 can be either neuroprotective or cytotoxic depending on its aggregation state.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; bcl-2-Associated X Protein; Cell Line; Cell Nucleus; Cytochromes c; Humans; Immunohistochemistry; Inclusion Bodies; Mice; Microscopy, Confocal; Mitochondria; Motor Neurons; Mutation; Neuroprotective Agents; Signal Transduction; Superoxide Dismutase; Superoxide Dismutase-1

Adenylate kinase 4 (AK4) is a unique member with no enzymatic activity in vitro in the adenylate kinase (AK) family although it shares high sequence homology with other AKs. It remains unclear what physiological function AK4 might play or why it is enzymatically inactive. In this study, we showed increased AK4 protein levels in cultured cells exposed to hypoxia and in an animal model of the neurodegenerative disease amyotrophic lateral sclerosis. We also showed that short hairpin RNA (shRNA)-mediated knockdown of AK4 in HEK293 cells with high levels of endogenous AK4 resulted in reduced cell proliferation and increased cell death. Furthermore, we found that AK4 over-expression in the neuronal cell line SH-SY5Y with low endogenous levels of AK4 protected cells from H(2)O(2) induced cell death. Proteomic studies revealed that the mitochondrial ADP/ATP translocases (ANTs) interacted with AK4 and higher amount of ANT was co-precipitated with AK4 when cells were exposed to H(2)O(2) treatment. In addition, structural analysis revealed that, while AK4 retains the capability of binding nucleotides, AK4 has a glutamine residue instead of a key arginine residue in the active site well conserved in other AKs. Mutation of the glutamine residue to arginine (Q159R) restored the adenylate kinase activity with GTP as substrate. Collectively, these results indicate that the enzymatically inactive AK4 is a stress responsive protein critical to cell survival and proliferation. It is likely that the interaction with the mitochondrial inner membrane protein ANT is important for AK4 to exert the protective benefits to cells under stress.

Topics: Adenylate Kinase; Amino Acid Sequence; Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cell Hypoxia; Cell Line; Cytochromes c; Humans; Hydrogen Peroxide; Mice; Mice, Transgenic; Mitochondria; Mitochondrial ADP, ATP Translocases; Molecular Sequence Data; Oxidative Stress; RNA, Small Interfering

We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Axons; Cytochromes c; Disease Models, Animal; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Mutation; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Superoxide Dismutase; Tyrosine; Ubiquitin

Glycogen synthase kinase (GSK)-3 has recently been implicated in the pathogenesis of neurodegenerative diseases. Although the neuroprotective effects of GSK-3 inhibitors in Alzheimer's disease have been established, their effects on amyotrophic lateral sclerosis (ALS) have not been well defined. This study was undertaken to evaluate the effects of GSK-3 inhibition in the G93A-SOD1 mouse model of ALS. Groups of G93A-SOD1 mice were treated with varying concentrations of GSK-3 inhibitor VIII, a specific GSK-3 inhibitor that crosses the BBB, intraperitoneally 5 days a week after 60 days of age. The GSK-3 inhibitor VIII treatment significantly delayed the onset of symptoms and prolonged the life span of the animals, and inhibited the activity of GSK-3 in a concentration-dependent manner. Furthermore, this treatment preserved survival signals and attenuated death and inflammatory signals. These data suggest that GSK-3 plays an important role in the pathogenic mechanisms of ALS and that inhibition of GSK-3 could be a potential therapeutic candidate for ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Caspase 3; Cell Death; Cyclooxygenase 2; Cytochromes c; Cytosol; Disease Progression; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Humans; In Situ Nick-End Labeling; Intercellular Adhesion Molecule-1; Mice; Mice, Transgenic; Motor Neurons; Poly(ADP-ribose) Polymerases; Postural Balance; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Thiazoles; Urea

There is evidence showing impaired mitochondrial energy production and increased oxidative damage to mitochondria in amyotrophic lateral sclerosis (ALS). It is known that a lack of cytochrome c (CyC) in the mitochondrial intermembrane space can increase free radical release from mitochondria through interruption of the electron transport. CyC also plays a role in the apoptotic cell death which is suspected in ALS. The aim of the study was to measure cerebrospinal fluid (CSF) and serum CyC levels in patients with ALS.. Forty ALS patients were diagnosed according to the El Escorial criteria of ALS. The clinical state of the patients was measured using the Amyotrophic Lateral Sclerosis Functional Rating Scale [ALSFRS].. It was shown that overall CyC levels were significantly decreased by 46 % in the CSF of patients with ALS compared with controls (p<0.05), and not affected in serum of patients with ALS (p>0.05). There was no significant difference in CyC levels in relation to the clinical parameters of the disease (p>0.05).. The study indicates that CyC may play a role in the pathogenesis of ALS. A possible mechanism is that increased neurodegeneration in ALS caused by free radical production decreases the concentrations of CyC.

Topics: Adult; Age Factors; Aged; Amyotrophic Lateral Sclerosis; Apoptosis; Case-Control Studies; Cerebrospinal Fluid Proteins; Cytochromes c; Down-Regulation; Electron Transport; Female; Humans; Male; Middle Aged; Mitochondria; Nerve Degeneration; Reference Values

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1(G93A) transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Binding, Competitive; Cell Enlargement; Cell Survival; Cells, Cultured; Cholestenones; Cytochromes c; Female; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria; Motor Neurons; Nerve Growth Factors; Nerve Regeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, GABA; Sciatic Nerve; Superoxide Dismutase; Superoxide Dismutase-1; Survival Analysis; Voltage-Dependent Anion Channels

Several studies have indicated that apoptotic pathways are responsible for the loss of motor neurons that constitute the hallmark of amyotrophic lateral sclerosis (ALS). In this study, we demonstrate that apoptosis induced by the expression of several mutant Cu,Zn superoxide dismutases (SOD1) typical of familial ALS is mediated by Apaf1, a scaffold protein involved in neural development. Using different cell lines of neuronal origin and modulating the expression of both mutant SOD1s and Apaf1, we show that the removal of Apaf1 prevents cells death. Interestingly, intercepting activation of the caspases cascade is also effective in preventing both the mitochondrial damage and the increase in the production of reactive oxygen species induced by fALS-SOD1, even in the presence of cytochrome c release. This death pathway may be crucial also for the pathogenesis of the sporadic form of the disease, where markers of increased oxidative stress and mitochondria damage have been found.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Cell Line, Tumor; Cytochromes c; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Mitochondria; Nerve Degeneration; Neuroblastoma; Oxidative Stress; Proteins; Superoxide Dismutase; Superoxide Dismutase-1

A "gain-of-function" toxic property of mutant Cu-Zn superoxide dismutase 1 (SOD1) is involved in the pathogenesis of some familial cases of amyotrophic lateral sclerosis (ALS). Expression of a mutant form of the human SOD1 gene in mice causes a degeneration of motor neurons, leading to progressive muscle weakness and hindlimb paralysis. Transgenic mice overexpressing a mutant human SOD1 gene (G93A-SOD1) were used to examine the mitochondrial involvement in familial ALS. We observed a decrease in mitochondrial respiration in brain and spinal cord of the G93A-SOD1 mice. This decrease was significant only at the last step of the respiratory chain (complex IV), and it was not observed in transgenic wild-type SOD1 and nontransgenic mice. Interestingly, this decrease was evident even at a very early age in mice, long before any clinical symptoms arose. The effect seemed to be CNS specific, because no decrease was observed in liver mitochondria. Differences in complex IV respiration between brain mitochondria of G93A-SOD1 and control mice were abolished when reduced cytochrome c was used as an electron donor, pinpointing the defect to cytochrome c. Submitochondrial studies showed that cytochrome c in the brain of G93A-SOD1 mice had a reduced association with the inner mitochondrial membrane (IMM). Brain mitochondrial lipids, including cardiolipin, had increased peroxidation in G93A-SOD1 mice. These results suggest a mechanism by which mutant SOD1 can disrupt the association of cytochrome c with the IMM, thereby priming an apoptotic program.

Topics: Aging; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Ascorbic Acid; Brain; Cytochromes c; Disease Models, Animal; Electron Transport; Electron Transport Complex IV; Female; Humans; Intracellular Membranes; Lipid Peroxidation; Male; Mice; Mice, Transgenic; Mitochondria; Nitric Oxide Synthase; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Tetramethylphenylenediamine

Multiple molecular defects trigger cell death in amyotrophic lateral sclerosis (ALS). Among these, altered transcriptional activity may perturb many cellular functions, leading to a cascade of secondary pathological effects. We showed that pharmacological treatment, using the histone deacetylase inhibitor sodium phenylbutyrate, significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice. Phenylbutyrate administration ameliorated histone hypoacetylation observed in G93A mice and induced expression of nuclear factor-kappaB (NF-kappaB) p50, the phosphorylated inhibitory subunit of NF-kappaB (pIkappaB) and beta cell lymphoma 2 (bcl-2), but reduced cytochrome c and caspase expression. Curcumin, an NF-kappaB inhibitor, and mutation of the NF-kappaB responsive element in the bcl-2 promoter, blocked butyrate-induced bcl-2 promoter activity. We provide evidence that the pharmacological induction of NF-kappaB-dependent transcription and bcl-2 gene expression is neuroprotective in ALS mice by inhibiting programmed cell death. Phenylbutyrate acts to phosphorylate IkappaB, translocating NF-kappaB p50 to the nucleus, or to directly acetylate NF-kappaB p50. NF-kappaB p50 transactivates bcl-2 gene expression. Up-regulated bcl-2 blocks cytochrome c release and subsequent caspase activation, slowing motor neuron death. These transcriptional and post-translational pathways ultimately promote motor neuron survival and ameliorate disease progression in ALS mice. Phenylbutyrate may therefore provide a novel therapeutic approach for the treatment of patients with ALS.

Topics: Acetylation; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Caspase Inhibitors; Cell Survival; Curcumin; Cytochromes c; Disease Progression; Gene Expression; Histones; Male; Mice; Mice, Transgenic; Motor Neurons; NF-kappa B; NF-kappa B p50 Subunit; Phenylbutyrates; Promoter Regions, Genetic; Protein Precursors; Proto-Oncogene Proteins c-bcl-2; Spinal Cord

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.

Topics: Amyotrophic Lateral Sclerosis; Animals; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Cytosol; DNA-Binding Proteins; Enzyme Activation; Green Fluorescent Proteins; Humans; Luminescent Proteins; Mice; Mitochondria; Mutation; Neuroblastoma; Neurons; Recombinant Fusion Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Ubiquitin-Protein Ligases