benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Nerve-Degeneration

In humans and many other mammalian species, the behavioural consequences of a cortical lesion tend to be milder when it occurs early in life, and there is evidence that an important factor contributing to the behavioural sparing in the young is the formation of new thalamo-cortical connections by thalamic neurons initially connected with the lesioned area. However, this plasticity may be hindered by the secondary death of many of these neurons owing to the elimination by the primary lesion of their trophic support from the cortex. With the long-term aim of preventing this neuronal death, we have here characterised its timing in the lateral geniculate nucleus of ferrets following lesions of the visual cortex on postnatal days 5, 10, 20 or 35. After the earliest lesions (P5 or P10), this cell death began rapidly and occurred synchronously, being maximal at 48 h and declining to zero over the next few days. Following later lesions the cell death began more slowly and continued for longer. The dying neurons contained activated caspase-3 and fragmented DNA and their number 2 days after a P5 lesion was reduced by the broad-band caspase inhibitor z-VAD.fmk. These experiments open the way for a concerted effort to enhance adaptive plasticity by neuroprotection in the hours or days following a cortical lesion.

Topics: Age Factors; Aging; Amino Acid Chloromethyl Ketones; Animals; Brain Damage, Chronic; Caspase 3; Caspase Inhibitors; Cell Death; Denervation; DNA Fragmentation; Enzyme Inhibitors; Female; Ferrets; Geniculate Bodies; Male; Nerve Degeneration; Neuronal Plasticity; Neuroprotective Agents; Time Factors; Visual Cortex; Visual Pathways

Emerging lines of evidence show that the mechanisms of neurite degeneration are convergent, with poor neuritic transport, mitochondrial dysfunction and an increase in intra-axonal calcium being the principal convergence points. Nevertheless, the details are unclear. Here, we revealed the induction of autophagy in degenerating neurites of sympathetic neuron initiated by three different experimental paradigms. Autophagosomes were colocalized with collapsed cytoskeletal proteins in neuritic beadings during degeneration. Accumulation of microtubule-associated protein light chain 3-II, which is the most reliable marker for autophagy, was observed in the early stage of neurite degeneration. The autophagy inhibitor 3-methyladenine efficiently suppressed neurite degeneration by protecting neurites from the loss of viability and mitochondrial function. Furthermore, knocking down the key autophagy-related genes Atg7 and Beclin1 significantly delayed axonal and dendritic degeneration after nerve growth factor deprivation. Reduced expression of Atg7 also suppressed neurite fragmentation after transection. Therefore, our present data suggest the critical role of autophagy in neurite degeneration and may provide a valuable clue in understanding the mechanism of axonal and dendritic degeneration.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 7; Beclin-1; Cells, Cultured; Gene Expression Regulation; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Nerve Degeneration; Nerve Growth Factor; Neurites; Neurons; Neuroprotective Agents; Proteins; Rats; RNA Interference; Superior Cervical Ganglion; Time Factors; Transfection

Glutaryl-CoA dehydrogenase deficiency is an inherited metabolic disease characterized by elevated concentrations of glutaric acid (GA) and its metabolites glutaconic acid (GC) and 3-hydroxy-glutaric acid (3-OH-GA). Its hallmarks are striatal and cortical degeneration, which have been linked to excitotoxic neuronal cell death. However, magnetic resonance imaging studies have also revealed widespread white matter disease. Correspondingly, we decided to investigate the effects of GA, GC, and 3-OH-GA on the rat immature oligodendroglia cell line, OLN-93. For comparison, we also exposed the neuroblastoma line SH-SY5Y and the microglia line BV-2 to GA, GC, and 3-OH-GA. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. Flow cytometry was used to assess apoptosis via annexin-V, anti-active caspase-3 antibody, and propidium iodide staining. GA, GC, and 3-OH-GA reduced OLN-93 oligodendroglia cell viability in a dose-dependent manner. Toxicity of GA, GC, and 3-OH-GA was abrogated by preincubation with the pan-caspase inhibitor z-VAD-fmk. Apoptosis but not necrosis was detected at various stages (early: annexin-V; effector: caspase-3) after 24-48 h of incubation with GA, GC, or 3-OH-GA in OLN-93 but not in neuroblastoma or microglia cells. OLN-93 lacked expression of N-methyl-d-aspartate receptors, making classical glutamatergic excitotoxicity an unlikely explanation for the selective toxicity of GA, GC, and 3-OH-GA for OLN-93 cells. GA, GC, and 3-OH-GA directly initiate the apoptotic cascade in oligodendroglia cells. This mechanism may contribute to the white matter damage observed in glutaryl-CoA dehydrogenase deficiency.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Base Sequence; Brain Diseases, Metabolic, Inborn; Caspase Inhibitors; Cell Differentiation; Cell Line; Glutarates; Glutaryl-CoA Dehydrogenase; Humans; Nerve Degeneration; Oligodendroglia; Oxidoreductases Acting on CH-CH Group Donors; Rats; Receptors, Glutamate; RNA, Messenger

Trauma triggers diffuse apoptotic neurodegeneration in the developing rat brain. To explore the pathogenesis of this phenomenon we investigated the involvement of three possible mechanisms: death receptor activation, activation of the intrinsic apoptotic pathway by cytochrome c release into the cytoplasm, and changes in trophic support provided by endogenous neurotrophins. We detected a decrease in the expression of bcl-2 and bcl-x(L), two antiapoptotic proteins that decrease mitochondrial membrane permeability, an increase in cytochrome c immunoreactivity in the cytosolic fraction, and an activation of caspase-9 in brain regions which show apoptotic neurodegeneration following percussion brain trauma in 7-day-old rats. Increase in the expression of the death receptor Fas was revealed by RT-PCR analysis, Western blotting, and immunohistochemistry, as was activation of caspase-8 in cortex and thalamus. Apoptotic neurodegeneration was accompanied by an increase in the expression of BDNF and NT-3 in vulnerable brain regions. The pancaspase inhibitor z-VAD.FMK ameliorated apoptotic neurodegeneration with a therapeutic time window of up to 8 h after trauma. These findings suggest involvement of intrinsic and extrinsic apoptotic pathways in neurodegeneration following trauma to the developing rat brain. Upregulation of neurotrophin expression may represent an endogenous mechanism that limits this apoptotic process.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; bcl-X Protein; Brain; Brain Injuries; Brain-Derived Neurotrophic Factor; Caspase 9; Caspases; Cyclin D1; Cytochrome c Group; Disease Models, Animal; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme Inhibitors; fas Receptor; Immunohistochemistry; Nerve Degeneration; Neurons; Neurotrophin 3; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction

The mechanisms underlying kainate (KA) neurotoxicity are still not well understood. We previously reported that KA-mediated neuronal damage in organotypic cultures of hippocampal slices was associated with p53 induction. Recently, both bax and caspase-3 have been demonstrated to be key components of the p53-dependent neuronal death pathway. Caspase activation has also been causally related to the release of mitochondrial cytochrome c (Cyto C) in the cytoplasm as a result of the collapse of the mitochondrial membrane potential (Deltapsi(M)) and the opening of mitochondrial permeability transition pores (mPTP). In the present study, we observed a rapid induction of bax in hippocampal slice cultures after KA treatment. In addition, the levels of Cyto C and caspase-3 were increased in the cytosol while the level of the caspase-9 precursor was decreased. There was also a complete reduction of Rhodamine 123 fluorescence after KA treatment, an indication of Deltapsi(M) dissipation. Furthermore, inhibition of mPTP opening by cyclosporin A partially prevented Cyto C release, caspase activation and neuronal death. These data suggest the involvement of bax, several caspases, as well as Cyto C release in KA-elicited neuronal death. Finally, inhibition of caspase-3 activity by z-VAD-fmk only partially protected neurons from KA toxicity, implying that multiple mechanisms may be involved in KA excitotoxicity.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; bcl-2-Associated X Protein; Brain Diseases; Caspase Inhibitors; Caspases; Cells, Cultured; Cyclosporine; Cytochrome c Group; Cytosol; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Hippocampus; Immunohistochemistry; Kainic Acid; Membrane Potentials; Mitochondria; Nerve Degeneration; Neurons; Neuroprotective Agents; Neurotoxins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyramidal Cells; Rats; RNA, Messenger

Mutations in the copper/zinc superoxide dismutase (SOD1) gene produce an animal model of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. To test a new therapeutic strategy for ALS, we examined the effect of caspase inhibition in transgenic mice expressing mutant human SOD1 with a substitution of glycine to alanine in position 93 (mSOD1(G93A)). Intracerebroventricular administration of zVAD-fmk, a broad caspase inhibitor, delays disease onset and mortality. Moreover, zVAD-fmk inhibits caspase-1 activity as well as caspase-1 and caspase-3 mRNA up-regulation, providing evidence for a non-cell-autonomous pathway regulating caspase expression. Caspases play an instrumental role in neurodegeneration in transgenic mSOD1(G93A) mice, which suggests that caspase inhibition may have a protective role in ALS.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Substitution; Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Disease Progression; Enzyme Activation; Gene Expression Regulation, Enzymologic; Humans; Injections, Intraventricular; Interleukin-1; Male; Mice; Mice, Transgenic; Motor Neurons; Nerve Degeneration; Neuroprotective Agents; Psychomotor Performance; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1

Activation of ionotropic glutamate receptors of the AMPA and NMDA subtypes likely contributes to neuronal injury and death in various neurodegenerative disorders. Excitotoxicity can manifest as either apoptosis or necrosis, but the mechanisms that determine the mode of cell death are not known. We now report that levels of AMPA receptor subunits GluR-1 and GluR-4 are rapidly decreased in cultured rat hippocampal neurons undergoing apoptosis in response to withdrawal of trophic support (WTS), whereas levels of NMDA receptor subunits NR1, NR2A, and NR2B are unchanged. Exposure of isolated synaptosomal membranes to "apoptotic" cytosolic extracts resulted in rapid degradation of AMPA receptor subunits. Treatment of cells and synaptosomal membranes with the caspase inhibitors prevented degradation of AMPA receptor subunits, demonstrating a requirement for caspases in the process. Calcium responses to AMPA receptor activation were reduced after withdrawal of trophic support and enhanced after treatment with caspase inhibitors. Vulnerability of neurons to excitotoxic necrosis was decreased after withdrawal of trophic support and potentiated by treatment with caspase inhibitors. Our data indicate that caspase-mediated degradation of AMPA receptor subunits occurs during early periods of cell stress and may serve to ensure apoptosis by preventing excitotoxic necrosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Caspases; Cell Survival; Cells, Cultured; Excitatory Amino Acid Agonists; Fluorescent Antibody Technique; Glutamic Acid; Hippocampus; N-Methylaspartate; Necrosis; Neocortex; Nerve Degeneration; Nerve Growth Factors; Neurons; Neuroprotective Agents; Neurotoxins; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Synaptosomes

Evidence has accumulated that Zn2+ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy. In the present study, we examined patterns and possible mechanisms of Zn2+ neurotoxicity. Inclusion of 30-300 microM Zn2+ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures. This Zn2+ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase. Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn2+ neurotoxicity. Interestingly, antioxidants, trolox or SKF38393, attenuated Zn(2+)-induced neuronal necrosis. Pretreatment with insulin or brain-derived neurotrophic factor increased the Zn(2+)-induced free radical injury. Kainate or AMPA facilitated Zn2+ entry and potentiated Zn2+ neurotoxicity in a way sensitive to trolox. Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn2+ neurotoxicity. These findings indicate that entry and accumulation of Zn2+ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Brain-Derived Neurotrophic Factor; Cells, Cultured; Cerebral Cortex; Chromans; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Dopamine Agonists; Drug Synergism; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Free Radicals; Hypoglycemic Agents; Insulin; Kainic Acid; Lipid Peroxidation; Mice; Microscopy, Electron; Mitochondrial Swelling; Necrosis; Nerve Degeneration; Neurons; Neurotoxins; Oligopeptides; Oxidative Stress; Pregnancy; Zinc

Hippocampal neurons from the trisomy 16 (Ts16) mouse, a potential animal model of Down's syndrome (trisomy 21) and neurodegenerative disorders such as Alzheimer's disease (AD), die at an accelerated rate in vitro. Here, we present evidence that the accelerated neuronal death in Ts16 occurs by apoptosis, as has been reported for neurons in AD. First, the nuclei of dying Ts16 neurons are pyknotic and undergo DNA fragmentation, as revealed by terminal transferase-mediated dUTP nick end-labeling. Second, the accelerated death of Ts16 neurons is prevented by inhibitors of the caspase family of proteases, which are thought to act at a late, obligatory step in the apoptosis pathway. In the presence of maximally effective concentrations of caspase inhibitors, Ts16 neuron survival was indistinguishable from that of control neurons. These results suggest that overexpression of one or more genes on mouse chromosome 16 leads to caspase-mediated apoptosis in Ts16 neurons.

Topics: Alzheimer Disease; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; Down Syndrome; Female; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nerve Degeneration; Neurons; Oligopeptides; Pregnancy; Trisomy

6-Hydroxydopamine (6-OHDA) administered intrastriatally to adult rats in a single injection causes neurodegeneration of the nigrostriatal pathway and loss of > 50% of dopamine neurons in substantia nigra pars compacta 30 days after administration. The death of nigral neurons occurs, at least partially, by a caspase-mediated mechanism. The nigral loss of dopaminergic neurons could be prevented by stereotaxical administration of zVAD.fmk, a caspase inhibitor, into the substantia nigra, indicating that 6-OHDA-induced nigrostriatal degeneration involves caspase activation. These results suggest that caspases are probably involved in neurodegenerative chronic processes such as Parkinson's disease and might be considered as possible targets in the treatment of such neurological disorders.

Topics: Amino Acid Chloromethyl Ketones; Animals; Caspase Inhibitors; Corpus Striatum; Cysteine Proteinase Inhibitors; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidopamine; Rats; Rats, Sprague-Dawley