RNAi knockdown of Par-4 inhibits neurosynaptic degeneration in ALS-linked mice

Evidence from human amyotrophic lateral sclerosis (ALS) patients and ALS-linked Cu/Zn superoxide dismutase (Cu/Zn-SOD) transgenic mice bearing the mutation of glycine to alanine at position 93 (G93A) suggests that the pro-apoptotic protein prostate apoptosis response-4 (Par-4) might be a critical link in the chain of events leading to motor neuron degeneration. We now report that Par-4 is enriched in synaptosomes and post-synaptic density from the ventral horn of the spinal cord. Levels of Par-4 in synaptic compartments increased significantly during rapid and slow declining stages of muscle strength in hSOD1 G93A mutant mice. In the pre-muscle weakness stage, hSOD1 G93A mutation sensitized synaptosomes from the ventral horn of the spinal cord to increased levels of Par-4 expression following excitotoxic and apoptotic insults. In ventral spinal synaptosomes, Par-4-mediated production of pro-apoptotic cytosolic factor(s) was significantly enhanced by the hSOD1 G93A mutation. RNA interference (RNAi) knockdown of Par-4 inhibited mitochondrial dysfunction and caspase-3 activation induced by G93A mutation in synaptosomes from the ventral horn of the spinal cord, and protected spinal motor neurons from apoptosis. These results identify the synapse as a crucial cellular site for the cell death promoting actions of Par-4 in motor neurons, and suggest that targeted inhibition of Par-4 by RNAi may prove to be a neuroprotective strategy for motor neuron degeneration.     

Partial rescue of neural apoptosis in the Lurcher mutant mouse through elimination of tissue plasminogen activator

Lurcher is a spontaneous mouse mutant characterized by premature and aberrant apoptosis in the cerebellum. The phenotype has been shown to be caused by a point mutation in the delta2 glutamate receptor subunit gene that results in a large constitutive inward current, which has proved that endogenous excitotoxicity can lead to apoptotic cell death. Additional studies have suggested a direct link between this endogenous excitotoxicity and the activation of intracellular cell death enzymes. We have previously shown that excitotoxic neuronal degeneration elicited through exogenous insults (e.g. excitotoxins, stroke) is promoted by an extracellular cascade involving the serine protease tissue plasminogen activator (tPA). However, whether it is through necrotic or apoptotic mechanisms that this excitotoxic cell death occurs has remained contested. We describe the attenuation of the Lurcher cell death progression in tPA-deficient mice. Elimination of tPA delayed the apoptotic death of Purkinje and granule neurons in Lurcher mice, and reduced the phosphorylation of Jun and the activation of caspase 8. These results indicate that not only does tPA-promoted excitotoxic cell death proceed through a receptor-mediated apoptotic pathway, but that neuronal cell death in the Lurcher mouse is facilitated by extracellular cascades in addition to the already described intracellular pathways. Finally, these findings suggest that therapeutic benefits may be achieved for a wide variety of insults to the CNS by regulating tPA activity to preserve neuronal viability.     

Neurodegenerative disorders and ischemic brain diseases

Degeneration and death of neurons is the fundamental process responsible for the clinical manifestations of many different neurological disorders of aging, incuding Alzheimer's disease, Parkinson's disease and stroke. The death of neurons in such disorders involves apoptotic biochemical cascades involving upstream effectors (Par-4, p53 and pro-apoptotic Bcl-2 family members), mitochondrial alterations and caspase activation. Both genetic and environmental factors, and the aging process itself, contribute to intiation of such neuronal apoptosis. For example, mutations in the amyloid precursor protein and presenilin genes can cause Alzheimer's disease, while head injury is a risk factor for both Alzheimer's and Parkinson's diseases. At the cellular level, neuronal apoptosis in neurodegenerative disorders may be triggered by oxidative stress, metabolic compromise and disruption of calcium homeostasis. Neuroprotective (anti-apoptotic) signaling pathways involving neurotrophic factors, cytokines and conditioning responses can counteract the effects of aging and genetic predisposition in experimental models of neurodegenerative disorders. A better understanding of the molecular underpinnings of neuronal death is leading directly to novel preventative and therapeutic approaches to neurodegenerative disorders.     

Prostate Apoptosis Response-4 Mediates Trophic Factor Withdrawal-Induced Apoptosis of Hippocampal Neurons

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.     

Beneficial effects of dietary restriction on cerebral cortical synaptic terminals: preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid beta-peptide, iron, and 3-nitropropionic acid

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.     

Excitotoxic versus apoptotic mechanisms of neuronal cell death in perinatal hypoxia/ischemia

Hypoxic/ischemic (H/I) neuronal degeneration in the developing central nervous system (CNS) is mediated by an excitotoxic mechanism, and it has also been reported that an apoptosis mechanism is involved. However, there is much disagreement regarding how excitotoxic and apoptotic cell death processes relate to one another. Some authors believe that an excitotoxic stimulus directly triggers apoptotic cell death, but this interpretation is largely speculative at the present time. Our findings support the interpretation that excitotoxic and apoptotic neurodegeneration are two separate and distinct cell death processes that can be distinguished from one another by ultrastructural evaluation. Here we review evidence supporting this interpretation, including evidence that H/I in the developing CNS triggers two separate waves of neurodegeneration, the first being excitotoxic and the second being apoptotic. The first (excitotoxic) wave destroys neurons that would normally provide synaptic inputs or synaptic targets for the neurons that die in the second (apoptotic) wave. Since neurons, during the developmental period of synaptogenesis, are programmed to commit suicide if they fail to achieve normal connectivity, this explains why neuroapoptosis occurs following H/I in the developing CNS. However, it does not support the interpretation that H/I directly triggers apoptotic neurodegeneration. Rather, it documents that H/I directly triggers excitotoxic neurodegeneration, and apoptotic neurodegeneration ensues subsequently as the natural response of developing neurons to a specific kind of deprivation - loss of the ability to form normal synaptic connections.     

Proteolytic cleavage of Beclin 1 exacerbates neurodegeneration

Background

Neuronal cell loss contributes to the pathology of acute and chronic neurodegenerative diseases, including Alzheimer’s disease (AD). It remains crucial to identify molecular mechanisms sensitizing neurons to various insults and cell death. To date, the multifunctional, autophagy-related protein Beclin 1 has been shown to be both necessary and sufficient for neuronal integrity in neurodegenerative models associated with protein aggregation. Interestingly, besides its role in cellular homeostasis, Beclin 1 has also been ascribed a role in apoptosis. This makes it critical to elucidate whether Beclin 1 regulates neuronal death and survival across neurodegenerative conditions independent of protein clearance. Here, we provide experimental evidence for a direct functional link between proteolytic cleavage of Beclin 1 and apoptotic neuronal cell loss in two independent models of neurodegeneration in vivo.

Methods

Proteolytic cleavage of Beclin 1 was characterized in lysates of human AD brain samples. We developed viral tools allowing for the selective neuronal expression of the various Beclin 1 forms, including Beclin 1 cleavage products as well as a cleavage-resistant form. The effect of these Beclin 1 forms on survival and integrity of neurons was examined in models of acute and chronic neurodegeneration in vitro and in vivo. Markers of neuronal integrity, neurodegeneration and inflammation were further assessed in a Kainic acid-based mouse model of acute excitotoxic neurodegeneration and in a hAPP-transgenic mouse model of AD following perturbation of Beclin 1 in the susceptible CA1 region of the hippocampus.

Results

We find a significant increase in caspase-mediated Beclin 1 cleavage fragments in brain lysates of human AD patients and mimic this phenotype in vivo using both an excitotoxic and hAPP-transgenic mouse model of neurodegeneration. Surprisingly, overexpression of the C-terminal cleavage-fragment exacerbated neurodegeneration in two distinct models of degeneration. Local inhibition of caspase activity ameliorated neurodegeneration after excitotoxic insult and prevented Beclin 1 cleavage. Furthermore, overexpression of a cleavage-resistant form of Beclin 1 in hippocampal neurons conferred neuroprotection against excitotoxic and Amyloid beta-associated insults in vivo.

Conclusions

Together, these findings indicate that the cleavage state of Beclin 1 determines its functional involvement in both neurodegeneration and neuroprotection. Hence, manipulating the cleavage state of Beclin 1 may represent a therapeutic strategy for preventing neuronal cell loss across multiple forms of neurodegeneration.

     

The neuronal death protein par-4 mediates dopaminergic synaptic plasticity

Par-4, discovered in a screen for genes whose expression is increased in prostate tumor cells undergoing apoptosis, participates in physiological and pathological nerve cell death. A new study, however, provides evidence for an unexpected role for Par-4 in regulating synaptic transmission in the brain: Par-4 binds to the D2 dopamine receptor (D2DR) and modulates its activity. Mice in which the function of Par-4 is disrupted exhibit impaired dopaminergic neurotransmission, resulting in a depression-like syndrome. Several other cell death-related proteins also appear to function in regulating synaptic plasticity, suggesting that a better understanding of the functions of these proteins may lead to novel therapeutic approaches for a psychiatric and neurodegenerative disorders.     

Down-regulation of ARC contributes to vulnerability of hippocampal neurons to ischemia/hypoxia

ARC is a caspase recruitment domain-containing molecule that plays an important role in the regulation of apoptosis. We examined ARC expression during neuronal cell death following ischemic injury in vivo and in vitro. After exposure to transient global ischemic conditions, the expression of ARC was substantially reduced in the CA1 region of hippocampus in a time-dependent manner with concomitant increase of TUNEL-positive cells. Quantitative analysis using Western blotting exhibited that most of ARC protein disappeared in the cultured hippocampal neurons exposed to hypoxia for 12 h and showing 60% cell viability. Forced expression of ARC in the primary cultures of hippocampal neurons or B103 neuronal cells significantly reduced hypoxia-induced cell death. Further, the C-terminal P/E rich region of ARC was effective to attenuate hypoxic insults. These results suggest that down-regulation of ARC expression in hippocampal neurons may contribute to neuronal death induced by ischemia/hypoxia.     

Human Bcl-2 protects against AMPA receptor-mediated apoptosis

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.     

Apoptosis, Excitotoxicity, and Neuropathology

While a high rate of cell loss is tolerated and even required to model the developing nervous system, an increased rate of cell death in the adult nervous system underlies neurodegenerative disease. Evolutionarily conserved mechanisms involving proteases, Bcl-2-related proteins, p53, and mitochondrial factors participate in the modulation and execution of cell death. In addition, specific death mechanisms, based on specific neuronal characteristics such as excitability and the presence of specific channels or enzymes, have been unraveled in the brain. Particularly important for various human diseases are excessive nitric oxide (NO) production and excitotoxicity. These two pathological mechanisms are closely linked, since excitotoxic stimulation of neurons may trigger enhanced NO production and exposure of neurons to NO may trigger the release of excitotoxins. Depending on the experimental situation and cell type, excitotoxic neuronal death may either be apoptotic or necrotic.     

A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid beta-peptide

The tumor suppressor protein p53 is essential for neuronal death in several experimental settings and may participate in human neurodegenerative disorders. Based upon recent studies characterizing chemical inhibitors of p53 in preclinical studies in the cancer therapy field, we synthesized the compound pifithrin-alpha and evaluated its potential neuroprotective properties in experimental models relevant to the pathogenesis of stroke and neurodegenerative disorders. Pifithrin-alpha protected neurons against apoptosis induced by DNA-damaging agents, amyloid beta-peptide and glutamate. Protection by pifithrin-alpha was correlated with decreased p53 DNA-binding activity, decreased expression of the p53 target gene BAX and suppression of mitochondrial dysfunction and caspase activation. Mice given pifithrin-alpha exhibited increased resistance of cortical and striatal neurons to focal ischemic injury and of hippocampal neurons to excitotoxic damage. These preclinical studies demonstrate the efficacy of a p53 inhibitor in models of stroke and neurodegenerative disorders, and suggest that drugs that inhibit p53 may reduce the extent of brain damage in related human neurodegenerative conditions.     

Selective and biphasic effect of the membrane lipid peroxidation product 4-hydroxy-2,3-nonenal on N-methyl-D-aspartate channels

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4-hydroxy-2,3-nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA-induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA-induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). The initial enhancement of NMDA-induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.     

The spinocerebellar ataxia 12 gene product and protein phosphatase 2A regulatory subunit Bbeta2 antagonizes neuronal survival by promoting mitochondrial fission

The neurodegenerative disorder spinocerebellar ataxia 12 (SCA12) is caused by CAG repeat expansion in the non-coding region of the PPP2R2B gene. PPP2R2B encodes Bbeta1 and Bbeta2, alternatively spliced and neuron-specific regulatory subunits of the protein phosphatase 2A (PP2A) holoenzyme. We show here that in PC12 cells and hippocampal neurons, cell stressors induced a rapid translocation of PP2A/Bbeta2 to mitochondria to promote apoptosis. Conversely, silencing of PP2A/Bbeta2 protected hippocampal neurons against free radical-mediated, excitotoxic, and ischemic insults. Evidence is accumulating that the mitochondrial fission/fusion equilibrium is an important determinant of cell survival. Accordingly, we found that Bbeta2 expression induces mitochondrial fragmentation, whereas Bbeta2 silencing or inhibition resulted in mitochondrial elongation. Based on epistasis experiments involving Bcl2 and core components of the mitochondrial fission machinery (Fis1 and dynamin-related protein 1), mitochondrial fragmentation occurs upstream of apoptosis and is both necessary and sufficient for hippocampal neuron death. Our data provide the first example of a proapoptotic phosphatase that predisposes to neuronal death by promoting mitochondrial division and point to a possible imbalance of the mitochondrial morphogenetic equilibrium in the pathogenesis of SCA12.     

Excitotoxic loss of post-synaptic sites is distinct temporally and mechanistically from neuronal death

Dendritic degeneration and loss of synaptic proteins are early events correlated with functional decline in neurodegenerative disease. The temporal and mechanistic relationship between synapse loss and cell death, however, remains unclear. We used confocal microscopy and image processing to count post-synaptic sites on rat hippocampal neurons by expressing post-synaptic density protein 95 fused to green fluorescent protein. Fluorescent puncta co-localized with neurotransmitter release sites, NMDA-induced Ca²⁺ increases and NMDA receptor immunoreactivity. During excitotoxic neurodegeneration, synaptic sites were lost and synaptic transmission impaired. These changes were mediated by NMDA receptors and required Ca²⁺-dependent activation of the proteasome pathway. Tracking synapses from the same cell following brief neurotoxic insult revealed transient loss followed by recovery. The time-course, concentration-dependence and mechanism for loss of post-synaptic sites were distinct from those leading to cell death. Cells expressing p14ARF, which inhibits ubiquitination of post-synaptic density protein 95 and prevents loss of synaptic sites, displayed an increased sensitivity to glutamate-induced cell death. Thus, excitotoxic synapse loss may be a disease-modifying process rather than an obligatory step leading to cell death. These results demonstrate the importance of assessing synaptic function independent of neuronal survival during neurodegeneration and indicate that this approach will be useful for identifying toxins that degrade synaptic connections and for screening for agents that protect synaptic function.