Hongmin Wang

Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death

Apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase, is released into the cytoplasm to induce cell death in response to poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation. How PARP-1 activation leads to AIF release is not known. Here we identify PAR polymer as a cell death signal that induces release of AIF. PAR polymer induces mitochondrial AIF release and translocation to the nucleus. PAR glycohydrolase, which degrades PAR polymer, prevents PARP-1-dependent AIF release. Cells with reduced levels of AIF are resistant to PARP-1-dependent cell death and PAR polymer cytotoxicity. These results reveal PAR polymer as an AIF-releasing factor that plays important roles in PARP-1-dependent cell death.

Poly(ADP-ribose) (PAR) polymer is a death signal

Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.

Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death

The profound neuroprotection observed in poly(ADP-ribose) polymerase-1 (PARP-1) null mice to ischemic and excitotoxic injury positions PARP-1 as a major mediator of neuronal cell death. We report here that apoptosis-inducing factor (AIF) mediates PARP-1-dependent glutamate excitotoxicity in a caspase-independent manner after translocation from the mitochondria to the nucleus. In primary murine cortical cultures, neurotoxic NMDA exposure triggers AIF translocation, mitochondrial membrane depolarization, and phosphatidyl serine exposure on the cell surface, which precedes cytochrome c release and caspase activation. NMDA neurotoxicity is not affected by broad-spectrum caspase inhibitors, but it is prevented by Bcl-2 overexpression and a neutralizing antibody to AIF. These results link PARP-1 activation with AIF translocation in NMDA-triggered excitotoxic neuronal death and provide a paradigm in which AIF can substitute for caspase executioners.

Poly(ADP-ribose) polymerase-1 and apoptosis inducing factor in neurotoxicity

Poly(ADP-ribose) polymerase-1 (PARP-1) is the guardian of the genome acting as a sentinel for genomic damage. However, PARP-1 is also mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. The biochemistry underlying PARP-1-mediated cell death has remained elusive, although NAD(+) consumption and energy failure have been thought to be one of the possible molecular mechanisms. Recent observations link PARP-1 activation with translocation of apoptosis-inducing factor (AIF) to the nucleus and indicate that AIF is an essential downstream effector of PARP-1-mediated cell death. PARP-1 activation signals AIF release from the mitochondria, resulting in a novel, caspase-independent pathway of programmed cell death. These recent findings suggest that AIF maybe a target for development of future therapeutic treatment for many neurological disorders involving excitotoxicity.

Effects of overexpression of Huntingtin proteins on mitochondrial integrity

Huntingtons disease (HD) is caused by an expansion of a CAG trinucleotide sequence that encodes a polyglutamine tract in the huntingtin (Htt) protein. Expansion of the polyglutamine tract above 35 repeats causes disease, with the age of onset inversely related to the degree of expansion above this number. Growing evidence suggests that mitochondrial function is compromised during HD pathogenesis, but how this occurs is not understood. We examined mitochondrial properties of HeLa cells that expressed green fluorescent protein (GFP)- or FLAG-tagged N-terminal portions of the Htt protein containing either, 17, 28, 74 or 138 polyglutamine repeats. Immunofluorescence staining of cells using antibodies against Tom20, a mitochondrion localized protein, revealed that cells expressing Htt proteins with 74 or 138 polyglutamine repeats were more sensitized to oxidative stress-induced mitochondria fragmentation and had reduced ATP levels compared with cells expressing Htt proteins with 17 or 28 polyglutamine repeats. By measuring changes in fluorescence of a photoactivated GFP protein targeted to mitochondria, we found that cells expressing red fluorescent protein (RFP)-tagged Htt protein containing 74 polyglutamine repeats had mitochondria that displayed reduced movement and fusion than cells expressing RFP-Htt protein with 28 polyglutamine repeats. Overexpression of Drp-1(K38A), a dominant-negative mitochondria-fission mutant, or Mfn2, a protein that promotes mitochondria fusion, suppressed polyglutamine-induced mitochondria fragmentation, the reduction of ATP levels and cell death. In a Caenorhabditis elegans model of HD, we found that reduction of Drp-1 expression by RNA interference rescued the motility defect associated with the expression of Htt proteins with polyglutamine repeats. These results suggest that the increase in cytotoxicity induced by Htt proteins containing expanded polyglutamine tracts is likely mediated, at least in part, by an alteration in normal mitochondrial dynamics, which results in increased mitochondrial fragmentation. Furthermore, our results suggest that it might be possible to reverse polyglutamine-induced cytotoxicity by preventing mitochondrial fragmentation.

Roles for Ephrins in Positionally Selective Synaptogenesis between Motor Neurons and Muscle Fibers

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.

Apoptosis inducing factor and PARP-mediated injury in the MPTP mouse model of Parkinson's disease.

Abstract: Experimental intoxication models are used to study the more common sporadic form of Parkinsons disease (PD). 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyrimidine (MPTP) animal models of PD provide a valuable and predictive tool to probe the molecular mechanisms of dopamine neuronal cell death in PD. MPTP is a powerful neurotoxin that induces neuronal degeneration in the substantia nigra pars compacta and produces PD‐like symptoms in several mammalian species tested, a feat not yet accomplished in genetically engineered mice expressing human genetic mutations. The mechanisms of MPTP‐induced neurotoxicity are not yet fully understood but involve activation of N‐methyl‐D‐aspartate (NMDA) receptors by glutamate, production of NO by nNOS and iNOS, oxidative injury to DNA, and activation of the DNA damage‐sensing enzyme poly (ADP‐ribose) polymerase (PARP). Recent experiments indicate that translocation of a mitochondrial protein apoptosis inducing factor (AIF) from mitochondria to the nucleus depends on PARP activation and plays an important role in excitotoxicity‐induced cell death. This article briefly reviews the experimental findings regarding excitotoxicity, PARP activation, and AIF translocation in MPTP toxicity and dopaminergic neuronal cell death.

Ubiquilin and p97/VCP bind erasin, forming a complex involved in ERAD

Loss of ubiquilin or erasin activates ER stress, increases accumulation of polyubiquitinated proteins, and shortens lifespan in worms.

Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease.

The ubiquitin proteasome system (UPS) is impaired in Huntingtons disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntingtons disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane‐mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntingtons disease and other intractable disorders.

Involvement of poly ADP ribosyl polymerase-1 in acute but not chronic zinc toxicity

We have previously suggested that zinc‐induced neuronal death may be mediated in part by inhibition of the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), secondary to depletion of the essential cosubstrate NAD+. Given convergent evidence implicating the NAD+‐catabolizing enzyme, poly ADP ribosyl polymerase (PARP) in mediating ATP depletion and neuronal death after excitotoxic and ischemic insults, we tested the specific hypothesis that the neuronal death induced by exposure to toxic levels of extracellular zinc might be partly mediated by PARP. PARP was activated in cultured mouse cortical astrocytes after a toxic acute Zn2+ exposure (350 µm Zn2+ for 15 min), but not in cortical neurons or glia after exposure to a toxic chronic Zn2+ exposure (40 µm Zn2+ for 1–4 h), an exposure sufficient to deplete NAD+ and ATP levels. Furthermore, the neurotoxicity induced by acute, but not chronic, Zn2+ exposure was reduced in mixed neuronal‐glial cultures prepared from mutant mice lacking the PARP gene. These data suggest PARP activation may contribute to more fulminant forms of Zn2+‐induced neuronal death.