Yingfei Wang

Poly(ADP-ribose) Signals to Mitochondrial AIF: A Key Event in Parthanatos

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in multiple neurologic diseases by mediating caspase-independent cell death, which has recently been designated parthanatos to distinguish it from other forms of cell death such as apoptosis, necrosis and autophagy. Mitochondrial apoptosis-inducing factor (AIF) release and translocation to the nucleus is the commitment point for parthanatos. This process involves a pathogenic role of poly(ADP-ribose) (PAR) polymer. It generates in the nucleus and translocates to the mitochondria to mediate AIF release following lethal PARP-1 activation. PAR polymer itself is toxic to cells. Thus, PAR polymer signaling to mitochondrial AIF is the key event initiating the deadly crosstalk between the nucleus and the mitochondria in parthanatos. Targeting PAR-mediated AIF release could be a potential approach for the therapy of neurologic disorders.

Poly(ADP-Ribose) (PAR) Binding to Apoptosis-Inducing Factor Is Critical for PAR Polymerase-1–Dependent Cell Death (Parthanatos)

Poly(ADP-ribose) binds to apoptosis-inducing factor to trigger its release from mitochondria and induce cell death. Scoring a PAR on Death’s Course Overactivation of the DNA repair enzyme PARP-1 [poly(ADP-ribose) (PAR) polymerase-1] leads to “parthanatos,” a form of cell death that is distinct from apoptosis and necrosis and that depends on release of apoptosis-inducing factor (AIF) from mitochondria. Here, Wang et al. showed that PAR bound directly to AIF, disrupting AIF’s association with the mitochondria and allowing it to translocate to the nucleus to mediate cell death. Moreover, mutation of the PAR binding site of AIF enabled the authors to separate AIF’s role in parthanatos from its function in mitochondrial respiration. Identification of AIF as a PAR-binding protein could potentially lead to the development of compounds that inhibit this interaction, protecting against parthanatos, or compounds that mimic it and thereby promote parthanatos as an agent of death in malignant cells. The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase–1 (PARP-1)–mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. We show that AIF is a high-affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site, and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH (reduced form of nicotinamide adenine dinucleotide) oxidase activity, its ability to bind FAD (flavin adenine dinucleotide) or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death after PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival–promoting functions are separate from its effects as a mitochondrially derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide notable opportunities for preventing cell death after activation of PARP-1.

Trypsin and trypsin-like proteases in the brain: Proteolysis and cellular functions

Abstract.Several serine proteases including thrombin, tissue-type plasminogen activator and urokinase-type plasminogen activator have been well characterized in the brain. In this article, we review the brain-related trypsin and trypsin-like serine proteases. Accumulating evidence demonstrates that trypsin and trypsin-like serine proteases play very important roles in neural development, plasticity, neurodegeneration and neuroregeneration in the brain. Neuropsin is able to hydrolyze the extracellular matrix components by its active site serine, and regulates learning and memory in normal brain. The mutant neurotrypsin contributes to mental retardation in children. Neurosin seems to be involved in the pathogenesis of neurodegenerative disorders, like Alzheimer’s disease, Parkinson’s disease or multiple sclerosis. Although mesotrypsin/trypsin IV is also implicated in neurodegeneration, its functional significance still remains largely unknown. Particularly, mesotrypsin/trypsin IV, P22 and neurosin exert their physiological and pathological functions through activation of certain protease-activated receptors (PARs). In the brain, the presence of serpins controls the activity of serine proteases. Therefore, understanding the interaction among brain trypsin, serpins and PARs will provide invaluable tools for regulating normal brain functions and for the clinical treatment of neural disorders.

Calpain activation is not required for AIF translocation in PARP‐1‐dependent cell death (parthanatos)

Apoptosis‐inducing factor (AIF) is critical for poly(ADP‐ribose) polymerase‐1 (PARP‐1)‐dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell‐free system in intact cells under conditions where endogenous calpain was activated by either NMDA or N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62‐kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA‐ or MNNG‐induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP‐1 inhibitor, 3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxyl]‐1(2H)‐iso‐quinolinone. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin‐induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up‐regulated secretion of the chemokine growth‐regulated oncogene/cytokine‐induced neutrophil chemoattractant‐1 (GRO/CINC‐1) in primary rat astrocytes in a concentration‐dependent manner. However, we found no increase of interleukin (IL)‐6, IL‐1β and tumour necrosis factor‐α secretion. Protease‐activated receptor 1 (PAR‐1)‐induced GRO/CINC‐1 release was mainly mediated by c‐Jun N‐terminal kinase (JNK) activation. Extracellular signal‐regulated kinase 1/2 might be partially involved, but not p38 mitogen‐activated protein kinase. Further studies demonstrated that PAR‐1 activation, as well as application of recombinant GRO/CINC‐1, protected astrocytes from C2‐ceramide‐induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB‐332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR‐1. These results demonstrate for the first time that PAR‐1 plays an important role in anti‐apoptosis in the brain by regulating the release of chemokine GRO/CINC‐1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.

Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release.

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

Proteinase-activated receptor-1 and -2 induce the release of chemokine GRO/CINC-1 from rat astrocytes via differential activation of JNK isoforms, evoking multiple protective pathways in brain.

Activation of both PAR-1 (proteinase-activated receptor-1) and PAR-2 resulted in release of the chemokine GRO (growth-regulated oncogene)/CINC-1 (cytokine-induced neutrophil chemoattractant-1), a functional counterpart of human interleukin-8, from rat astrocytes. Here, we investigate whether the two PAR receptor subtypes can signal separately. PAR-2-induced GRO/CINC-1 release was independent of protein kinase C, phosphoinositide 3-kinase and MEK (mitogen-activated protein kinase kinase)-1/2 activation, whereas these three kinases were involved in PAR-1-induced GRO/CINC-1 release. Despite such clear differences between PAR-1 and PAR-2 signalling pathways, JNK (c-Jun N-terminal kinase) was identified in both signalling pathways to play a pivotal role. By isoform-specific loss-of-function studies using small interfering RNA against JNK1-3, we demonstrate that different JNK isoforms mediated GRO/CINC-1 secretion, when it was induced by either PAR-1 or PAR-2 activation. JNK2 and JNK3 isoforms were both activated by PAR-1 and essential for chemokine GRO/CINC-1 secretion, whereas PAR-1-mediated JNK1 activation was mainly responsible for c-Jun phosphorylation, which was not involved in GRO/CINC-1 release. In contrast, PAR-2-induced JNK1 activation, which failed to phosphorylate c-Jun, uniquely contributed to GRO/CINC-1 release. Therefore our results show for the first time that JNK-mediated chemokine GRO/CINC-1 release occurred in a JNK isoform-dependent fashion and invoked PAR subtype-specific mechanisms. Furthermore, here we demonstrate that activation of PAR-2, as well as PAR-1, rescued astrocytes from ceramide-induced apoptosis via regulating chemokine GRO/CINC-1 release. Taken together, our results suggest that PAR-1 and PAR-2 have overlapping functions, but can activate separate pathways under certain pathological conditions to rescue neural cells from cell death. This provides new functional insights into PAR/JNK signalling and the protective actions of PARs in brain.

p24A, a Type I Transmembrane Protein, Controls ARF1-dependent Resensitization of Protease-activated Receptor-2 by Influence on Receptor Trafficking

Protease-activated receptor-2 (PAR-2), the second member of the G protein-coupled PAR family, is irreversibly activated by trypsin or tryptase and then targeted to lysosomes for degradation. Intracellular presynthesized receptors stored at the Golgi apparatus repopulate the cell surface after trypsin stimulation, thereby leading to rapid resensitization to trypsin signaling. However, the molecular mechanisms of the exocytic trafficking of PAR-2 from the Golgi apparatus to the plasma membrane remain largely unclear. Here we show that p24A, a type I transmembrane protein, which is a crucial constituent of the Golgi apparatus, associates with PAR-2 at the Golgi apparatus. The protein interaction occurs between the N-terminal region of p24A (residues 1-105; p24A-GL (GOLD domain with a small linker)) and the second extracellular loop of PAR-2. After receptor activation, PAR-2 dissociates from p24A. Importantly, we found that ADP-ribosylation factor 1 regulated the dissociation process and initiated PAR-2 trafficking to the plasma membrane. Conversely, overexpression of the fragment p24A-GL, but not other mutants containing the functional coiled-coil domain of p24A, arrested PAR-2 at the Golgi apparatus and inhibited receptor trafficking to the plasma membrane, which consequently prevented resensitization of PAR-2. These findings identify a new function of p24A as a regulator of signal-dependent trafficking that regulates the life cycle of PAR-2, Thus, we reveal a new molecular mechanism underlying resensitization of PAR-2.

Activation of protease-activated receptors in astrocytes evokes a novel neuroprotective pathway through release of chemokines of the growth-regulated oncogene/cytokine-induced neutrophil chemoattractant family

Activation of protease‐activated receptors (PARs) is known to exert neuroprotection when low concentrations of the agonist protease thrombin are applied. However, the mechanism of protection is still unclear. Here, we showed that activation of multiple PARs, including PAR‐1, PAR‐2 and PAR‐4, was able to elevate the release of the chemokine cytokine‐induced neutrophil chemoattractant (CINC)‐3 from rat astrocytes, in addition to evoking CINC‐1 secretion. Different molecular mechanisms were identified as being involved in the secretion of CINC‐1 and CINC‐3, upon activation of different PARs. Importantly, we found that both CINC‐1 and CINC‐3 could signal to rat cortical neurons. Both chemokines acted via CXCR2 to prevent C2‐ceramide‐induced cytochrome c release from mitochondria. Consequently CINC‐1 and CINC‐3 protected neurons from apoptosis. We further revealed that conditioned media obtained from PAR‐activated astrocytes similarly protected cortical neurons against C2‐ceramide‐induced cell death. The neuroprotection was considerably suppressed by a CXCR2 antagonist. CXCR2 is the cognate receptor for CINC. Therefore, our findings demonstrate that PAR‐activated astrocytes are able to protect neurons against neurodegeneration and cell death via regulation of the secretion of chemokines CINC‐1 and CINC‐3. These data indicate a previously unknown mechanism for astrocyte‐mediated neuroprotection achieved by PAR activation.

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

DNA damage-activated nuclease identified Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by a nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and activates one or more nucleases to cleave DNA. Wang et al. found that macrophage migration inhibitory factor (MIF) is an AIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIF could thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive. Science, this issue p. 82; see also p. 36 An endonuclease that functions in a disease-associated form of cell death is identified. [Also see Perspective by Jonas] INTRODUCTION Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a nuclear enzyme responding to oxidative stress and DNA damage. Excessive activation of PARP-1 causes an intrinsic caspase-independent cell death program designated parthanatos, which occurs in many organ systems because of toxic or stressful insults, including ischemia-reperfusion injury after stroke and myocardial infarction, inflammatory injury, reactive oxygen species–induced injury, glutamate excitotoxicity, and neurodegenerative diseases. Inhibition or genetic deletion of PARP-1 is profoundly protective against such cellular injury in models of human disease. RATIONALE The molecular mechanisms underlying parthanatos involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis into 20- to 50-kb large DNA fragments—a commitment point for parthanatos. Because AIF itself has no obvious nuclease activity, we propose that AIF recruits a nuclease or a nuclease complex to the nucleus to trigger DNA cleavage and parthanatos. Although the endonuclease G (EndoG) homolog may promote DNA degradation in Caenorhabditis elegans through cooperating with the AIF homolog, our group and others showed that EndoG does not have an essential role in PARP-dependent chromatinolysis and cell death in mammals. Thus, the identity of the nuclease responsible for large DNA fragmentation following AIF entry to the nucleus during parthanatos has been a long-standing mystery. RESULTS Using two sequential unbiased screens, including a human protein array and a small interfering RNA screen, we discovered that macrophage migration inhibitory factor (MIF) binds AIF and is required for parthanatos. Three-dimensional modeling of MIF revealed that the MIF trimer has the same core topology structure as PD-D/E(X)K superfamily nucleases. In the presence of Mg2+ or Ca2+, MIF has both 3′ exonuclease and endonuclease activity. It binds to 5′ unpaired bases of single-stranded DNA with stem loop structure and cleaves its 3′ unpaired bases. These nuclease activities allow MIF to cleave genomic DNA into large fragments. Depletion of MIF markedly reduced chromatinolysis and cell death induced by N-methyl-d-aspartate (NMDA) receptor–activated glutamate excitotoxicity in primary neuronal cultures, DNA damage caused by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or focal stroke in mice. Mutating key amino acid residues in the PD-D/E(X)K nuclease domain of MIF eliminated its nuclease activity and prevented parthanatos. Disrupting the AIF and MIF interaction prevented the translocation of MIF from the cytosol to the nucleus and protected against parthanatos. Moreover, depletion of MIF, disruption of AIF and MIF interaction, and eliminating MIF’s nuclease activity has long-lasting histological and behavioral rescue in the focal ischemia model of stroke. CONCLUSION We identified MIF as a PARP-1–dependent AIF-associated nuclease that is required for parthanatos. In response to oxidative stress or DNA damage, PARP-1 activation triggers AIF release from the mitochondria. AIF then recruits MIF to the nucleus where MIF cleaves genomic DNA into large fragments and causes cell death. Depletion of MIF, disruption of AIF and MIF interaction, or blocking MIF nuclease activity inhibited chromatinolysis and parthanatos. Targeting MIF nuclease activity may offer an important therapeutic opportunity for a variety of disorders with excessive PARP-1 activation. Stressors lead to DNA damage, PARP-1 activation, and PAR formation. PAR facilitates the release of AIF from mitochondria where it binds MIF. This complex translocates to the nucleus to bind DNA; the result is DNA fragmentation and cell death. Interference with this cascade by preventing the formation of the AIF-MIF complex or by a nuclease-deficient MIF prevents DNA fragmentation and promotes cell survival. Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF’s nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.