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Aggregation of α-synuclein contributes to the formation of Lewy bodies and neurites, the pathologic hallmarks of Parkinson disease (PD) and α-synucleinopathies. Although a number of human mutations have been identified in familial PD, the mechanisms that promote α-synuclein accumulation and toxicity are poorly understood. Here, we report that hyperactivity of the nonreceptor tyrosine kinase c-Abl critically regulates α-synuclein-induced neuropathology. In mice expressing a human α-synucleinopathy-associated mutation (hA53Tα-syn mice), deletion of the gene encoding c-Abl reduced α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Conversely, overexpression of constitutively active c-Abl in hA53Tα-syn mice accelerated α-synuclein aggregation, neuropathology, and neurobehavioral deficits. Moreover, c-Abl activation led to an age-dependent increase in phosphotyrosine 39 α-synuclein. In human postmortem samples, there was an accumulation of phosphotyrosine 39 α-synuclein in brain tissues and Lewy bodies of PD patients compared with age- matched controls. Furthermore, in vitro studies show that c-Abl phosphorylation of α-synuclein at tyrosine 39 enhances α-synuclein aggregation. Taken together, this work establishes a critical role for c-Abl in α-synuclein-induced neurodegeneration and demonstrates that selective inhibition of c-Abl may be neuroprotective. This study further indicates that phosphotyrosine 39 α-synuclein is a potential disease indicator for PD and related α-synucleinopathies., Introduction Parkinson disease (PD) is the second most common neurodegenerative disorder and is characterized by the preferential loss of dopamine (DA) neurons leading to motoric dysfunction including bradykinesia, rest tremor, [...]

Glutamate acting on N-methyl-D-aspartate (NMDA) receptors induces neuronal injury following stroke, through activation of poly(ADPribose) polymerase-1 (PARP-1) and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a previously undescribed NMDA receptor-induced survival protein that is neuroprotective against glutamate NMDA receptor- mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer-induced cell death (parthanatos). Iduna's protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer-binding protein, and mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion-induced stroke in mice. To our knowledge, these results define Iduna as the first known endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling could be a new therapeutic strategy for the treatment of neurologic disorders., Glutamate is the major excitatory neurotransmitter regulating normal physiologic activity in the brain. Excessive glutamate release leads to excitotoxicity, which has a prominent role in many disorders of the nervous [...]

Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function, coAbl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium ([Mpp.sup.+]) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571's protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD. AIMP2 | Parkinson disease | STI-571 | ubiquitin doi/ 10.1073/pnas.1006083107

Activation of poly(ADP-ribose) polymerase (PARP) and subsequent translocation of apoptosis-inducing factor contribute to caspase-independent neuronal injury from N-methyl-D-aspartate, oxygen-glucose deprivation, and ischemic stroke. Some studies have implicated endonuclease G in the DNA fragmentation associated with caspase-independent cell death. Here, we compared wild-type and endonuclease G null mice to investigate whether endonuclease G plays a role in the PARP-dependent injury that results from transient focal cerebral ischemia. Latex casts did not reveal differences in the cerebral arterial distribution territory or posterior communicating arterial diameter, and the decrease in laser-Doppler flux during middle cerebral artery occlusion was similar in wild-type and endonuclease G null mice. After 90 min of occlusion and 1 day of reperfusion, similar degrees of nuclear translocation of apoptosis-inducing factor and DNA degradation were evident in male wild-type and null mice. At 3 days of reperfusion, infarct volume and neurological deficit scores were not different between male wild-type and endonuclease G null mice or between female wild-type and endonuclease G null mice. These data demonstrate that endonuclease G is not required for the pathogenesis of transient focal ischemia in either male or female mice. Treatment with a PARP inhibitor decreased infarct volume and deficit scores equivalently in male wild-type and endonuclease G null mice, indicating that the injury in endonuclease G null mice remains dependent on PARP. Thus endonuclease G is not obligatory for executing PARP-dependent injury during ischemic stroke. apoptosis; DR2313; mouse; middle cerebral artery doi: 10.1152/ajpregu.00747.2009.

Identification of the signaling pathways that mediate neuronal survival signaling could lead to new therapeutic targets for neurologic disorders and stroke. Sublethal doses of NMDA can induce robust endogenous protective mechanisms in neurons. Through differential analysis of primary library expression and microarray analyses, here we have shown that nuclear factor I, subtype A (NFI-A), a member of the NFI/CAAT-box transcription factor family, is induced in mouse neurons by NMDA receptor activation in a NOS- and ERK-dependent manner. Knockdown of NFI-A induction using siRNA substantially reduced the neuroprotective effects of sublethal doses of NMDA. Further analysis indicated that NFI-A transcriptional activity was required for the neuroprotective effects of NMDA receptor activation. Additional evidence of the neuroprotective effects of NFI-A was provided by the observations that [Nfia.sup.[-/-]] neurons were highly sensitive to NMDA-induced excitotoxicity and were more susceptible to developmental cell death than wild-type neurons and that [Nfia.sup.±] mice were more sensitive to NMDA-induced intrastriatal lesions than were wild-type animals. These results identify NFI-A as what we believe to be a novel neuroprotective transcription factor with implications in neuroprotection and neuronal plasticity following NMDA receptor activation., Introduction During mammalian development and in adulthood, a variety of molecular programs support the survival of neurons. Support from neurotrophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor [...]

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential ([DELTA][[PSI].sub.m]) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of [DELTA][[PSI].sub.m] relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal [DELTA][[PSI].sub.m]. We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease. autophagy | Parkinson's disease | phosphatase and tensin homolog-induced putative kinase 1 www.pnas.org/cgi/doi/10.1073/pnas.0911187107

Inhibitors of apoptosis (IAPs) are a family of highly-conserved proteins that regulate cell survival through binding to caspases, the final executioners of apoptosis. X-linked IAP (XIAP) is the most widely expressed IAP and plays an important function in regulating cell survival. XIAP contains 3 baculoviral IAP repeats (BIRs) followed by a RING finger domain at the C terminal. The BIR domains of XIAP possess anticaspase activities, whereas the RING finger domain enables XIAP to function as an E3 ubiquitin ligase in the ubiquitin and proteasomal system. Our previous study showed that parkin, a protein that is important for the survival of dopaminergic neurons in Parkinson's disease (PD), is S-nitrosylated both in vitro and in vivo in PD patients. S-nitrosylation of parkin compromises its ubiquitin E3 ligase activity and its protective function, which suggests that nitrosative stress is an important factor in regulating neuronal survival during the pathogenesis of PD. In this study we show that XIAP is S-nitrosylated in vitro and in vivo in an animal model of PD and in PD patients. Nitric oxide modifies mainly cysteine residues within the BIR domains. In contrast to parkin, S-nitrosylation of XIAP does not affect its E3 ligase activity, but instead directly compromises its anticaspase-3 and antiapoptotic function. Our results confirm that nitrosative stress contributes to PD pathogenesis through the impairment of prosurvival proteins such as parkin and XIAP through different mechanisms, indicating that abnormal S-nitrosylation plays an important role in the process of neurodegeneration. nitric oxide | apoptosis | neurodegeneration | inhibitors of apoptosis

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD. LRRK2 | Parkinson's disease | proteasome | ubiquitin

Author(s): David Sulzer (corresponding author) [1, 2, 3]; Roy N. Alcalay [2]; Francesca Garretti [1]; Lucien Cote [2]; Ellen Kanter [1]; Julian Agin-Liebes [1]; Christopher Liong [2]; Curtis McMurtrey [4]; [...]

Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimers, Huntingtons and Parkinsons disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases., Author(s): Shane A. Liddelow (corresponding author) [1, 2]; Kevin A. Guttenplan [1]; Laura E. Clarke [1]; Frederick C. Bennett [1, 3]; Christopher J. Bohlen [2]; Lucas Schirmer [4, 5]; Mariko [...]

Parkinson's disease (PD) is a common neurodegenerative movement disorder. Whereas the majority of PD cases are sporadic, rare genetic defects have been linked to this prevalent movement disorder. Mutations in DJ-1 are associated with autosomal recessive early-onset PD. The exact biochemical function of DJ-1 has remained elusive. Here we report the generation of DJ-1 knockout (KO) mice by targeted deletion of exon 2 and exon 3. There is no observable degeneration of the central dopaminergic pathways, and the mice are anatomically and behaviorally similar to WT mice. Fluorescent Amplex red measurements of [H.sub.2][O.sub.2] indicate that isolated mitochondria from young and old DJ-1 KO mice have a 2-fold increase in [H.sub.2][O.sub.2]. DJ-1 KO mice of 2-3 months of age have a 60% reduction in mitochondrial aconitase activity without compromising other mitochondrial processes. At an early age there are no differences in antioxidant enzymes, but in older mice there is an up-regulation of mitochondrial manganese superoxide dismutase and glutathione peroxidase and a 2-fold increase in mitochondrial glutathione peroxidase activity. Mutational analysis and mass spectrometry reveal that DJ-1 is an atypical peroxiredoxin-like peroxidase that scavenges [H.sub.2][O.sub.2] through oxidation of Cys-106. In vivo there is an increase of DJ-1 oxidized at Cys-106 after 1-methyl4-phenyl-1,2,3,6 tetrahydropyridine intoxication of WT mice. Taken together these data indicate that the DJ-1 KO mice have a deficit in scavenging mitochondrial [H.sub.2][O.sub.2] due to the physiological function of DJ-1 as an atypical peroxiredoxin-like peroxidase. glutathione peroxidase | mitochondria | manganese superoxide dismutase | PARK7 | Parkinson's disease

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-l-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. excitotoxicity | poly(ADP-ribose)glycohydrolase | poly(ADP-ribose) polymerase | stroke

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. excitotoxicity | poly(ADP-ribose)polymerase

Parkinson disease (PD) is a relatively common disorder of the nervous system that afflicts patients later in life with tremor, slowness of movement, gait instability, and rigidity. Treatment of these [...]

Parkinson's disease (PD) is a disorder of movement, cognition, and emotion, and it is characterized pathologically by neuronal degeneration with Lewy bodies, which are cytoplasmic inclusion bodies containing deposits of aggregated proteins. Most PD cases appear to be sporadic, but genetic forms of the disease, caused by mutations in [alpha]-synuclein, parkin, and other genes, have helped elucidate pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with clinical features of PD and with pleomorphic pathology including deposits of aggregated protein. To study expression and interactions of LRRK2, we synthesized cDNAs and generated expression constructs coding for human WT and mutant LRRK2 proteins. Expression of full-length LRRK2 in cells in culture suggests that the protein is predominately cytoplasmic, as is endogenous protein by subcellular fractionation. Using coimmunoprecipitation, we find that LRRK2, expressed in cells in culture, interacts with parkin but not with [alpha]-synuclein, DJ-1, or tau. A small proportion of the cells overexpressing LRRK2 contain protein aggregates, and this proportion is greatly increased by coexpression of parkin. In addition, parkin increases ubiquitination of aggregated protein. Also, mutant LRRK2 causes neuronal degeneration in both SH-SY5Y cells and primary neurons. This cell model may be useful for studies of PD cellular pathogenesis and therapeutics. These findings suggest a gain-of-function mechanism in the pathogenesis of LRRK2-1inked PD and suggest that LRRK2 may be involved in a pathogenic pathway with other PD-related proteins such as parkin, which may help illuminate both familial and sporadic PD. cell death | Parkinson's disease | protein aggregation | protein interaction | ubiquitin

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease (PD) with a clinical appearance indistinguishable from idiopathic PD. Initial studies suggest that LRRK2 mutations are the most common yet identified determinant of PD susceptibility, transmitted in an autosomal-dominant mode of inheritance. Herein, we characterize the LRRK2 gene and transcript in human brain and subclone the predominant ORF. Exogenously expressed LRRK2 protein migrates at [approximately equal to] 280 kDa and is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Familial-linked mutations G2019S or R1441C do not have an obvious effect on protein steady-state levels, turnover, or localization. However, in vitro kinase assays using full-length recombinant LRRK2 reveal an increase in activity caused by familial-linked mutations in both autophosphorylation and the phosphorylation of a generic substrate. These results suggest a gain-of-function mechanism for LRRK2-linked disease with a central role for kinase activity in the development of PD. dardarin | parkinsonism | PARK8

Epidemiological evidence suggests a reduced incidence of many common types of non-smoking-related cancers in individuals with Parkinson's disease (PD). Genes that underlie familial forms of PD are often abnormally expressed in cancer, owing to their differential regulation or mutation. Functional studies implicate these genes in maintenance of the cell cycle, in some cases through interaction in the ubiquitin-proteasome system. Variation in genes associated with familial-linked PD could therefore modify susceptibility to both cancer and PD, implying some degree of overlap in the underlying biochemical dysfunction. When considering the normal function of these PD-linked genes in the periphery and their potential role in cancer, further emphasis might be placed on protein handling relating to cell-cycle control in the etiology of PD.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tins.2005.05.002 Byline: Andrew B. West (a), Valina L. Dawson (a)(b)(c), Ted M. Dawson (a)(b) Abstract: Epidemiological evidence suggests a reduced incidence of many common types of non-smoking-related cancers in individuals with Parkinson's disease (PD). Genes that underlie familial forms of PD are often abnormally expressed in cancer, owing to their differential regulation or mutation. Functional studies implicate these genes in maintenance of the cell cycle, in some cases through interaction in the ubiquitin-proteasome system. Variation in genes associated with familial-linked PD could therefore modify susceptibility to both cancer and PD, implying some degree of overlap in the underlying biochemical dysfunction. When considering the normal function of these PD-linked genes in the periphery and their potential role in cancer, further emphasis might be placed on protein handling relating to cell-cycle control in the etiology of PD. Author Affiliation: (a) Institute for Cell Engineering, and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (b) Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA (c) Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

The molecular pathways involved in the pathogenesis of Parkinson's disease (PD) are discussed. The principle molecular pathways underlying the pathogenesis of sporadic and familial forms of PD are represented by mitochondrial dysfunction, oxidative and nitrosative stress, the accumulation of aberrant or misfolded proteins and impairment of ubiquitin-proteasome system.

The metabolism of poly(ADP-ribose) (PAR) is critical for genomic stability in multicellular eukaryotes. Here, we show that the failure to degrade PAR by means of disruption of the murine poly(ADP-ribose) glycohydrolase (PARG) gene unexpectedly causes early embryonic lethality and enhanced sensitivity to genotoxic stress. This lethality results from the failure to hydrolyze PAR, because PARG null embryonic day (E) 3.5 blastocysts accumulate PAR and concurrently undergo apoptosis. Moreover, embryonic trophoblast stem cell lines established from early PARG null embryos are viable only when cultured in medium containing the poly(ADP-ribose) polymerase inhibitor benzamide. Cells lacking PARG also show reduced growth, accumulation of PAR, and increased sensitivity to cytotoxicity induced by N-methyl-N'-nitro-N-nitrosoguanidine and menadione after benzamide withdrawal. These results provide compelling evidence that the failure to degrade PAR has deleterious consequences. Further, they define a role for PARG in embryonic development and a protective role in the response to genotoxic stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by degeneration of catecholaminergic neurons of the substantia nigra pars compacta and locus coeruleus, among other regions. Autosomal-recessive juvenile Parkinsonism (ARJP) is caused by mutations in the PARK2 gene coding for parkin and constitutes the most common familial form of PD. The majority of ARJP-associated parkin mutations are thought to be loss of function-mutations; however, the pathogenesis of ARJP remains poorly understood. Here, we report the generation of parkin null mice by targeted deletion of parkin exon 7. These mice show a loss of catecholaminergic neurons in the locus coeruleus and an accompanying loss of norepinephrine in discrete regions of the central nervous system. Moreover, there is a dramatic reduction of the norepinephrine-dependent startle response. The nigrostriatal dopaminergic system does not show any impairment. This mouse model will help gain a better understanding of parkin function and the mechanisms underlying parkin-associated PD.

Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in five in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by 5-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates., Parkinson's disease (PD) is a common neurodegenerative disorder that leads to the progressive loss of dopamine (DA) neurons (1). The majority of PD is sporadic and is thought to be [...]

Different cell-death mechanisms control many physiological and pathological processes in humans. Mitochondria play important roles in cell death through the release of pro-apoptotic factors such as cytochrome c and apoptosis-inducing factor (AIF), which activate caspase-dependent and caspase-independent cell death, respectively. Poly(ADP-ribose) polymerase 1 (PARP-1) is emerging as an important activator of caspase-independent cell death. PARP-1 generates the majority of long, branched poly(ADP-ribose) (PAR) polymers following DNA damage. Overactivation of PARP-1 initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF. AIF then shuttles from mitochondria to the nucleus and induces peripheral chromatin condensation, large-scale fragmentation of DNA and, ultimately, cytotoxicity. Identification of the pro-death and pro-survival signals in the PARP-1-mediated cell-death program might provide novel therapeutic targets in human diseases.

The excitatory neurotransmitter, glutamate, activates N-methyi-Daspartate (NMDA) receptors to induce long-lasting synaptic changes through alterations in gene expression. It is believed that these long-lasting changes contribute to learning and memory, drug tolerance, and ischemic preconditioning. To identify NMDA-induced late-response genes, we used a powerful gene-identification method, differential analysis of primary cDNA library expression (DAzLE), and cDNA microarray from primary cortical neurons. We report here that a variety of genes, which we have named plasticity-induced genes (PLINGs), are up-regulated with differential expression patterns after NMDA receptor activation, indicating that there is a broad and dynamic range of long-lasting neuronal responses that occur through NMDA receptor activation. Our results provide a molecular dissection of the activity-dependent long-lasting neuronal responses induced by NMDA receptor activation.

Using a method of expression profiling called differential analysis of cDNA library expression (DAzLE), we report the expression profile of late response genes in a model of activity-dependent neuronal survival and neurite outgrowth. Using DAzLE, we isolated differentially expressed genes from cultured rat embryonic cortical neurons after KCI (50 mM)-mediated membrane depolarization. We identified 469 activity-dependent regulated genes, of which 174 are genes of unknown function. The regulation of 63 genes was found to be nitric oxide (NO)-dependent. Identifiable genes fell into several major categories, including signal transduction pathways, neuronal development, DNA replication, gene transcription, protein metabolism, energy regulatory proteins, and antiapoptotic proteins. These genes may be important in activity-dependent neuron survival and development. Furthermore, these late response genes provide the tools to begin to investigate downstream events in activity-dependent neuronal survival and development. The major advantage of DAzLE is that it provides a nearly complete and relatively comprehensive differential screening profile that has the potential to be a powerful and useful tool in other fields of study.

Parkinson's disease (PD) is a complex disorder with many different causes, yet they may intersect in common pathways, raising the possibility that neuroprotective agents may have broad applicability in the treatment of PD. Current evidence suggests that mitochondrial complex I inhibition may be the central cause of sporadic PD and that derangements in complex I cause α-synuclein aggregation, which contributes to the demise of dopamine neurons. Accumulation and aggregation of α-synuclein may further contribute to the death of dopamine neurons through impairments in protein handling and detoxificatinn. Dysfunction of parkin (a ubiquitin E3 ligase) and DJ-1 could contribute to these deficits. Strategies aimed at restoring complex I activity, reducing oxidative stress and α-synuclein aggregation, and enhancing protein degradation may hold particular promise as powerful neuroprotective agents in the treatment of PD., PD is the most common neurodegerterative movement disorder. It affects more than 0.1% of the population older than 40 years of age (1). Clinically, most patients present with a motoric [...]

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.

Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, [H.sub.2][O.sub.2], and N-methyl-D-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death., PARP-1 is a nuclear enzyme that responds to DNA damage and facilitates DNA repair (1-3). Once activated, PARP-1 transfers 50 to 200 molecules of ADP-ribose to a variety of nuclear [...]

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in [alpha]-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the [alpha]-synuclein-interacting protein, synphilin-1. Co-expression of [alpha]-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and [alpha]-synuclein in a common pathogenic mechanism through their interaction with synphilin-1., Author(s): Kenny K.K. Chung [1, 5]; Yi Zhang [2, 5]; Kah Leong Lim [1]; Yuji Tanaka [4]; Hui Huang [1]; Jun Gao [1]; Christopher A. Ross [2, 4]; Valina L. [...]

Drugs that inhibit the inducible form of nitric oxide synthase (iNOS) may be beneficial in treating Parkinson's disease. Giving the neurotoxin MPTP to mice increases the production of iNOS and causes neurodegeneration in the substantia nigra.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes parkinsonism in humans and nonhuman animals, and its use has led to greater understanding of the pathogenesis of Parkinson's disease. However, its molecular targets have not been defined. We show that mice lacking the gene for poly(ADP-ribose) polymerase (PARP), which catalyzes the attachment of ADP ribose units from NAD to nuclear proteins after DNA damage, are dramatically spared from MPTP neurotoxicity. MPTP potently activates PARP exclusively in vulnerable dopamine containing neurons of the substantia nigra. MPTP elicits a novel pattern of poly(ADP-ribosyl)ation of nuclear proteins that completely depends on neuronally derived nitric oxide. Thus, NO, DNA damage, and PARP activation play a critical role in MPTP-induced parkinsonism and suggest that inhibitors of PARP may have protective benefit in the treatment of Parkinson's disease.

Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ABP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ABP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. In this article, Csaba Szabo and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.

N-methyl-D-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p[21.sup.ras] (Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor, L-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate, L-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4] oxadiazolo[4,3-alpha] quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cell-permeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO/non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.

Indirect mechanisms are implicated in the pathogenesis of the dementia associated with human immunodeficiency virus-type 1 (HIV-1) infection. Proinflammatory molecules such as tumor necrosis factor α and eicosanoids are elevated in the central nervous system of patients with HIV-1-related dementia. Nitric oxide (NO) is a potential mediator of neuronal injury, because cytokines may activate the immunologic (type II) isoform of NO synthase (iNOS). The levels of (iNOS in severe HIV-1-associated dementia coincided with increased expression of the HIV-1 coat protein gp41. Furthermore, gp41 induced iNOS in primary cultures of mixed rat neuronal and glial cells and killed neurons through a NO-dependent mechanism. Thus, gp41-induced NO formation may contribute to the severe cognitive dysfunction associated with HIV-1 infection., Neurocognitive deficits are common in HIV-1 infection. Twenty to 30% of patients with acquired immunodeficiency syndrome (AIDS) develop dementia during the course of their illness (1). HIV-1 frequently enters the [...]

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide ([Mathematical Expression Omitted]) at lower L-Arg concentrations. By using electron para-magnetic resonance spin-trapping techniques, we monitored NO and [Mathematical Expression Omitted] formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the [Ca.sup.2+] ionophore A23187 triggered NO generation but no [Mathematical Expression Omitted] was seen. With cells in L-Arg-free medium, we observed [Mathematical Expression Omitted] formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. [Mathematical Expression Omitted] formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating [Mathematical Expression Omitted] and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving [Mathematical Expression Omitted]/NO-mediated cellular injury.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.

Mice suffering from a deficiency of neuronal nitric oxide synthase have been observed to exhibit abnormal appearance, movement and breeding habits. They have also demonstrated the tendency to long-term depression. In a particular experiment, mice have shown such aggressive behavior as offensive attack, biting, wrestling and chasing. The effects of the injection of nitric oxide in such mice are hard to diagnose as the behavioral aspect cannot be easily distinguished from the physiological one.

Effect of wild concentrations of immunosuppressants FK506 and cyclosporin A lead to attenuation of N-methyl-D-aspartate neurotoxicity, augmented levels of phosphorylation of nitric oxide synthase (NOS), inactivation of NOS-induced catalysis and NO-stimulated elevated levels of cyclic guanine monophosphate, cGMP. NOS-mediated catalysis is effected through dephosphorylation or through activation of calcineurin. FK506 fails to influence enhanced concentrations of cGMP in cortical cultures and inactivates calcium ionophore A23187-mediated enhancement of nitrite. It also renders inactivation of nitrite production stimulated by phorbol-ester.

It was demonstrated that the human immunodeficiency virus type 1 coat protein gp120 in extremely low concentrations elicits neurotoxicity in primary cortical neurons. This toxicity requires extracellular glutamate and calcium and involves glutamate receptors and N-methyl-D-aspartate receptors. Nitric oxide contributes to gp120 toxicity. Moreover, superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.

Leucine-rich repeat kinase-2 (LRRK2) mutations are a common cause of Parkinson's disease. Here we identify inhibitors of LRRK2 kinase that are protective in in vitro and in vivo models of LRRK2-induced neurodegeneration. These results establish that LRRK2-induced degeneration of neurons in vivo is kinase dependent and that LRRK2 kinase inhibition provides a potential new neuroprotective paradigm for the treatment of Parkinson's disease., Parkinson's disease is a very common neurodegenerative disorder with no proven neuroprotective or neurorestorative therapies. Recent advances in identifying genetic causes of Parkinson's disease have provided new opportunities for discovery [...]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) participates in a cell death cascade wherein a variety of stimuli activate nitric oxide (NO) synthases with NO nitrosylating GAPDH, conferring on it the ability to bind to Siah, an E3-ubiquitin-ligase, whose nuclear localization signal enables the GAPDH/Siah protein complex to translocate to the nucleus where degradation of Siah targets elicits cell death. R-(-)-Deprenyl (deprenyl) ameliorates the progression of disability in early Parkinson's disease and also has neuroprotective actions. We show that deprenyl and a related agent, TCH346, in subnanomolar concentrations, prevent S-nitrosylation of GAPDH, the binding of GAPDH to Siah, and nuclear translocation of GAPDH. In mice treated with the dopamine neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), low doses of deprenyl prevent binding of GAPDH and Siah1 in the dopamine-enriched corpus striatum. nitric oxide | apoptosis | Parkinson's disease | S-nitrosylation | Siah

Poly(adenosine 5'-diphosphoribose) synthetase (PARS) is a nuclear enzyme which, when activated by DNA strand breaks, adds up to 100 adenosine 5'-diphosphoribose (ADP-ribose) units to nuclear proteins such as histones and PARS itself. This activation can lead to cell death through depletion of β-nicotinamide adenine dinucleotide (the source of ADP-ribose) and adenosine triphosphate. Nitric oxide (NO) stimulated ADP-ribosylation of PARS in rat brain. Benzamide and other derivatives, which inhibit PARS, blocked N-methyl-D-aspartate NO-mediated neurotoxicity with relative potencies paralleling their ability to inhibit PARS. Thus, NO appeared to elicit neurotoxicity by activating PARS., Nitric oxide is a messenger molecule that regulates macrophage killing of tumor cells and bacteria (1) and blood vessel relaxation (2) and also is a neurotransmitter (3). When produced in [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.09.026 Byline: Joo-Ho Shin, Valina L. Dawson, Ted M. Dawson

Author(s): David Sulzer; Roy N. Alcalay; Francesca Garretti; Lucien Cote; Ellen Kanter; Julian Agin-Liebes; Christopher Liong; Curtis McMurtrey; William H. Hildebrand; Xiaobo Mao; Valina L. Dawson; Ted M. Dawson; Carla [...]

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders., Huntington's disease (HD) and dentatorubral and pallidoluysian atrophy (DRPLA) are neurodegenerative disorders caused by polyglutamine expansions in the huntingtin and atrophin-1 proteins, respectively (1-4). Huntingtin with expanded polyglutamine aggregates in [...]

Over a century ago, an influenza epidemic caused widespread parkinsonism; thus, it seems ironic that viral vectors might provide a new therapy for patients with Parkinson's disease (PD). However, a recent study presents a series of elegant experiments that demonstrate the restorative and protective effects of a glial cell line-derived neurotrophic factor (GDNF), delivered by lentiviral vectors, in the brains of both old and parkinsonian primates. These techniques might hold promise for PD and other neurodegenerative disorders.

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor [Beta] (TGF-[Beta]) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP[12.sup.-/-]) mice manifest cell cycle arrest in [G.sub.1] phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-[Beta]1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-[Beta] receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-[Beta] receptor construct. TGF-[Beta] receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP[12.sup.-/-] cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP[12.sup.-/-] cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-[Beta] receptor signaling.

Genetically engineered mice with targeted disruption of the neuronal nitric oxide synthase (nNOS) gene established the inhibitory role of nitric oxide (NO) in male impulsive aggressive behavior. This was later confirmed by using selective nNOS inhibitors in male wild-type mice. The molecular mechanisms accounting for the aggressive behavior caused by the lack of neuronally derived NO is not known. Recent studies suggest that central serotonergic neuronal circuits and particularly 5-[HT.sub.1A] and 5-[HT.sub.1B] receptors play a prominent role in the regulation of aggression. Accordingly, we investigated whether the aggressiveness caused by the lack of nNOS might be because of alterations in serotonergic function. We now demonstrate that the excessive aggressiveness and impulsiveness of nNOS knockout mice is caused by selective decrements in serotonin (5-HT) turnover and deficient 5-[HT.sub.1A] and 5-[HT.sub.1B] receptor function in brain regions regulating emotion. These results indicate an important role for NO in normal brain 5-HT function and may have significant implications for the treatment of psychiatric disorders characterized by aggressiveness and impulsivity.