Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.

Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia.

Nitric oxide (NO) and peroxynitrite, formed from NO and superoxide anion, have been implicated as mediators of neuronal damage following focal ischemia, but their molecular targets have not been defined. One candidate pathway is DNA damage leading to activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP), which catalyzes attachment of ADP ribose units from NAD to nuclear proteins following DNA damage. Excessive activation of PARP can deplete NAD and ATP, which is consumed in regeneration of NAD, leading to cell death by energy depletion. We show that genetic disruption of PARP provides profound protection against glutamate-NO-mediated ischemic insults in vitro and major decreases in infarct volume after reversible middle cerebral artery occlusion. These results provide compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia and suggest that therapies designed towards inhibiting PARP may provide benefit in the treatment of cerebrovascular disease.

Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) damages dopaminergic neurons as seen in Parkinson disease. Here we show that after administration of MPTP to mice, there was a robust gliosis in the substantia nigra pars compacta associated with significant upregulation of inducible nitric oxide synthase (iNOS). These changes preceded or paralleled MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking the iNOS gene were significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that iNOS is important in the MPTP neurotoxic process and indicates that inhibitors of iNOS may provide protective benefit in the treatment of Parkinson disease.

Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease.

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.

Urinary bladder-urethral sphincter dysfunction in mice with targeted disruption of neuronal nitric oxide synthase models idiopathic voiding disorders in humans.

Idiopathic voiding disorders affect up to 10-15% of men and women. We describe bladder abnormalities in mice with targeted deletion of the gene for neuronal nitric oxide synthase which model the clinical disorders. The mice possess hypertrophic dilated bladders and dysfunctional urinary outlets which do not relax in response to electrical field stimulation or L-arginine. The mice also display increased urinary frequency.

Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity.

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 beta-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- and NO-mediated neurotoxicity with relative potencies paralleling their ability to inhibit PARS. Thus, NO appeared to elicit neurotoxicity by activating PARS.

Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41.

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 alpha 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.

Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.

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.

Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor.

The endogenous polyamines spermine and spermidine increase the binding of [3H]MK-801 to NMDA receptors. This effect is antagonized by diethylenetriamine (DET). We report here that spermine increases the rates of both association and dissociation of binding of [3H]MK-801, suggesting that it increases the accessibility of the binding site for MK-801 within the ion channel of the receptor complex. 1,10-Diaminodecane (DA10) inhibited the binding of [3H]MK-801. This effect was due to a decrease in the rate of association with no change in the rate of dissociation of [3H]MK-801. The effect of DA10 was not mediated by an action of DA10 at the binding sites for glutamate, glycine, Mg2+, or Zn2+, and was attenuated by DET. This suggests that DA10 acts at the polyamine recognition site. In hippocampal neurons the NMDA-elicited current was decreased by DA10, an effect opposite to that of spermine. The effects of spermine and DA10 were selectively blocked by DET. It is concluded that DA10 acts as a negative allosteric modulator or inverse agonist at the polyamine recognition site of the NMDA receptor.

EndoG is dispensable in embryogenesis and apoptosis.

The mitochondrial protein, endonuclease G (EndoG), is one of the endonucleases implicated in DNA fragmentation during apoptosis. It has been shown to translocate from the mitochondria to the nucleus upon cell death stimuli. These observations suggest that EndoG is a mitochondrial cell death effector, and that it possibly acts as a cell death nuclease, similar to DNA fragmentation factor. To better understand the role of EndoG in development and apoptosis, we generated EndoG null mice by homologous gene targeting without disruption of D2Wsu81e. EndoG null mice are viable and develop to adulthood with no obvious abnormalities. Fibroblasts generated from the EndoG null mice show no difference in susceptibility when induced to die by a variety of intrinsic and extrinsic apoptotic stimuli. Additionally, EndoG null mice are equally sensitive to excitotoxic stress. These data suggest that EndoG is not essential for early embryogenesis and apoptosis.

Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice.

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus.

Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.

Spatial and functional relationship between poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in the brain.

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.

The role of the ubiquitin-proteasomal pathway in Parkinson's disease and other neurodegenerative disorders.

A unifying feature of neurodegenerative diseases is the abnormal accumulation and processing of mutant or damaged intra- and extracellular proteins; this leads to selective neuronal vulnerability and dysfunction. The ubiquitin-proteasomal pathway (UPP) is poised to play a central role in the processing of damaged and toxic proteins by ubiquitin-dependent proteolysis. The UPP can be overwhelmed in several neurodegenerative diseases. This results in the accumulation of toxic proteins and the formation of inclusions, and ultimately to neuronal dysfunction and cell death. Further analysis of the cellular and molecular mechanisms by which the UPP influences the detoxification of damaged and toxic proteins in neurodegenerative diseases could provide novel concepts and targets for the treatment and understanding of the pathogenesis of these devastating disorders.

NMDA but not non-NMDA excitotoxicity is mediated by Poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP-1), a nuclear enzyme that facilitates DNA repair, may be instrumental in acute neuronal cell death in a variety of insults including, cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, and CNS trauma. Excitotoxicity is thought to underlie these and other toxic models of neuronal death. Different glutamate agonists may trigger different downstream pathways toward neurotoxicity. We examine the role of PARP-1 in NMDA- and non-NMDA-mediated excitotoxicity. NMDA and non-NMDA agonists were stereotactically delivered into the striatum of mice lacking PARP-1 and control mice in acute (48 hr) and chronic (3 week) toxicity paradigms. Mice lacking PARP-1 are highly resistant to the excitoxicity induced by NMDA but are as equally susceptible to AMPA excitotoxicity as wild-type mice. Restoring PARP-1 protein in mice lacking PARP-1 by viral transfection restored susceptibility to NMDA, supporting the requirement of PARP-1 in NMDA neurotoxicity. Furthermore, Western blot analyses demonstrate that PARP-1 is activated after NMDA delivery but not after AMPA administration. Consistent with the theory that nitric oxide (NO) and peroxynitrite are prominent in NMDA-induced neurotoxicity, PARP-1 was not activated in mice lacking the gene for neuronal NO synthase after NMDA administration. These results suggest a selective role of PARP-1 in glutamate excitoxicity, and strategies of inhibiting PARP-1 in NMDA-mediated neurotoxicity may offer substantial acute and chronic neuroprotection.

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.

Free radicals and neuronal cell death.

Production of oxygen free radicals is a natural consequence of aerobic metabolism and they are constantly generated in vivo by chemical reactions and metabolic processes. Antioxidant defence systems scavenge and minimise the formation of oxygen-radical-derived biochemical products, however, these defences are not completely effective even under normal physiological conditions. In pathologic situations, oxygen free radicals can be generated in excess of a cell's antioxidant capacity resulting in severe damage to cellular constituents including proteins, DNA and lipids. The inherent biochemical and physiological charateristics of the brain, including high lipid concentrations and energy requirements, make it particularly susceptible to free radical mediated insult. Increasing evidence indicates that many neurological disorders may have components of free radical and oxidative stress induce injury.

Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion.

Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-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 Szabó 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.

Nitric oxide, S-nitrosylation and neurodegeneration.

Nitric oxide is a critically important signaling molecule, controlling a wide range of pathways and biological processes. Highly reactive nitric oxide mediates its function through reaction with different molecules directly or indirectly. One of these modifications is the S-nitrosylation of cysteine residues in proteins. S-nitrosylation is emerging as an important redox signaling mechanism and has been found to regulate a broad range of biologic, physiologic and cellular functions. One of the major findings in this area recently is the linkage of nitrosative stress to various neurodegenerative disorders. Oxidative stress has long been regarded as a prime mediator in the development of neurodegeneration as various indices of oxidative stress are readily observed in postmortem studies. A causative role for nitrosative stress in neurodegeneration is just now being appreciated. The direct connection of S-nitrosylation to the pathogenesis of Parkinson's disease in recent studies further provide insights into how imbalance in nitric oxide metabolism can contribute to the development of selective injury and disease.

Circadian locomotor analysis of male mice lacking the gene for neuronal nitric oxide synthase (nNOS-/-)

Nitric oxide (NO) is an endogenous gas that functions as a neurotransmitter. Because NO is very labile with a half-life of less than 5 sec, most functional studies of NO have manipulated its synthetic enzyme, NO synthase (NOS). Three isoforms of NOS have been identified: (1) in the endothelial lining of blood vessels (eNOS), (2) an inducible form found in macrophages (iNOS), and (3) in neurons (nNOS). Most pharmacological studies to date have blocked all three isoforms of NOS. Previous studies using such agents have revealed that NO might be necessary for photic entrainment of circadian rhythms; general NOS inhibitors attenuate phase shifts of free-running behavior, light-induced c-fos expression in the suprachiasmatic nucleus (SCN), and phase shifts of neural firing activity in SCN maintained in vitro. To assess the specific role of nNOS in mediating entrainment of circadian rhythms, mice with targeted deletion of the gene encoding the neuronal isoform of NOS (nNOS-/-) were used. Wild-type (WT) and nNOS-/- mice initially were entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was either phase advanced or phase delayed. After an additional 3 weeks, animals were held in either constant dim light or constant dark. WT and nNOS-/- animals did not differ in their ability to entrain to the LD cycle, phase shift locomotor activity, or free run in constant conditions. Animals held in constant dark were killed after light exposure during either the subjective day or subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos expression in the SCN of both WT and nNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that NO from neurons might not be necessary for photic entrainment.