Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/andersenLab © 2011 Buck Institute, All Rights Reserved Increased oxidative stress in the Parkinsonian substantia nigra is believed to contribute to neurodegeneration, in part due to regionally elevated levels of the enzyme monoamine oxidase B (MAOB). Increased oxidative stress has also been reported to be associated with the inhibition of E3 ligase activity of the Parkinson's diseaserelated protein parkin. In an inducible MAOB cell model, losses in parkin E3 ligase activity were found to occur in conjunction with reduced mitochondrial turnover and decreased mitochondrial function, although this did not inhibit parkin's ability to translocation to damaged mitochondria. The mTOR inhibitor rapamycin was found to restore both mitophagy and mitochondrial function in these cells. These data suggest that MAOB induction can interfere with mitochondrial quality control via losses in parkin activity that in turn impact on mitochondrial turnover. Rapamycin may be an effective means of counteracting the effects of lost parkin function by independently enhancing autophagic removal of damaged mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/22329629 Sat, 31 Dec 2011 00:00:00 -0800 Microglial activation and subsequent release of toxic proinflammatory factors are believed to play an important role in neuronal cell death associated with Parkinson's disease (PD). Compounds that inhibit microglia activation and suppress proinflammatory factor release have been reported to have neuroprotective effects in animal models of PD. In this study, we tested whether diadzein, a natural isoflavone found in soybean, attenuated lipopolysaccharide (LPS)induced release of inflammatory mediators in BV2, a murine microglial cell line. Diadzein pretreatment was found to significantly suppress the production of the proinflammatory factors nitric oxide and IL6 as well as their mRNA expression in conjunction with reductions in ROS production, p38 MAPK phosphorylation, and NFB activation. Furthermore, transfer of conditioned media (CM) from BV2 cells pretreated with diadzein resulted in a significantly reduction in dopaminergic neurotoxicity compared with CM from microglia stimulated with LPS alone. Together, our results suggest that diadzein's neuroprotective properties may be due to its ability to dampen induction of microglial activation and the subsequent release of soluble proinflammatory factors. This appears to be via inhibition of oxidative induction of the p38 MAP kinaseNFB pathway, resulting in reduced expression of proinflammatory genes and release of their corresponding gene products. http://www.ncbi.nlm.nih.gov/pubmed/22573480 Sat, 31 Dec 2011 00:00:00 -0800 Differential cysteine oxidation within mitochondrial Complex I has been quantified in an in vivo oxidative stress model of Parkinsons disease (PD). We developed a strategy that incorporates rapid and efficient immunoaffinity purification of Complex I followed by differential alkylation and quantitative detection using sensitive mass spectrometry (MS) techniques. This method allowed us to quantify the reversible cysteine oxidation status of 34 distinct cysteine residues out of a total 130 present in murine Complex I. Six Complex I cysteine residues were found to display an increase in oxidation relative to controls in brains from mice undergoing in vivo glutathione depletion. Three of these residues were found to reside within ironsulfur clusters of Complex I, suggesting that their redox state may affect electron transport function. http://www.ncbi.nlm.nih.gov/pubmed/21196577 Fri, 31 Dec 2010 00:00:00 -0800 Dopaminergic neurons of the substantia nigra pars compacta are defective in Parkinson's disease, but the specificity of this dysfunction is not understood. One hypothesis is that mitochondrial bioenergetic capacity is intrinsically lower in striatal dopaminergic presynaptic nerve varicosities, making them unusually susceptible to inhibition of electron transport by oxidative damage. To test this hypothesis, we separated isolated synaptosomes bearing dopamine transporters using immunomagnetic beads and compared their respiration with that of the residual nondopaminergic synaptosomes. As predicted, dopaminergic synaptosomes from striatum had lower respiratory rates. However, so did dopaminergic synaptosomes from cortex, indicating a lack of the predicted striatal specificity. We used fluorescent probes to analyze the bioenergetic competence of individual synaptosomes in the two fractions. The respiratory differences became nonsignificant when respiration rates were normalized to the number of respirationcompetent synaptosomes, suggesting that differences reflected the quality of the different fractions. To circumvent damage induced by synaptosomal separation, we monitored membrane potentials in whole unseparated single synaptosomes using fluorescent imaging, and then identified the dopaminergic subpopulation using a fluorescent dopamine transporter substrate (ASP() 4(4diethylaminostyryl)Nmethylpyridinium iodide). The capacity of dopaminergic and nondopaminergic synaptosomes to maintain plasma membrane and mitochondrial membrane potential under several stresses did not differ. In addition, this capacity did not decline in either subpopulation with age, a risk factor for Parkinson's disease. We conclude that the intrinsic bioenergetic capacities of dopaminergic and nondopaminergic presynaptic synaptosomes from mice do not differ. http://www.ncbi.nlm.nih.gov/pubmed/21430153 Fri, 31 Dec 2010 00:00:00 -0800 Lithium has recently been suggested to have neuroprotective properties in relation to several neurodegenerative diseases. In this study, we examined the potential cytoprotective effect of lithium in preventing oxidative stressinduced protein accumulation and neuronal cell death in the presence of increased synuclein levels in vitro and in vivo. Specifically, lithium administration was found to protect against cell death in a hydrogen peroxidetreated, stable synucleinenhanced green fluorescent protein (EGFP)overexpressing dopaminergic N27 cell line. Lithium feeding (0.255 lithium chloride) of 9monthold panneuronal synuclein transgenic mice over a 3month period was also sufficient to prevent accumulation of oxidized/nitrated synuclein as a consequence of chronic paraquat/maneb administration in multiple brain regions, including the glomerular layer, mitral cells, and the granule cell layer of the olfactory bulb (OB), striatum, substantia nigra pars compacta (SNpc) and Purkinje cells of the cerebellum. Lithium not only prevented synucleinmediated protein accumulation/aggregation in these brain regions but also protected neuronal cells including mitral cells and dopaminergic SNpc neurons against oxidative stressinduced neurodegeneration. These results suggest that lithium can prevent both synuclein accumulation and neurodegeneration in an animal model of PD, suggesting that this drug, already FDAapproved for use in bipolar disorder, may constitute a novel therapy for another human disease. 2011 WileyLiss, Inc. http://www.ncbi.nlm.nih.gov/pubmed/21710541 Fri, 31 Dec 2010 00:00:00 -0800 We previously demonstrated that elevation of astrocytic monoamine oxidase B (MAOB) levels in adoxycycline (dox)inducible transgenic mouse model following 14 days of dox induction results in several neuropathologic features similar to those observed in the Parkinsonian midbrain (Mallajosyula et al., 2008).These include a specific, selective and progressive loss of dopaminergic neurons of the substantia nigra (SN),selective decreases in mitochondrial complex I (CI) activity and increased oxidative stress. Here, we report that the temporal sequence of events following MAOB elevation initially involves increased oxidative stress followed by CI inhibition and finally neurodegeneration. Furthermore, dox removal (DR) at days 3 and 5 of MAOB induction was sufficient to arrest further increases in oxidative stress as well as subsequent neurodegenerative events. In order to assess the contribution of MAOBinduced oxidative stress to later events, we compared the impact of DR which reverses the MAOB increase with treatment of animals with the lipophilic antioxidant compound EUK189. EUK189 was found to be as effective as DR in halting downstream CI inhibition and also significantly attenuated SN DA cell loss as a result of astrocytic MAOB induction. This suggests that MAOBmediated ROS contributes to neuropathology associated with this model and that antioxidant treatment can arrest further progression of dopaminergic cell death. This has implications for early intervention therapies. http://www.ncbi.nlm.nih.gov/pubmed/21809503 Fri, 31 Dec 2010 00:00:00 -0800 Cellular senescence is an established cellular stress response that acts primarily to prevent the proliferation of cells that experience potentially oncogenic stress. In recent years, it has become increasingly apparent that the senescence response is a complex phenotype, which has a variety of cell nonautonomous effects. The senescenceassociated secretory phenotype, or SASP, entails the secretion of numerous cytokines, growth factors and proteases. The SASP can have beneficial or detrimental effects, depending on the physiological context. One recently described beneficial effect is to aid tissue repair. Among the detrimental effects, the SASP can disrupt normal tissue structures and function, and, ironically, can promote malignant phenotypes in nearby cells. These detrimental effects in many ways recapitulate the degenerative and hyperplastic pathologies that develop during aging. Because the SASP is largely a response to genomic or epigenomic damage, we suggest it may be a model for a cellular damage response that can propagate damage signals both within and among tissues. We propose that both the degenerative and hyperplastic diseases of aging may be fueled by such damage signals. http://www.ncbi.nlm.nih.gov/pubmed/21925603 Fri, 31 Dec 2010 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/22157187 Fri, 31 Dec 2010 00:00:00 -0800 Parkinson's disease (PD) involves both motor and nonmotor disturbances. Nonmotor features include alterations in sensory olfactory function which may constitute a viable biomarker for the disorder. It is not clear what causes olfactory dysfunction but it appears to coincide with the development of synucleopathy within the olfactory bulb (OB). Elevation in alphasynuclein is indeed a risk factor for development of the sporadic disorder. The multifactorial nature of the idiopathic disease combined with variability in its presentation suggests that it is likely to be influenced by several factors and that in vivo models that explore the synergistic effect of alphasynuclein elevation with other potential contributing factors are likely to be of importance in understanding the disease etiology. Using a dual transgenic mouse model of dopaminergic alphasynuclein overexpression coupled with doxycycline (Dox)inducible glutathione depletion in these same cells, we demonstrate an agerelated loss in behavioral olfactory function coupled with a significant neurodegeneration of glomerular dopaminergic neurons. This is accompanied by increases in alphasynuclein levels in nondopaminergic cells in the granule cell layer. In addition, isolated olfactory bulb synaptosomes from dual transgenic lines with Dox consistently showed a slight but significant reduction in maximum mitochondrial respiration compared to controls. These results suggest that in the presence of increased oxidative stress, increased alphasynuclein expression within dopaminergic OB neurons results in neurodegeneration in the glomerular layer and increased alphasynuclein levels in the granular cell layer which coincide with olfactory dysfunction. http://www.ncbi.nlm.nih.gov/pubmed/21055449 Sun, 31 Oct 2010 00:00:00 -0700 Neurogenesis, the production of new neurons from less differentiated precursor cells, normally occurs in adult brains in the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Neurogenesis declines with aging. In previous studies, neurogenesis was stimulated by 1methyl4phenyl1,2,3,6tetrahydropyridine hydrochloride (MPTP) in young animals. In the present study, we examined the effect of acute MPTP administration and mutant synuclein A53T on neurogenesis and migration of newborn neurons in the aged (23month) versus young (2month) rodent brain. Cell proliferation and neurogenesis were assessed via bromodeoxyuridine (BrdU) labeling and immunostaining for cell typespecific markers. In the aged brain, neural precursor cells in the rostral subventricular zone retained the capacity for proliferation and migration in response to MPTPinduced Parkinsonism, although the response is less robust than in younger animals. Furthermore, in transgenic mice that overexpress mutant synuclein (A53T), brains examined day 21 after MPTP administration showed markedly decreased olfactory bulb and substantia nigra neurogenesis. Our data suggest that in addition to aging effects associated with decline in the number of newly generated cells, mutant synuclein reduces MPTPinduced neurogenesis. This could provide a novel therapeutic target for chronic brain repair in this condition. http://www.ncbi.nlm.nih.gov/pubmed/21108729 Sun, 31 Oct 2010 00:00:00 -0700 Alphasynuclein is the major protein component of Lewy bodies, a cardinal pathological feature of the degenerating Parkinsonian brain. Alphasynuclein has been reported to be able to intercalate into membranes via formation of an alphahelical structure at its Nterminal end. Recent in vitro studies from various laboratories have demonstrated that alphasynuclein can physically associate with mitochondria and interfere with mitochondrial function. Syn predominantly associates with the inner mitochondrial membrane, where it can apparently interact with complex I resulting in reduced mitochondrial complex I activity and increased free radical production. However, the effect of in vivo alphasynuclein accumulation within dopaminergic neurons on mitochondrial function has not been thoroughly studied. Examination of transgenic animals which overexpress the familial mutant A53T form of the protein selectively within dopaminergic neurons reveals that A53T localizes to the mitochondrial membranes as monomers and oligomers particularly under conditions of proteasomal inhibitory stress, and that this localization coincides with a selective agerelated mitochondrial complex I inhibition and decreased substratespecific respiration along with increases in mitochondrial autophagy (mitophagy). http://www.ncbi.nlm.nih.gov/pubmed/20887775 Thu, 30 Sep 2010 00:00:00 -0700 As Parkinson's disease (PD) appears to be a multifactoral disorder, the use of animal models to investigate combined effects of genetic and environmental risk factors are of great importance especially in the context of aging which is the single major risk factor for the disorder. Here we assessed the combined effects of neonatal iron feeding and environmental paraquat exposure on agerelated nigrostriatal degeneration in transgenic mice expressing the A53T familial mutant form of human synuclein within these neurons. We report here that A53T synuclein mice exhibit greater susceptibility to paraquat. Increased oral intake of iron in the neonatal period leads to a progressive agerelated enhancement of dopaminergic neurodegeneration associated with paraquat neurotoxicity. Furthermore, neurodegeneration associated with these combined genetic and environmental risk factors could be attenuated by systemic treatment with the bioavailable antioxidant compound EUK189. These data suggest that environmental factors previously identified as contributors to neurodegeneration associated with sporadic PD may also be candidates for observed variations in symptoms and disease progression in monogenic forms and that this may mechanistically involve increased levels of oxidativelyinduced posttranslational nitration of alphasynuclein. http://www.ncbi.nlm.nih.gov/pubmed/21039522 Thu, 30 Sep 2010 00:00:00 -0700 We previously demonstrated that elevation of astrocytic monoamine oxidase B (MAOB) levels in a doxycycline (dox)inducible transgenic mouse model following 14 days of dox induction results in several neuropathologic features similar to those observed in the Parkinsonian midbrain (Mallajosyula et al., 2008). These include a specific, selective and progressive loss of dopaminergic neurons of the substantia nigra (SN), selective decreases in mitochondrial complex I (CI) activity and increased oxidative stress. Here, we report that the temporal sequence of events following MAOB elevation initially involves increased oxidative stress followed by CI inhibition and finally neurodegeneration. Furthermore, dox removal (DR) at days 3 and 5 of MAOB induction was sufficient to arrest further increases in oxidative stress as well as subsequent neurodegenerative events. In order to assess the contribution of MAOBinduced oxidative stress to later events, we compared the impact of DR which reverses the MAOB increase with treatment of animals with the lipophilic antioxidant compound EUK189. EUK189 was found to be as effective as DR in halting downstream CI inhibition and also significantly attenuated SN DA cell loss as a result of astrocytic MAOB induction. This suggests that MAOBmediated ROS contributes to neuropathology associated with this model and that antioxidant treatment can arrest further progression of dopaminergic cell death. This has implications for early intervention therapies. http://www.ncbi.nlm.nih.gov/pubmed/20655384 Tue, 31 Aug 2010 00:00:00 -0700 Abstract Impairment of the mitochondrial electron transport chain has been suggested to be a critical factor in the neuropathogenesis of Parkinson's disease (PD), as inhibition of mitochondrial complex I (CI) activity is consistently detected in PD patients as well as in mitochondrial toxin models of the disorder. Increased levels of various reactive oxygen and nitrogen species appear to contribute to CI inhibition and mitochondrial dysfunction in PD. Reactive nitrogen species (RNS) such as nitric oxide (NO) and its metabolite peroxynitrite (PN) may inhibit CI activity via several different mechanisms including Snitrosylation, nitration, and protein thiol formation. Studies using various cell and animal PD models have demonstrated that selective mitochondrial CI inhibition in dopaminergic cells may be due to both NOmediated Snitrosylation and nitration of CI subunits. Strategies to modulate mitochondrial NO levels will therefore likely be a promising approach to enhance mitochondrial function and protect dopaminergic neurons against oxidative or nitrosative insult. http://www.ncbi.nlm.nih.gov/pubmed/20815786 Tue, 31 Aug 2010 00:00:00 -0700 Brain iron promotes neurodegeneration in Parkinson's disease (PD). While hemoglobin (Hb) is the most abundant source of peripheral iron in humans, its relationship with PD is uncertain. This report examines the association between Hb in late life and PD incidence. From 1991 to 1993, Hb was measured in 3507 men in the HonoluluAsia Aging Study. Men were aged 7193 years and without PD. Participants were followed until 2001 for incident PD. Hb levels declined markedly with age. For men aged 7175 years, 14.8 had levels 14 g/dL versus 53.6 in those aged 86 and older (p 0.001). During followup, 47 men developed PD (19.8/10,000 personyears). After age adjustment, PD incidence rose significantly from 10.3 to 34.9/10,000 personyears as Hb increased from 14 to /= 16 g/dL (p = 0.024 relative hazard 3.2 95 confidence interval, 1.28.9). Associations persisted after accounting for early mortality and adjustments for concomitant risk factors. While Hb declines with advancing age, evidence suggests that Hb that remains high in elderly men is associated with an increased risk of PD. http://www.ncbi.nlm.nih.gov/pubmed/20709430 Sat, 31 Jul 2010 00:00:00 -0700 The goal of the present investigation was to determine the persistence of striatal dopaminergic dysfunction after a mild chemicallyinduced hypoxic event in Fisher 344 rats. To this end, we gave a single injection of the mitochondrial complex II inhibitor 3nitropropionic acid (3NP 16.5 mg/kg, i.p.) to 2 month old male F344 rats and measured various indices of striatal dopaminergic functioning and lipid peroxidation over a 3 month span. Separate groups of rats were used to measure rod walking, evoked dopamine (DA) release, DA content, MDA accumulation, DA receptor binding, and tyrosine hydroxylase activity. The results showed that 3NP exposure reduced most measures of DA functioning including motoric ability, DA release, and D(2) receptor densities for 1 to 3 months post drug administration. Interestingly, DA content was reduced 1 week after 3NP exposure, but rose to 147 of control values 1 month after 3NP treatment. MDA accumulation, a measure of lipid peroxidation activity, was increased 24 hr and 1 month after 3NP treatment. 3NP did not affect tyrosine hydroxylase activity, suggesting that alterations in DA functioning were not the result of nigrostriatal terminal loss. These data demonstrate that a brief mild hypoxic episode caused by 3NP exposure has longterm detrimental effects on the functioning of the nigrostriatal DA system. Synapse, 2010. (c) 2010 WileyLiss, Inc. http://www.ncbi.nlm.nih.gov/pubmed/20730800 Sat, 31 Jul 2010 00:00:00 -0700 Translational medicine has recently experienced an upsurge in interest and funding, yet the idea is not new. More than half a century ago, the Swedish scientist Arvid Carlsson performed basic research on the neurotransmitter dopamine that was rapidly translated into the first clinical treatment for Parkinson 's disease. For his contributions, Carlsson shared the Nobel Prize in Physiology or Medicine in the year 2000. http://www.ncbi.nlm.nih.gov/pubmed/20368162 Wed, 31 Mar 2010 00:00:00 -0700 Impairment of the ubiquitin proteasome system (UPS) and iron accumulation in the substantia nigra (SN) have both been implicated in the pathogenesis of Parkinson's disease (PD). We previously reported that chemical iron chelation can protect against proteasome inhibitor lactacystininduced dopamine (DA) neurodegeneration in vivo. Here, we tested potential neuroprotection via genetic expression of the iron chelator human ferritin heavy chain (Hferritin). We found that overexpression of Hferritin in DA neurons significantly reduced lactacystininduced nigral DA neuron loss and striatal DA depletion. Overexpression of Hferritin also attenuated elevated levels of total and ferrous iron as well as the divalent metal ion transporter 1 (DMT1) in the SN following lactacystin treatment. In addition, overexpression of Hferritin alleviated the inhibitory effects of lactacystin on proteasome activity in the nigral tissues. These results suggest that Hferritin exerts neuroprotection possibly by modulating iron homeostasis and restoring proteasome activity. http://www.ncbi.nlm.nih.gov/pubmed/19818853 Sun, 31 Jan 2010 00:00:00 -0800 The contribution of iron dysregulation to the etiology of a variety of neuronal diseases comes as no surprise given its necessity in numerous general cellular and neuronspecific functions, its abundance, and its highly reactive nature. Homeostatic mechanisms such as the iron regulatory protein and hypoxiainducible factor pathways are firmly evolutionarily set in place to prevent 'free' iron from participating in chemical Fenton and HaberWeiss reactions which can result in subsequent generation of toxic hydroxyl radicals. However, given the multiple layers of complexity in cellular iron regulation, disruption of any number of genetic and environmental components can disturb the delicate balance between the various molecular players involved in maintaining an appropriate metabolic iron homeostasis. In this review, we will primarily focus on: (i) the impact of aging and gender on iron dysfunction as these are important criteria in the determination of ironrelated disorders such as Parkinson's disease (PD), (ii) how iron mismanagement via disruption of cellular entry and exit pathways may contribute to these disorders, and (iii) how the availability of noninvasive measurement of brain iron may aid in PD diagnosis. http://www.ncbi.nlm.nih.gov/pubmed/20085612 Thu, 31 Dec 2009 00:00:00 -0800 Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathionedependent oxidoreductases (Grxs) have previously been demonstrated to be involved in ironsulfur (FeS) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in ironsulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dosedependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (maconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and maconitase activities. It also results in significant increases in ironregulatory protein (IRP) binding, likely as a consequence of conversion of FeScontaining cellular aconitase to its nonFeScontaining IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial FeS biogenesis. http://www.ncbi.nlm.nih.gov/pubmed/19290777 Mon, 30 Nov 2009 00:00:00 -0800 Hypoxiainducible factor (HIF) plays an important role in cell survival by regulating iron, antioxidant defense, and mitochondrial function. Pharmacological inhibitors of the irondependent enzyme class prolyl hydroxylases (PHD), which target alpha subunits of HIF proteins for degradation, have recently been demonstrated to alleviate neurodegeneration associated with stroke and hypoxicischemic injuries. Here we report that inhibition of PHD by 3,4dihydroxybenzoate (DHB) protects against 1methyl4phenyl1,2,3,6tetrahydropyridine (MPTP)induced nigral dopaminergic cell loss and upregulates HIF1alpha within these neurons. Elevations in mRNA and protein levels of HIFdependent genes heme oxygenase1 (Ho1) and manganese superoxide dismutase (Mnsod) following DHB pretreatment alone are also maintained in the presence of MPTP. MPTPinduced reductions in ferroportin and elevations in nigral and striatal iron levels were reverted to levels comparable with that of untreated controls with DHB pretreatment. Reductions in pyruvate dehydrogenase mRNA and activity resulting from MPTP were also found to be attenuated by DHB. In vitro, the HIF pathway was activated in N27 cells grown at 3 oxygen treated with either PHD inhibitors or an iron chelator. Concordant with our in vivo data, the MPP()elicited increase in total iron as well as decreases in cell viability were attenuated in the presence of DHB. Taken together, these data suggest that protection against MPTP neurotoxicity may be mediated by alterations in iron homeostasis and defense against oxidative stress and mitochondrial dysfunction brought about by cellular HIF1alpha induction. This study provides novel data extending the possible therapeutic utility of HIF induction to a Parkinson disease model of neurodegeneration, which may prove beneficial not only in this disorder itself but also in other diseases associated with metalinduced oxidative stress. http://www.ncbi.nlm.nih.gov/pubmed/19679656 Wed, 30 Sep 2009 00:00:00 -0700 While ferritin elevation within dopaminergic (DA) neurons of the substantia nigra (SN) is protective against neurodegeneration elicited by two toxin models of Parkinson's disease (PD), MPTP and paraquat, in young animals, its prolonged elevation results in a selective agerelated neurodegeneration. A similar agerelated neurodegeneration has been reported in iron regulatory protein 2deficient (IRP2 /) mice coinciding with increased ferritin levels within degenerating neurons. This has been speculated to be due to subsequent reductions in the labile iron pool (LIP) needed for the synthesis of ironsulfurcontaining enzymes. In order to assess whether LIP reduction is responsible for agerelated neurodegeneration in our ferritin transgenics, we examined LIP levels in ferritinexpressing transgenics with increasing age. While LIP levels were reduced within DA SN nerve terminals isolated from young ferritin transgenics compared to wildtype littermate controls, they were found to be increased in older transgenic animals at the age at which selective neurodegeneration is first noted. Furthermore, administration of the bioavailable iron chelator, clioquinol (CQ), to older mice was found to protect against both increased LIP and subsequent dopaminergic neurodegeneration. This suggests that agerelated neurodegeneration in these mice is likely due to increased iron availability rather than its reduction. This may have important implications for PD and other related neurodegenerative conditions in which iron and ferritin have been implicated. http://www.ncbi.nlm.nih.gov/pubmed/19699718 Wed, 30 Sep 2009 00:00:00 -0700 Resveratrol, a naturally occurring polyphenol, exhibits antioxidant, antiaging, and anticancer activity. Resveratrol has also been shown to inhibit tumor initiation, promotion, and progression in a variety of cell culture systems. Earlier, we showed that paraquat, a bipyridyl herbicide, triggers endoplasmic reticulum stress, cell dysfunction, and dopaminergic cell death. Due to its antioxidant activity, we assessed the ability of resveratrol to rescue cells from the toxic effects of paraquat. While resveratrol did not have any protective effect at low concentrations, it triggered endoplasmic reticulum (ER) stressinduced cell death at higher concentrations (50250 microM). The present study was carried out to determine the mechanism by which resveratrol triggers ER stress and cell death in dopaminergic N27 cells. Our studies demonstrate that resveratrol triggers ER stress and cell dysfunction, caspase activation, p23 cleavage and inhibition of proteasomal activity in dopaminergic N27 cells. While over expression of uncleavable p23 was associated with decreased cell death, downregulation of p23 protein expression by siRNA resulted in enhancement of ER stressinduced cell death triggered by resveratrol indicating a protective role for the small cochaperone p23 in dopaminergic cell death. http://www.ncbi.nlm.nih.gov/pubmed/19145491 Mon, 31 Aug 2009 00:00:00 -0700 Alphasynuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alphasynuclein tyrosine residues. We found a 9fold increase (relative to controls) in levels of 3NT at Tyr39 of alphasynuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAOB) is overexpressed and which emulates several features of PD. Increased nitration of Tyr39 on endogenous alphasynuclein via elevations in MAOB levels could be abrogated by the addition of deprenyl, a specific MAOB inhibitor. The increased levels of 3NT was selective for Tyr39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr125, Tyr133, and Tyr136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein. http://www.ncbi.nlm.nih.gov/pubmed/19697948 Mon, 31 Aug 2009 00:00:00 -0700 We previously demonstrated that spare respiratory capacity of the TCA cycle enzyme alphaketoglutarate dehydrogenase (KGDH) was completely abolished upon increasing levels of MAOB activity in a dopaminergic cell model system (Kumar et al., J Biol Chem 278:4643246439, 2003). MAOB mediated increases in H(2)O(2) also appeared to result in direct oxidative inhibition of both mitochondrial complex I and aconitase. In order to elucidate the contribution that each of these components exerts over metabolic respiratory control as well as the impact of MAOB elevation on their spare respiratory capacities, we performed metabolic respiratory control analysis. In addition to KGDH, we assessed the activities and substratemediated respiration of complex I, pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and mitochondrial aconitase in the absence and presence of complexspecific inhibitors in specific and mixed substrate conditions in mitochondria from our MAOB elevated cells versus controls. Data from this study indicates that Complex I and KGDH are the most sensitive to inhibition by MAOB mediated H(2)O(2) generation, and could be instrumental in determining the fate of mitochondrial metabolism in this cellular PD model system. http://www.ncbi.nlm.nih.gov/pubmed/19526285 Fri, 31 Jul 2009 00:00:00 -0700 Apoptosisinducing factor (AIF)deficient harlequin (Hq) mice undergo neurodegeneration associated with a 4050 reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H2O2 removal, or ROS production stimulated by complex I inhibitors rotenone or 1methyl4phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by approximately 50 in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenonestimulated rates of H2O2 release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested. http://www.ncbi.nlm.nih.gov/pubmed/19280713 Sat, 28 Feb 2009 00:00:00 -0800 Parkinson's disease (PD) features oxidative stress and accumulation of misfolded (unfolded, alternatively folded, or mutant) proteins with associated loss of dopaminergic neurons. Oxidative stress and the accumulated misfolded proteins elicit cellular responses that include an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Chronic ER stress and accumulation of misfolded proteins in excessive amounts, however, overwhelm the cellular 'quality control' system and impair the protective mechanisms designed to promote correct folding and degrade faulty proteins, ultimately leading to organelle dysfunction and neuronal cell death. Paraquat belongs to a class of bipyridyl herbicides and triggers oxidative stress and dopaminergic cell death. Epidemiological studies suggest an increased risk for developing PD following chronic exposure to paraquat. The present study was carried out to determine the role of paraquat in triggering cellular stress particularly ER stress and to elucidate the pathways that couple ER stress to dopaminergic cell death. We demonstrate that paraquat triggers ER stress, cell dysfunction, and dopaminergic cell death. p23, a small cochaperone protein, is cleaved during ER stressinduced cell death triggered by paraquat and blockage of the caspase cleavage site of p23 was associated with decreased cell death. Paraquat also inhibits proteasomal activity that may further trigger accumulation of misfolded proteins resulting in ER stress. Our results indicate a protective role for p23 in PDrelated programmed cell death. The data also underscore the involvement of ER, caspases, and the proteasomal system in ER stressinduced cell death process. http://www.ncbi.nlm.nih.gov/pubmed/18773310 Sat, 31 Jan 2009 00:00:00 -0800 Glutathione depletion is one of the earliest detectable events in the Parkinsonian substantia nigra (SN), but whether it is causative for ensuing molecular events associated with the disease is unknown. Here we report that reduction in levels of glutathione in immortalized midbrainderived dopaminergic neurons results in increases in the cellular labile iron pool (LIP). This increase is independent of either iron regulatory protein/iron regulatory element (IRP/IRE) or hypoxia inducible factor (HIF) induction but is both H(2)0(2) and protein synthesisdependent. Our findings suggest a novel mechanistic link between dopaminergic glutathione depletion and increased iron levels based on translational activation of TfR1. This may have important implications for neurodegeneration associated with Parkinson's disease in which both glutathione reduction and iron elevation have been implicated. http://www.ncbi.nlm.nih.gov/pubmed/19118623 Sat, 31 Jan 2009 00:00:00 -0800 Environmental paraquat and neonatal iron exposure have both separately been suggested as potential risk factors for sporadic forms of Parkinson's disease (PD). In this study, we demonstrate that combined environmental exposure to these two agents results in modulations in microglial activation state. Apocynin, an NADPH oxidase inhibitor, was found to attenuate the release of superoxide from microglia stimulated by combined paraquat and iron and blocked paraquatinduced dopaminergic neuronal death. Furthermore, pretreatment with the synthetic superoxide dismutase/catalase mimetic, EUK189, significantly decreased microglial activation mediated by combined paraquat and iron treatment. These findings support the notion that environmental PD risk factors may act synergetically to produce neurodegeneration associated with the disorder and that iron and paraquat may act via common oxidative stressmediated mechanism involving microglial activation. http://www.ncbi.nlm.nih.gov/pubmed/19027846 Wed, 31 Dec 2008 00:00:00 -0800 Dopaminergic neurodegeneration during Parkinson disease (PD) involves several pathways including proteasome inhibition, alphasynuclein (alphasyn) aggregation, mitochondrial dysfunction, and glutathione (GSH) depletion. We have utilized a systems biology approach and built a dynamic model to understand and link the various events related to PD pathophysiology. We have corroborated the modeling data by examining the effects of alphasyn expression in the absence and presence of proteasome inhibition on GSH metabolism in dopaminergic neuronal cultures. We report here that the expression of the mutant A53T form of alphasyn is neurotoxic and causes GSH depletion in cells after proteasome inhibition, compared to wildtype alphasynexpressing cells and vector control. Modeling data predicted that GSH depletion in these cells was due to ATP loss associated with mitochondrial dysfunction. ATP depletion elicited by combined A53T expression and proteasome inhibition results in decreased de novo synthesis of GSH via the ratelimiting enzyme gammaglutamyl cysteine ligase. Based on these data and other recent reports, we propose a novel dynamic model to explain how the presence of mutated alphasyn protein or proteasome inhibition may individually impact on mitochondrial function and in combination result in alterations in GSH metabolism via enhanced mitochondrial dysfunction. http://www.ncbi.nlm.nih.gov/pubmed/18761401 Tue, 30 Sep 2008 00:00:00 -0700 Parkinson's disease (PD) is an adultonset neurodegenerative disorder characterized by preferential loss of dopaminergic neurons in an area of the midbrain called the substantia nigra (SN) along with occurrence of intraneuronal inclusions called Lewy bodies. The majority of cases of PD are sporadic in nature with late onset (95 of patients) however a few PD cases (5) are seen in familial clusters with generally earlier onset. Although PD has been heavily researched, so far the exact cause of the rather selective cell death is unknown. Multiple lines of evidence suggest an important role for oxidative stress. Dopaminergic neurons (DA) are particularly prone to oxidative stress due to DA metabolism and autooxidation combined with increased iron, decreased total glutathione levels and mitochondrial complex I inhibitioninduced ROS production in the SN which can lead to cell death by exceeding the oxidative capacity of DAcontaining cells in the region. Enhancing antioxidant capabilities and chelating labile iron pools in this region therefore constitutes a rational approach to prevent or slow ongoing damage of DA neurons. In this review, we summarize the various sources of reactive oxygen species that may cause redox imbalance in PD as well as potential therapeutic targets for attenuation of oxidative stress associated with PD. http://www.ncbi.nlm.nih.gov/pubmed/18358848 Thu, 31 Jul 2008 00:00:00 -0700 In response to injury, endogenous precursors in the adult brain can proliferate and generate new neurons, which may have the capacity to replace dysfunctional or dead cells. Although injuryinduced neurogenesis has been demonstrated in animal models of stroke, Alzheimer's disease (AD) and Huntington's disease (HD), studies of Parkinson's disease (PD) have produced conflicting results. In this study, we investigated the ability of adult mice to generate new neurons in response to the parkinsonian toxin 1methyl4phenyl1,2,3,6tetrahydropyridine (MPTP), which causes selective degeneration of nigrostriatal dopamine neurons. MPTP lesions increased the incorporation of 5bromo2'deoxyuridine5'monophosphate (BrdU), as well as the number of cells that coexpressed BrdU and the immature neuronal marker doublecortin (DCX), in two neuroproliferative regionsthe subgranular zone of the dentate gyrus (DG) and the rostral subventricular zone (SVZ). BrdUlabeled, DCXexpressing cells were not found in the substantia nigra (SN) of MPTPtreated mice, where neuronal cell bodies are destroyed, but were present in increased numbers in the striatum, where SN neurons lost in PD normally project. Fibroblast growth factor2 (FGF2), which enhances neurogenesis in a mouse model of HD, also increased the number of BrdU/DCXimmunopositive cells in the SN of MPTPtreated mice. Thus, MPTPinduced brain injury increases striatal neurogenesis and, in combination with FGF2 treatment, also stimulates neurogenesis in SN. http://www.ncbi.nlm.nih.gov/pubmed/18407421 Wed, 30 Apr 2008 00:00:00 -0700 Parkinson's disease (PD) is a complex neurodegenerative syndrome likely involving contributions from various factors in individuals including genetic susceptibility, exposure to environmental toxins, and the aging process itself. Increased oxidative stress appears to be a common causative aspect involved in the preferential loss of dopaminergic neurons in a region of the brain prominently affected by the disorder, the substantia nigra (SN). Loss of dopaminergic SN neurons is responsible for the classic clinical motor symptoms associated with PD. Several oxidative and nitrative posttranslational modifications (PTMs) have been identified on proteins pertinent to PD that may affect this or other aspects of disease progression. In this review, we discuss several examples of such PTMs to illustrate their potential consequences in terms of initiation or progression of PD neuropathophysiology. http://www.ncbi.nlm.nih.gov/pubmed/18395015 Mon, 31 Mar 2008 00:00:00 -0700 Agerelated increases in monoamine oxidase B (MAOB) may contribute to neurodegeneration associated with Parkinson's disease (PD). The MAOB inhibitor deprenyl, a longstanding antiparkinsonian therapy, is currently used clinically in concert with the dopamine precursor LDOPA. Clinical studies suggesting that deprenyl treatment alone is not protective against PD associated mortality were targeted to symptomatic patients. However, dopamine loss is at least 60 by the time PD is symptomatically detectable, therefore lack of effect of MAOB inhibition in these patients does not negate a role for MAOB in presymptomatic dopaminergic loss. In order to directly evaluate the role of agerelated elevations in astroglial MAOB in the early initiation or progression of PD, we created genetically engineered transgenic mice in which MAOB levels could be specifically induced within astroglia in adult animals. Elevated astrocytic MAOB mimicking age related increase resulted in specific, selective and progressive loss of dopaminergic neurons in the substantia nigra (SN), the same subset of neurons primarily impacted in the human condition. This was accompanied by other PDrelated alterations including selective decreases in mitochondrial complex I activity and increased mitochondrial oxidative stress. Along with a global astrogliosis, we observed local microglial activation within the SN. These pathologies correlated with decreased locomotor activity. Importantly, these events occurred even in the absence of the PDinducing neurotoxin MPTP. Our data demonstrates that elevation of murine astrocytic MAOB by itself can induce several phenotypes of PD, signifying that MAOB could be directly involved in multiple aspects of disease neuropathology. Mechanistically this may involve increases in membrane permeant H(2)O(2) which can oxidize dopamine within dopaminergic neurons to dopaminochrome which, via interaction with mitochondrial complex I, can result in increased mitochondrial superoxide. Our inducible astrocytic MAOB transgenic provides a novel model for exploring pathways involved in initiation and progression of several key features associated with PD pathology and for therapeutic drug testing. http://www.ncbi.nlm.nih.gov/pubmed/18286173 Thu, 31 Jan 2008 00:00:00 -0800 Parkinson's disease is a neurodegenerative disorder characterized by the preferential loss of midbrain dopaminergic neurons in the substantia nigra (SN). One of the earliest detectable biochemical alterations that occurs in the Parkinsonian brain is a marked reduction in SN levels of total glutathione (glutathione plus glutathione disulfide), occurring before losses in mitochondrial complex I (CI) activity, striatal dopamine levels, or midbrain dopaminergic neurodegeneration associated with the disease. Previous in vitro data from our laboratory has suggested that prolonged depletion of dopaminergic glutathione results in selective impairment of mitochondrial complex I activity through a reversible thiol oxidation event. To address the effects of depletion in dopaminergic glutathione levels in vivo on the nigrostriatal system, we created genetically engineered transgenic mouse lines in which expression of gammaglutamyl cysteine ligase, the ratelimiting enzyme in de novo glutathione synthesis, can be inducibly downregulated in catecholaminergic neurons, including those of the SN. A novel method for isolation of purified dopaminergic striatal synaptosomes was used to study the impact of dopaminergic glutathione depletion on mitochondrial events demonstrated previously to occur in vitro as a consequence of this alteration. Dopaminergic glutathione depletion was found to result in a selective reversible thioloxidationdependent mitochondrial complex I inhibition, followed by an agerelated nigrostriatal neurodegeneration. This suggests that depletion in glutathione within dopaminergic SN neurons has a direct impact on mitochondrial complex I activity via increased nitric oxiderelated thiol oxidation and agerelated dopaminergic SN cell loss. http://www.ncbi.nlm.nih.gov/pubmed/18094238 Fri, 30 Nov 2007 00:00:00 -0800 Oxidative/nitrosative stress and mitochondrial dysfunction have been implicated in the degeneration of dopaminergic neurons in the substantia nigra during Parkinson's disease (PD). During early stages of PD, there is a significant depletion of the thiol antioxidant glutathione (GSH), which may lead to oxidative stress, mitochondrial dysfunction, and ultimately neuronal cell death. Mitochondrial complex I (CI) is believed to be the central player to the mitochondrial dysfunction occurring in PD. We have generated a dynamic, mechanistic model for mitochondrial dysfunction associated with PD progression that is activated by rotenone, GSH depletion, increased nitric oxide and peroxynitrite. The potential insults independently inhibit CI and other complexes of the electron transport chain, drop the proton motive force, and reduce ATP production, ultimately affecting the overall mitochondrial performance. We show that mitochondrial dysfunction significantly affects glutathione synthesis thereby increasing the oxidative damage and further exacerbating the toxicities of these mitochondrial agents resulting in neurodegeneration. Rat dopaminergic neuronal cell culture and in vitro experiments using mouse brain mitochondria were employed to validate important features of the model. MAJOR CONCLUSIONS: Using a combination of experimental and in silico modeling approaches, we have demonstrated the interdependence of mitochondrial function with GSH metabolism in relation to neurodegeneration in PD. http://www.ncbi.nlm.nih.gov/pubmed/17936517 Wed, 31 Oct 2007 00:00:00 -0700 Extensive epidemiological data in humans and studies in animal models of Parkinson's disease (PD) suggest that sporadic forms of the disorder are not strictly genetic in nature but most likely because of combined environmental exposures over the period of the lifespan coupled with increased genetic susceptibilities. Environmental paraquat and neonatal iron exposure have both been separately suggested as potential risk factors for sporadic forms of the disease. In this study, we demonstrate that combined environmental exposure to these two agents results in accelerated agerelated degeneration of nigrostriatal dopaminergic neurons. Furthermore, pretreatment with the synthetic superoxide dismutase/catalase mimetic, EUK189, significantly attenuated neuronal death mediated by combined paraquat and iron treatment. These findings support the notion that environmental PD risk factors may act synergistically to produce neurodegeneration associated with the disorder and that iron and paraquat may act via common oxidative stressmediated mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/17596439 Thu, 31 May 2007 00:00:00 -0700 Iron elevation is welldocumented in the Parkinsonian midbrain but its cause and contribution to subsequent neurodegeneration remain unknown. Mice administered iron at doses equivalent to those found in ironfortified human infant formula during a developmental period equivalent to the first human year of life display progressive midbrain neurodegeneration and enhanced vulnerability to toxic injury. This may have major implications for the impact of neonatal iron intake as a potential risk factor for later development of Parkinson's disease (PD). http://www.ncbi.nlm.nih.gov/pubmed/16765489 Sat, 31 Mar 2007 00:00:00 -0700 Ferritin elevation has been reported by some laboratories to occur within the substantia nigra (SN), the area of the brain affected in Parkinson's disease (PD), but whether such an increase could be causatively involved in neurodegeneration associated with the disorder is unknown. Here, we report that chronic ferritin elevation in midbrain dopaminecontaining neurons results in a progressive agerelated neurodegeneration of these cells. This provides strong evidence that chronic ferritin overload could be directly involved in agerelated neurodegeneration such as occurs in Parkinson's and other related diseases. http://www.ncbi.nlm.nih.gov/pubmed/16631136 Wed, 28 Feb 2007 00:00:00 -0800 Selective damage of mitochondrial complex I within the dopaminergic neurons of the substantia nigra is the central event during Parkinson disease. Peroxynitrite is one of the important free radicals probably mediating complex I damage. Peroxynitrite inhibits brain complex I mainly by 3nitrotyrosine and nitrosothiol formation, but how these modifications alter the structurefunction relation of complex I is unclear. Curcumin pretreatment protects brain mitochondria against peroxynitrite in vitro by direct detoxification and prevention of 3nitrotyrosine formation and in vivo by elevation of total cellular glutathione levels. These results suggest a potential therapeutic role for curcumin against nitrosative stress in neurological disorders. http://www.ncbi.nlm.nih.gov/pubmed/17184173 Wed, 28 Feb 2007 00:00:00 -0800 A disruption in optimal iron levels within different brain regions has been demonstrated in several neurodegenerative disorders. Although iron is an essential element that is required for many processes in the human body, an excess can lead to the generation of free radicals that can damage cells. Iron levels are therefore stringently regulated within cells by a host of regulatory proteins that keep iron levels in check. The iron regulatory proteins (IRPs) have the ability to sense and control the level of intracellular iron by binding to iron responsive elements (IREs) of several genes encoding key proteins such as the transferrin receptor (TfR) and ferritin. Concurrently, the hypoxiainducible factor (HIF) has also been shown in previous studies to regulate intracellular iron by binding to HIFresponsive elements (HREs) that are located within the genes of ironrelated proteins such as TfR and heme oxygenase1 (HO1). This review will focus on the interactions between the IRP/IRE and HIF/HRE systems and how cells utilize these intricate networks to regulate intracellular iron levels. Additionally, since iron chelation has been suggested to be a therapeutic treatment for disorders such as Parkinson's and Alzheimer's disease, understanding the exact mechanisms by which iron acts to cause disease and how the brain would be impacted by iron chelation could potentially give us novel insights into new therapies directed towards preventing or slowing neuronal cell loss associated with these disorders. http://www.ncbi.nlm.nih.gov/pubmed/17168739 Thu, 30 Nov 2006 00:00:00 -0800 The murine mutant weaver (gene symbol, wv) mouse, which carries a mutation in the gene encoding the Gprotein inwardly rectifying potassium channel Girk2, exhibits a diverse range of defects as a result of postnatal cell death in several different brain neuron subtypes. Loss of dopaminergic nigrostriatal neurons in the weaver, unlike cerebellar granule neuronal loss, is via a noncaspasemediated mechanism. Here, we present data demonstrating that degeneration of midbrain dopaminergic neurons in weaver is mediated via neuroinflammation. Furthermore, in vivo administration of the antiinflammatory agent minocycline attenuates nigrostriatal dopaminergic neurodegeneration. This has novel implications for the use of the weaver mouse as a model for Parkinson's disease, which has been associated with increased neuroinflammation. http://www.ncbi.nlm.nih.gov/pubmed/17093086 Tue, 31 Oct 2006 00:00:00 -0800 The pathogenesis underlying the selective degeneration of nigral dopaminergic neurons in Parkinson's disease is not fully understood but several lines of evidence implicate the role of oxidative stress and mitochondrial dysfunction. Depletion in levels of the thiol reducing agent glutathione (GSH GSSG) is the earliest reported biochemical event to occur in the Parkinsonian substantia nigra prior to selective loss of complex I (CI) activity associated with the disease believed to contribute to subsequent dopaminergic cell death. Recent studies from our laboratory have demonstrated that acute reduction in both cellular and mitochondrial glutathione levels results in increased oxidative stress and a decrease in mitochondrial function linked to a selective decrease in CI activity through an NOmediated mechanism (Jha, N. Jurma, O. Lalli, G. Liu, Y. Pettus, E. H. Greenamyre, J. T. Liu, R. M. Forman, H. J. Andersen, J. K. Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease J. Biol. Chem. 275: 2609626101 2000. Hsu, M. Srinivas, B. Kumar, J. Subramanian, R. Andersen, J. Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NOmediated pathway not involving peroxynitrite: implications for Parkinson's disease J. Neurochem. 92: 10911103.2005.). However, the effect of prolonged glutathione depletion on dopaminergic cells is not known. In this present study, using low concentrations of buthionineSsulfoximine, a chemical inhibitor of the de novo glutathione synthesizing enzyme glutamate cysteine ligase, we developed a chronic model in which glutathione depletion in dopaminergic N27 cells for a 7day period was found to lead to inhibition of CI activity via a peroxynitritemediated event which is reversible by the thiol reducing agent, dithiothreitol, and coincides with increased Snitrosation of mitochondrial proteins. http://www.ncbi.nlm.nih.gov/pubmed/17023271 Sat, 30 Sep 2006 00:00:00 -0700 Glutathione is an abundant intracellular thiol antioxidant whose levels are reduced both in Parkinson's disease itself and in a widely used animal model of the disorder, systemic MPTP administration. Previous in vitro work from our laboratory has suggested that glutathione depletion may be directly responsible for mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with the disease. Here, we demonstrate the ability of gammaglutamylcysteine ethyl ester, a lipid permeable derivative of the major substrate for scavenger glutathione synthesis, to counteract glutathione loss and neurodegeneration associated with in vitro and in vivo administration of MPTP or its derivatives. This data suggests that prevention of glutathione depletion is a likely therapeutic target for the disease. http://www.ncbi.nlm.nih.gov/pubmed/16644116 Sun, 30 Apr 2006 00:00:00 -0700 Upregulation of activity of gammaglutamyl transpeptidase (GGT) has been reported to occur in the Parkinsonian substantia nigra, the area of the brain affected by the disease. Increased GGT activity has been hypothesized to play a role in subsequent mitochondrial complex I (CI) inhibition by increasing cysteine as substrate for cellular uptake. Intracellular cysteine has been proposed to form toxic adducts with dopamine which can be metabolized to compounds which inhibit CI activity. We have demonstrated that in addition to CI inhibition, GGT activity is upregulated in dopaminergic cells as a consequence of glutathione depletion. Inhibition of GGT rather than resulting in increased CI inhibition results in exacerbation of this inhibitory effect. This suggests that increased GGT activity is likely an adaptive response to the loss of glutathione to conserve intracellular glutathione content and results in a compensatory effect on CI activity rather than in its inhibition as has been previously widely hypothesized. http://www.ncbi.nlm.nih.gov/pubmed/16632116 Fri, 31 Mar 2006 00:00:00 -0800 Iron is essential for many biological processes however excess concentrations can be harmful to many tissues. Its amounts must therefore be carefully regulated in all cells of the body including those in the brain. Increased amounts of iron have been reported in many neurodegenerative disorders. Whether this increased iron contributes to neurodegeneration has been considered controversial. In this review, we discuss some recently identified anomalies in proteins linked with iron metabolism which signify a critical role for iron dysregulation in neurodegeneration. http://www.ncbi.nlm.nih.gov/pubmed/16565471 Tue, 28 Feb 2006 00:00:00 -0800 Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK134 and EUK189) in protecting against paraquatinduced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK134 or EUK189 significantly attenuates paraquatinduced neurotoxicity in vitro in a concentrationdependent manner. Furthermore, systemic administration of EUK189 decreases paraquatmediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquatinduced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD. http://www.ncbi.nlm.nih.gov/pubmed/15946937 Sun, 31 Jul 2005 00:00:00 -0700 Oxidative stress and mitochondrial dysfunction signify two important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models and in affected tissues from the disease itself have demonstrated a selective inhibition of mitochondrial complex I activity that appears to affect normal mitochondrial physiology leading to neuronal cell death. Earlier experiments from our laboratory have demonstrated that induced depletion of glutathione (GSH GSSG) in cultured dopaminergic cells resulted in increased oxidative stress and a decrease in mitochondrial function. Furthermore, this dysfunction was linked to a selective decrease in mitochondrial complex I activity that appears to be due to oxidation of this complex. Glutathione depletion is the earliest detectable biochemical event during PD progression and occurs prior to complex I inhibition. Recent observations have also indicated that oxidative damage to complex I via naturally occurring free radicals such as peroxynitrite leads to modification of tyrosine and/or cysteine residues resulting in complex I inhibition. Using the sucrose gradient method, we detected in complex Ienriched fractions from a glutathionedepleted dopaminergic cell line two bands corresponding to approximately 25kDa and approximately 30kDa polypeptides that demonstrate antinitrotyrosine immunoreactivity, suggesting the possible involvement of protein nitration by peroxynitrite in glutathione depletionmediated complex I inhibition. http://www.ncbi.nlm.nih.gov/pubmed/15998245 Thu, 30 Jun 2005 00:00:00 -0700 Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the timecourse of neurodegeneration was correlated to changes in 4hydroxy2nonenal (4HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4HNEpositive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4HNEimmunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquatinduced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquattreated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4HNEpositive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage. http://www.ncbi.nlm.nih.gov/pubmed/15857406 Thu, 31 Mar 2005 00:00:00 -0800 Dopaminergic neurons of the midbrain are the main source of dopamine (DA) in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). Dopaminergic neurons are found in a 'harsh' region of the brain, the substantia nigra pars compacta, which is DArich and contains both redox available neuromelanin and a high iron content. Although their numbers are few, these dopaminergic neurons play an important role in the control of multiple brain functions including voluntary movement and a broad array of behavioral processes such as mood, reward, addiction, and stress. Studies into the developmental pathways which are involved in the generation of dopaminergic neurons in the brain have led to the identification of several specific transcription factors including Nurr1, Lmx1b and Pitx3, all shown to be important in the development of the mesencephalic dopaminergic system. The selective degeneration of these dopaminergic neurons in the substantia nigra pars compacta leads to PD but the exact cause for this nigral cell loss is still unknown. http://www.ncbi.nlm.nih.gov/pubmed/15743669 Mon, 28 Feb 2005 00:00:00 -0800 An early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss, and cell death. We have demonstrated using dopaminergic PC12 cell lines genetically engineered to inducibly downregulate glutathione synthesis that total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Here, we demonstrate that inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitritemediated inhibition of complex I or through H2O2mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme alphaketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex activity of both enzymes are reduced in PD. While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. Our data suggest that NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells. This may have major implications in terms of understanding mechanisms of dopamine cell death associated with PD especially as they relate to complex I inhibition. http://www.ncbi.nlm.nih.gov/pubmed/15715660 Mon, 28 Feb 2005 00:00:00 -0800 Glutathione peroxidase (GSHPx) has been demonstrated in several in vivo studies to reduce both the risk and severity of oxidativelyinduced tissue damage. The seizureinducing neurotoxin kainic acid (KA) has been suggested to elicit its toxic effects in part via generation of oxidative stress. In this study, we report that expression of elevated levels of murine GSHPx1 in transgenic mice surprisingly results in increased rather than decreased KA susceptibility including increased seizure activity and neuronal hippocampal damage. Isolated transgenic primary hippocampal culture neurons also display increased susceptibility to KA treatment compared with those from wildtype animals. This could be due to alterations in the redox state of the glutathione system resulting in elevated glutathione disulfide (GSSG) levels which, in turn, may directly activate NMDA receptors or enhanced response of the NMDA receptor. http://www.ncbi.nlm.nih.gov/pubmed/15698635 Mon, 28 Feb 2005 00:00:00 -0800 We have recently demonstrated that chelation of in vivo brain iron in a form which is not available to participate in oxidative events protects against a toxininduced form of Parkinsonism in rodents, the wellestablished MPTP model 32. These data strongly suggest that iron elevations observed in the Parkinsonian substantia nigra (SN), the brain region which undergoes selective neurodegeneration in the disease, are actively involved in subsequent neurodegenerative events. However the mechanism(s) by which iron levels become elevated in the Parkinsonian SN are still unclear. We hypothesize that increased oxidative stress associated with the disease may result in dysregulation of iron homeostasis in midbrain dopaminergic neurons via alterations in binding of iron regulatory proteins (IRPs). This would mechanistically explain the noted increase in cellular iron levels in the Parkinsonian SN which appear to contribute to subsequent neurodegeneration. http://www.ncbi.nlm.nih.gov/pubmed/15665414 Fri, 31 Dec 2004 00:00:00 -0800 Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 2609626101). Complex I is a approximately 980kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, onedimensional and twodimensional SDSPAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction. http://www.ncbi.nlm.nih.gov/pubmed/15591592 Fri, 31 Dec 2004 00:00:00 -0800 Oxidative stress has long been linked to the neuronal cell death that is associated with certain neurodegenerative conditions. Whether it is a primary cause or merely a downstream consequence of the neurodegenerative process is still an open question, however. The advent of a growing number of in vitro and in vivo models that emulate human disease pathology is aiding scientists in deciphering just where oxidative stress intersects with other cellular events in the emerging roadmap leading to neurodegeneration. Here I review the evidence for oxidative stress in neurodegeneration and how this relates to other cellular events. http://www.ncbi.nlm.nih.gov/pubmed/15298006 Sat, 31 Jul 2004 00:00:00 -0700 The catecholamineoxidizing enzyme monoamine oxidaseB (MAOB) has been hypothesized to be an important determining factor in the etiology of both normal aging and agerelated neurological disorders such as Parkinson's disease (PD). Catalysis of substrate by the enzyme produces H2O2 which is a primary originator of oxidative stress which in turn can lead to cellular damage. MAOB increases with age as does predisposition towards PD which has also been linked to increased oxidative stress. Inhibition of MAOB, along with supplementation of lost dopamine via LDOPA, is one of the major antiparkinsonian therapies currently in use. In this review, we address several factors contributing to a possible role for MAOB in normal brain aging and neurological disease and also discuss the use of MAOB inhibitors as drug therapy for these conditions. http://www.ncbi.nlm.nih.gov/pubmed/15247489 Wed, 30 Jun 2004 00:00:00 -0700 Environmental exposure to the oxidantproducing herbicide paraquat has been implicated as a risk factor in Parkinson's disease. Although intraperitoneal paraquat injections in mice cause a selective loss of dopaminergic neurons in the substantia nigra pars compacta, the exact mechanism involved is still poorly understood. Our data show that paraquat induces the sequential phosphorylation of cJun Nterminal kinase (JNK) and cJun and the activation of caspase3 and sequential neuronal death both in vitro and in vivo. These effects are diminished by the specific JNK inhibitor SP600125 and the antioxidant manganese(III) tetrakis (4benzoic acid) porphyrin in vitro. Furthermore, JNK pathway inhibitor CEP11004 effectively blocks paraquatinduced dopaminergic neuronal death in vivo. These results suggest that the JNK signaling cascade is a direct activator of the paraquatmediated nigral dopaminergic neuronal apoptotic machinery and provides a molecular linkage between oxidative stress and neuronal apoptosis. http://www.ncbi.nlm.nih.gov/pubmed/15155744 Wed, 30 Jun 2004 00:00:00 -0700 Selective dopaminergic cell loss in Parkinson's disease is correlated with increased levels of cellular iron. It is still hotly debated as to whether the increase in iron is an upstream event which acts to promote neurodegeneration via formation of oxidative stress or whether iron accumulates as a byproduct of the neuronal cell loss. Here we review evidence for loss of iron homeostasis as a causative factor in diseaseassociated neurodegeneration and the primary players which may be involved. A series of recent studies suggest that iron regulatory proteins (IRPs) coordinate both cellular iron levels and energy metabolism, both of which are disrupted in Parkinson's disease (PD) and may in turn contribute to increased levels of oxidative stress associated with the disease. Iron has also been recently been implicated in promotion of alphasynuclein aggregation either directly or via increasing levels of oxidative stress suggesting an important role for it in Lewy body formation, another important hallmark of the disease. http://www.ncbi.nlm.nih.gov/pubmed/15231240 Wed, 30 Jun 2004 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/14715449 Wed, 31 Dec 2003 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/15312295 Wed, 31 Dec 2003 00:00:00 -0800 Agerelated increases in brain monoamine oxidase B (MAOB) and its ability to produce reactive oxygen species as a byproduct of catalysis could contribute to neurodegeneration associated with Parkinson's disease. This may be via increased oxidative stress and/or mitochondrial dysfunction either on its own or through its interaction with endogenous or exogenous neurotoxic species. We have created genetically engineered dopaminergic PC12 cell lines with subtly increased levels of MAOB mimicking those observed during normal aging. In our cells, increased MAOB activity was found to result in increased H2O2 production. This was found to correlate with a decrease in mitochondrial complex I activity which may involve both direct oxidative damage to the complex itself as well as oxidative effects on the tricarboxylic acid cycle enzyme alphaketoglutarate dehydrogenase (KGDH) which provides substrate for the complex. Both complex I and KGDH activities have been reported to be decreased in the Parkinsonian brain. These in vitro events are reversible by catalase addition. Importantly, MAOB elevation was found to abolish the spare KGDH threshold capacity, which can normally be significantly inhibited before it affects maximal mitochondrial oxygen consumption rates. Our data suggest that H2O2 production via subtle elevations in MAOB levels can result in oxidative effects on KGDH that can compromise the ability of dopaminergic neurons to cope with increased energetic stress. http://www.ncbi.nlm.nih.gov/pubmed/12963742 Fri, 31 Oct 2003 00:00:00 -0800 Given the critical role that the cJun Nterminal kinase (JNK) pathway plays in regulating many of the cellular processes which are affected in Parkinson's disease (PD), the possible importance of JNK in disease pathogenesis is being increasingly recognized. Here we review recent findings implicating the JNK signaling pathway in animal models of Parkinson's disease and discuss the relationship between this pathway and the prominent pathological processes observed in the disease state. We suggest that regulation of the JNK signaling pathway may be a central facet in potential treatments for the disease. http://www.ncbi.nlm.nih.gov/pubmed/12880208 Mon, 30 Jun 2003 00:00:00 -0700 Levels of iron are increased in the brains of Parkinson's disease (PD) patients compared to agematched controls. This has been postulated to contribute to progression of the disease via several mechanisms including exacerbation of oxidative stress, initiation of inflammatory responses and triggering of Lewy body formation. In this minireview, we examine the putative role of iron in PD and its pharmacological chelation as a prospective therapeutic for the disease. http://www.ncbi.nlm.nih.gov/pubmed/12882349 Mon, 30 Jun 2003 00:00:00 -0700 Although the causative genetic mutations for a subset of some of the most prevalent human motor neurodegenerative diseases have been identified, the exact molecular mechanisms behind motor neuron and associated muscular loss in these disorders remain an unsolved mystery. In a recent issue of Science, two mutagenesisderived mouse mutants are described that contain missense mutations in the gene encoding the cytoplasmic dynein heavychain protein, which is part of a major cellular motor complex involved in retrograde axonal transport. These mutations result in progressive motor neuron degeneration in heterozygous animals and Lewylike inclusion bodies in the homozygotes resembling those that occur in related human pathologies such as amyotrophic lateral sclerosis, spinal muscular atrophy, and spinalbulbar muscular atrophy. This discovery opens up the exciting possibility that similar mutations may be involved in these human disease states. http://www.ncbi.nlm.nih.gov/pubmed/12844533 Wed, 30 Apr 2003 00:00:00 -0700 Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson'sinducing agent 1methyl4phenyl1,2,3,6tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease. http://www.ncbi.nlm.nih.gov/pubmed/12670420 Fri, 28 Feb 2003 00:00:00 -0800 The homozygous mouse mutant weaver exhibits a massive loss of cerebellar granule neurons postnatally. The death of these cells is associated with a single amino acid mutation in the G proteinactivated inwardly rectifying potassium channel, Girk2. Evidence suggests that both the mutated Girk2 channel and the calcium channelassociated Nmethyldaspartate receptor play important roles in the apoptotic death of weaver cerebellar granule cells, but the downstream events associated with this process are unknown. In this study, we demonstrate that the consequences of the mutation result in caspase activation. In addition, our results show that caspase inhibition in vivo decreases caspase activation and granule cell apoptosis and significantly improves behavioral deficits associated with the weaver's phenotype. http://www.ncbi.nlm.nih.gov/pubmed/12221097 Thu, 31 Oct 2002 00:00:00 -0800 Parkinson's disease (PD) is a progressive neurodegenerative disease involving neurodegeneration of dopaminergic neurons of the substantia nigra (SN), a part of the midbrain. Oxidative stress has been implicated to play a major role in the neuronal cell death associated with PD. Importantly, there is a drastic depletion in cytoplasmic levels of the thiol tripeptide glutathione within the SN of PD patients. Glutathione (GSH) exhibits several functions in the brain chiefly acting as an antioxidant and a redox regulator. GSH depletion has been shown to affect mitochondrial function probably via selective inhibition of mitochondrial complex I activity. An important biochemical feature of neurodegeneration during PD is the presence of abnormal protein aggregates present as intracytoplasmic inclusions called Lewy bodies. Oxidative damage via GSH depletion might also accelerate the buildup of defective proteins leading to cell death of SN dopaminergic neurons by impairing the ubiquitinproteasome pathway of protein degradation. Replenishment of normal glutathione levels within the brain may hold an important key to therapeutics for PD. Several reports have suggested that iron accumulation in the SN patients might also contribute to oxidative stress during PD. http://www.ncbi.nlm.nih.gov/pubmed/12213603 Sat, 31 Aug 2002 00:00:00 -0700 OBJECTIVE: Transgenic mice that overexpress a human gene encoding mutant cytosolic superoxide dismutase (SOD1) develop a progressive motor neuron loss that resembles human ALS. Why mutant SOD1 initiates motor neuron death is unknown. One hypothesis proposes that the mutant molecule has enhanced peroxidase activity, reducing hydrogen peroxide (H2O2) to form toxic hydroxyl adducts on critical targets. To test this hypothesis, the authors generated transgenic ALS mice with altered levels of glutathione peroxidase (GSHPx), the major soluble enzyme that detoxifies H2O2. METHODS: SOD1(G93A) ALS mice were bred with mice bearing a murine GSHPx transgene that have a fourfold elevation in brain GSHPx levels and with mice having targeted inactivation of the GSHPx gene and reduced brain GSHPx activity. RESULTS: Survival was not prolonged in ALS mice with elevated brain GSHPx activity (p = 0.09). ALS mice with decreased GSHPx brain activity (20 of normal) showed no acceleration of the disease course (p = 0.89). The age at disease onset in the ALS mice was unaffected by brain GSHPx activity. CONCLUSION: The level of GSHPx activity in the CNS of transgenic ALS mice does not play a critical role in the development of motor neuron disease. http://www.ncbi.nlm.nih.gov/pubmed/12221165 Sat, 31 Aug 2002 00:00:00 -0700 Aging is a threestage process: metabolism, damage, and pathology. The biochemical processes that sustain life generate toxins as an intrinsic side effect. These toxins cause damage, of which a small proportion cannot be removed by any endogenous repair process and thus accumulates. This accumulating damage ultimately drives agerelated degeneration. Interventions can be designed at all three stages. However, intervention in metabolism can only modestly postpone pathology, because production of toxins is so intrinsic a property of metabolic processes that greatly reducing that production would entail fundamental redesign of those processes. Similarly, intervention in pathology is a "losing battle" if the damage that drives it is accumulating unabated. By contrast, intervention to remove the accumulating damage would sever the link between metabolism and pathology, and so has the potential to postpone aging indefinitely. We survey the major categories of such damage and the ways in which, with current or foreseeable biotechnology, they could be reversed. Such ways exist in all cases, implying that indefinite postponement of agingwhich we term "engineered negligible senescence"may be within sight. Given the major demographic consequences if it came about, this possibility merits urgent debate. http://www.ncbi.nlm.nih.gov/pubmed/11976218 Sun, 31 Mar 2002 00:00:00 -0800 Parkinson's disease (PD) is characterized by the presence of proteinaceous neuronal inclusions called Lewy bodies in susceptible dopaminergic midbrain neurons. Inhibition of the ubiquitinproteasome protein degradation pathway may contribute to protein buildup and subsequent cell death. Ubiquitin is normally activated for transfer to substrate proteins by interaction with the E1 ubiquitin ligase enzyme via a thiol ester bond. Parkinson's disease is also characterized by decreases in midbrain levels of total glutathione which could impact on E1 enzyme activity via oxidation of the active site sulfhydryl. We have demonstrated that increasing reductions in total glutathione in dopaminergic PC12 cells results in corresponding decreases in ubiquitinprotein conjugate levels suggesting that ubiquitination of proteins is inhibited in a glutathionedependent fashion. Decreased ubiquitinated protein levels appears to be due to inhibition of E1 activity as demonstrated by reductions in endogenous E1ubiquitin conjugate levels as well as decreases in the production of de novo E1ubiquitin conjugates when glutathione is depleted. This is a reversible process as E1 activity increases upon glutathione restoration. Our data suggests that decreases in cellular glutathione in dopaminergic cells results in decreased E1 activity and subsequent disruption of the ubiquitin pathway. This may have implications for neuronal degeneration in PD. http://www.ncbi.nlm.nih.gov/pubmed/11841562 Thu, 31 Jan 2002 00:00:00 -0800 Parkinson's disease (PD) and 1methyl4phenyl1,2,3,6tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra (SN). Apoptosis has been implicated in this cell loss however, whether or not it is a major component of disease pathology remains controversial. Caspases are a major class of proteases involved in the apoptotic process. To evaluate the role of caspases in PD, we analyzed caspase activation in MPTPtreated mice, in cultured dopaminergic cells, and in postmortem PD brain tissue. MPTP was found to elicit not only the activation of the effector caspase3 but also the initiators caspase8 and caspase9, mitochondrial cytochrome c release, and Bid cleavage in the SN of wildtype mice. These changes were attenuated in transgenic mice neuronally expressing the general caspase inhibitor protein baculoviral p35. These mice also displayed increased resistance to the cytotoxic effects of the drug. MPTPassociated toxicity in culture was found temporally to involve cytochrome c release, activation of caspase9, caspase3, and caspase8, and Bid cleavage. Caspase9 inhibition prevented the activation of both caspase3 and caspase8 and also inhibited Bid cleavage, but not cytochrome c release. Activated caspase8 and caspase9 were immunologically detectable within MPP()treated mesencephalic dopaminergic neurons, dopaminergic nigral neurons from MPTPtreated mice, and autopsied Parkinsonian tissue from lateonset sporadic cases of the disease. These data demonstrate that MPTPmediated activation of caspase9 via cytochrome c release results in the activation of caspase8 and Bid cleavage, which we speculate may be involved in the amplification of caspasemediated dopaminergic cell death. These data suggest that caspase inhibitors constitute a plausible therapeutic for PD. http://www.ncbi.nlm.nih.gov/pubmed/11739563 Fri, 30 Nov 2001 00:00:00 -0800 Protein aggregation appears to be the common denominator in a series of distinct neurodegenerative diseases yet its role in the associated neuronal pathology in these various conditions remains elusive. In Parkinson's disease, localization of alpha synuclein aggregates within intracellular Lewy body occlusions represent a major hallmark of this disorder and suggest that such aggregation may play a causative role in the resulting dopaminergic cell loss. In this Viewpoint article, recent data is reviewed related to how alpha synuclein aggregation may occur, what cellular events might be responsible, and how this may interfere with normal cellular function(s). It appears likely that while aggregation of alpha synuclein may interfere with its normal function in the cell, this is not the primary cause of the related neurodegeneration. http://www.ncbi.nlm.nih.gov/pubmed/11511392 Fri, 31 Aug 2001 00:00:00 -0700 Genetically engineered animal models have been and will continue to be invaluable for exploring the basic mechanisms involved in the aging process as well as in extending our understanding of diseases found to be more prevalent in the older human population. Continued development of such in vivo systems will allow scientists to further dissect the role genetic and environmental factors play in aging and in agerelated disease states and to enhance our understanding of these processes. In this article we discuss techniques involved in the development of such models and review some examples of laboratory mouse strains that have been used to study either normal aging or select diseases associated with aging. http://www.ncbi.nlm.nih.gov/pubmed/11697220 Fri, 31 Aug 2001 00:00:00 -0700 Recently it has been hypothesized that apoptotic cell death is involved in several neuropathological conditions including Parkinson's disease (PD). Initial morphological studies assessing the presence of apoptosis in Parkinsonian brain tissues yielded mixed results. Based on more recent studies in human PD brains as well in animal and cell culture models of the disease, a picture is emerging, however, that strongly suggests that many of the molecular players thought to participate in this type of neuronal cell death are active in the disease. The task of researchers in the field is now to deduce how these players may be interacting with one another to bring about cell death in PD and to design effective therapies to interfere with these processes. http://www.ncbi.nlm.nih.gov/pubmed/11462217 Sat, 30 Jun 2001 00:00:00 -0700 Glutathione (GSH) is considered one of the primary antioxidant compounds in the brain, important for the removal of peroxides from this organ. GSH levels have been reported to be significantly lower in the substantia nigra (SN) of Parkinson patients vs. agematched controls. Curiously, GSH has been proposed to be present in brain astrocytes rather than in neurons even though these cells are not lost in Parkinson disease. We report that the catalytic and regulatory subunit proteins of glutamyl cysteine synthetase (GCS), the primary enzyme involved in GSH synthesis, are present not only in astrocytes but also in dopaminergic neurons of the SN. This may have important implications in terms of GSH loss associated with Parkinson disease. http://www.ncbi.nlm.nih.gov/pubmed/11288148 Sat, 31 Mar 2001 00:00:00 -0800 Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)mediated cell death is unknown. We examined H(2)O(2)induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time and concentrationdependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic upregulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)mediated cell death. http://www.ncbi.nlm.nih.gov/pubmed/11259492 Wed, 28 Feb 2001 00:00:00 -0800 A growing body of evidence has implicated oxidative stress as an important factor in the neuropathology associated with Parkinson's disease. Dopaminergic nigrostriatal neurons, the predominant cells lost in Parkinson's, are believed to be highly prone to oxidative damage due to the propensity for dopamine to autooxidize and thereby produce elevated levels of hydrogen peroxide and catecholamine quinones. Hydrogen peroxide formed during this process can either be converted by iron to form highly reactive hydroxyl radicals or removed through reduction by glutathione. Glutathione can also conjugate with quinones formed during dopamine oxidation preventing them from facilitating the release of iron from the ironstorage molecule ferritin. Alterations in both iron and glutathione levels in the substantia nigra have been correlated with the neuronal degeneration accompanying Parkinson's disease but a direct causative role for either has yet to be definitively proved. We will discuss the use of genetically engineered cell and mouse lines generated in our laboratory as models to examine the role that alterations in iron and glutathione levels may play in neurodegeneration of dopaminergic neurons of the substantia nigra associated with Parkinson's disease, and how these two parameters may interact with one another to bring this about. http://www.ncbi.nlm.nih.gov/pubmed/11280021 Sun, 31 Dec 2000 00:00:00 -0800 Parkinson's disease (PD) is characterized by the presence of neuronal inclusions in the midbrain known as Lewy bodies. These proteinaceous intracellular deposits may contribute to subsequent dopaminergic neurodegeneration in this brain region. While it is assumed by many investigators that immunopositive ubiquitin staining in Parkinson'sassociated Lewy bodies represents the presence of elevated levels of intracellular ubiquitinprotein conjugates, this has yet to be definitively proven. Increases in oxidative stress along with decreased levels of the thiol regulatory molecule glutathione in the midbrain in PD may cause Sthiolation of important components of the ubiquitination system drastically reducing their activities and in fact impairing the cell's ability to ubiquitinate proteins at all. Here, we review evidence for free versus proteinbound ubiquitin in Parkinsonianassociated Lewy bodies and discuss future directions towards resolving this important question as well as its implication for future treatment therapies. http://www.ncbi.nlm.nih.gov/pubmed/10989121 Thu, 31 Aug 2000 00:00:00 -0700 Genetic variants with greatly extended lifespan are proving invaluable in uncovering signal transduction pathways that influence the rates of normal ageing. These studies have so far been confined to invertebrate models such as Caenorhabditis elegans and Drosophila, but there has been much speculation as to whether a similar approach could be applied to mammals. The recent publication of results on a mouse strain, mutant in a gene encoding the signaling molecule p66(shc), gives cause for optimism. The mutation renders the mouse resistant to the action of oxygen radical generators and appears to increase mean lifespan by 30. This approach may provide a boost for the modeling of human agerelated diseases. http://www.ncbi.nlm.nih.gov/pubmed/10797480 Sun, 30 Apr 2000 00:00:00 -0700