Glutamate, excitotoxicity, and programmed cell death in parkinson disease

Experimental Neurology 220 (2009) 230–233

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Commentary

Glutamate, excitotoxicity, and programmed cell death in parkinson disease W. Michael Caudle, Jing Zhang ⁎ Department of Pathology, University of Washington School of Medicine, HMC Box 359635, 325 9th Ave., Seattle, WA 98104, USA

a r t i c l e

i n f o

Article history: Received 11 August 2009 Revised 17 September 2009 Accepted 29 September 2009 Available online 6 October 2009

Parkinson disease (PD) is a progressive and chronic neurodegenerative disorder that currently affects millions of people in the United States and many more worldwide (Dauer and Przedborski, 2003). The cardinal clinical symptoms associated with PD include slowness of movement (bradykinesia), tremor, postural instability, and rigidity of the limbs and trunk. In addition, patients also present with a spectrum of nonmotor signs, such as hypo- or anosmia, constipation, depression, and cognitive impairment. While motor phenotypes are primarily due to damage and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), nonmotor signs involve neuronal loss in many other brain regions, e.g., the locus ceoruleus (norepinephrine), raphe (serotonin), and isocortex (Braak et al., 2003). These reductions are accompanied by accumulations of α-synuclein inclusions called Lewy bodies or Lewy neurites (Dauer and Przedborski, 2003). Both genetic and idiopathic etiologies of PD have been demonstrated. Currently, there are five PD-causing genes that have been identified, α-synuclein, DJ-1, LRRK2, Parkin, and PINK1 (Farrer, 2006). Although rare, these inherited cases have broadened our understanding of PD and the pathogenic cascade. In contrast to genetic causes, the vast majority of PD cases (N90%), at least in the Caucasian population, are sporadic in nature and present the research and clinical community with a much more complex set of problems, in terms of disease etiopathogenesis. Although the cause of PD is generally viewed as a multifactorial cascade of deleterious events, the most prominent evidence points to oxidative stress, decrements in mitochondrial function, protein mishandling and aggregation, and excitotoxicity as potential causative factors, and programmed cell death as a primary mode of cell death in PD (Levy et al., 2009). A recent paper by Meredith et al. (2009) (Impaired glutamate homeostasis and programmed cell death in a chronic MPTP mouse model of Parkinson's disease) published in a recent issue of Expe-

⁎ Corresponding author. Fax: +1 206 897 5249. E-mail address: [email protected] (J. Zhang). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.09.027

rimental Neurology (2009 Sep; 219 (1): 334–340) has investigated the contributions of glutamatergic excitotoxicity in the pathogenesis of PD as well as apoptosis and autophagy (a relatively new form of programmed cell death) as mechanisms of neurodegeneration. Thus, our commentary will serve to address the importance of the author's findings and to place them in the context of our current understanding of excitotoxicity and dopaminergic neurodegeneration in PD. Glutamate is the major excitatory neurotransmitter in the central nervous system where it acts upon ionotropic (N-methyl-D-aspartate (NMDA) and α-amino-3-hyroxy-5-methylisoxazole proprionic acid (AMPA)) or metabotropic (mGlu1-mGlu8) receptors (Dingledine et al., 1999; Greenamyre and Porter, 1994). Although glutamate plays a central role in excitatory neurotransmission, alterations in glutamate homeostasis can have significant repercussions on neurons through the generation of neurotoxic or excitotoxic cascades (Olney, 1990). These cascades are primarily initiated following activation of the NMDA, AMPA receptors, and voltage gated calcium channels resulting in a massive influx of extracellular calcium. In addition, intracellular stores of calcium are liberated from the endoplasmic reticulum. It is believed that excitotoxicity can damage neurons directly through the overstimulation of NMDA receptors as a result of increased release of extracellular glutamate or a reduction in its removal from the synaptic cleft, thus propagating the influx of calcium. However, studies have suggested that the role of excitotoxicity in neurodegenerative diseases such as PD follows an indirect pathway (Albin and Greenamyre, 1992; Greene and Greenamyre, 1996). A change in membrane polarity, such as through alterations in mitochondrial respiration results in the voltage dependent removal of extracellular magnesium, which blocks the calcium influx under normal conditions, and can enhance the neurons' vulnerability to glutamate. This is especially interesting given the evidence of impaired mitochondrial function associated with PD. Glutamate is also the predominant excitatory transmitter in the basal ganglia, which is the seat of the motor deficits seen in PD (Greenamyre and Porter, 1994). In addition to sending glutamatergic projects to the striatum, the cortex also sends projections to the subthalamic nucleus (STN), thalamus, and SNpc, in addition to other

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nuclei in the brainstem and spinal cord (Albin et al., 1989). The SNpc receives further glutamatergic innervation from the STN in the indirect basal ganglia pathway. Evidence has demonstrated that the dopaminergic projection from the SNpc to various nuclei in the basal ganglia circuit exerts an important regulatory function on the firing pattern of certain glutamatergic pathways. Indeed, dopamine depletion, as seen in PD causes a complex set of changes to the functioning of the basal ganglia (Wichmann and DeLong, 1993). Most notably, the reduction in the dopaminergic message being sent to the striatum results in an overactivation of the STN, causing an increase in the release of glutamate onto the dopaminergic cell bodies located in the SNpc, which are rich in NMDA and AMPA receptors, as well as mGluR1 and mGluR2/3 (Rodriguez et al., 1998). It has been hypothesized that the increase in firing of STN neurons in PD functions as a compensatory mechanism aimed at elevating the release of dopamine from the surviving dopaminergic neurons in the SNpc in order to maintain dopamine homeostasis (Bezard et al., 1999). However, a sustained increase in glutamate released onto an already compromised dopaminergic cell population could elicit an excitotoxic cascade and potentiate neurodegeneration. The fact that glutamatergic pathways have such a significant role in the pathophysiology and pathogenesis of PD suggests that these glutamatergic circuits may hold potential for therapeutic intervention. Indeed, evidence from mouse and nonhuman primate models of PD demonstrated that certain NMDA and AMPA receptor antagonists alleviate many of the parkinsonian symptoms (Greenamyre et al., 1994; Klockgether et al. 1991; Starr, 1995). Treatment with the mGlu2/3 receptor agonists has also been shown to attenuate MPTPinduced dopaminergic neurodegenration in mice (Battaglia et al., 2003; Corti et al., 2007). Motor symptoms and cell loss were also alleviated in parkinsonian animals following treatment with mGlu1 and mGlu5 receptor antagonists (Battaglia et al., 2004; Breysse et al., 2002; Ossowska et al., 2001; Spooren et al., 2001). In addition to pharmaceutical, surgical interventions involving ablation of the STN as well as inactivation of the STN by deep brain stimulation has been shown to ameliorate the motor dysfunction associated with PD (Bergman et al., 1990; Limousin et al., 1998). Moreover, a reduction in firing of the STN and the subsequent blunted release of glutamate onto neurons of the SNpc could be neuroprotective by alleviating the potential for excitotoxicity. A recent study demonstrated a 20–24% increase in TH-immunoreactive (TH-ir) neurons in the SNpc of monkeys given MPTP followed by STN lesioning or DBS, compared with monkeys that did not receive surgical interventions (Wallace et al., 2007). The recent research report by Meredith et al. (2009) sought to determine whether alterations in glutamate signaling in the SNpc in a chronic mouse model of PD contribute to the precipitous loss of dopamine neurons in this region. The model utilized in this report has been well-characterized as a chronic mouse model of PD, recapitulating many of the neurochemical and behavioral features associated with PD in human patients, including reductions in striatal dopamine, dopaminergic neurons in the SNpc, locomotor behaviors, as well as the presence of α-synuclein-positive inclusions in the SNpc (Meredith et al., 2002, 2008; Petroske et al., 2001). The model is based upon the idea of inhibiting the metabolism and clearance of MPP+ from the brain through the use of the adjuvant, probenecid. Use of probenecid has been shown to potentiate MPTP neurotoxcity resulting in pathological signs that last upwards of 6 months (Lau et al., 1990). As seen in previous studies, chronic exposure of mice to MPTP and probenecid caused an approximately 68% reduction in TH-ir neurons in the SNpc 3 weeks following cessation of MPTP compared with animals that received saline or probenecid alone. The loss of dopaminergic cells in the MPTP animals was accompanied by TUNEL-positive cells, denoting apoptosis, as well as the presence of organelle- and proteinaceous-filled secondary lysosomes and autophagosomes, suggesting the stimulation of autophagy. In order to

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investigate whether chronic treatment with MPTP induced disruption of glutamate homeostasis, Meredith et al. (2009) evaluate the extracellular levels of glutamate in the SNpc. Interestingly, a significant increase in glutamate levels was seen in the MPTP/ probenecid treated mice compared with control animals. Replacement of dopamine by L-DOPA caused a significant reduction in extracellular glutamate, presumably via a negative feedback inhibition of glutamatergic inputs to the SNpc. To determine if the expression and function of the midbrain glutamate transporters could contribute to the elevated synaptic glutamate levels, Meredith et al. (2009) perform uptake assays using D-aspartate. Surprisingly, they found the rate of transport, Vmax, of D-aspartate unchanged between MPTP/probenecid and control groups. However, an increase in transporter affinity, Km, for D-aspartate in the MPTP/probenecid treatment group compared with control was noted. An increase in substrate affinity without a change in uptake velocity would suggest that in the SNpc MPTP/probenecid treatment enhances the interaction of extracellular glutamate with the transporter, yet does not potentiate the ability of the transporters to sequester glutamate from the extracellular space. As suggested by the authors, these events could attenuate the clearance of extracellular glutamate from the synapse. Taken in concert, these results are exciting as this is the first study to demonstrate an alteration in glutamate homeostasis in the SNpc of a mouse model of PD. Another significant contribution of Meredith's investigation relates to the discovery that both apoptosis and autophagy are linked to increased glutamate excitotoxicity in a mouse model of PD. Apoptosis, one of the well-known consequence of excitoxicity (Ankarcrona et al., 1995; Gobbel and Chan, 2001; Liu et al., 2001), is a classical cascade leading to cell death that is characterized by increased mitochondrial membrane potential, leading to the release of cytochrome c, resulting in the activation of caspase 9 and 3. This cascade facilitates the morphological changes that define apoptosis, such as cell shrinkage, membrane blebbing, and chromatin condensation and fragmentation. Apoptosis has been viewed as the predominant cell death mechanism associated with neurodegeneration in PD until recently (Hirsch et al., 1999). Animal models of PD unequivocally demonstrate the presence of apoptotic mechanisms in the SNpc of mice exposed to MPTP, as well as rats treated with 6OHDA (Silva et al., 2005; Yu et al., 2003, 2002). However, the results in human postmortem SNpc have been less convincing. While some studies show an increase in apoptosis in 1–10% of dopaminergic cells in the SNpc of PD tissue, others report no change from age matched controls (Kingsbury et al., 1998; Kosel et al., 1997; Tatton et al., 1998). While some suggest that these discrepancies can be attributed to experimental shortcomings (Tatton et al., 1998), they may also be due to the slow progression of neuronal loss over the course of the disease, with a small number of neurons undergoing active apoptosis at any one time. Thus, a snapshot of apoptosis may not provide an accurate representation of cell death at a single time point. In addition to apoptosis, it has been demonstrated that other cell death mechanisms may contribute to the neurodegeneration seen in the SNpc. Anglade et al. (1997) reported the presence of markers of autophagy in the SNpc of postmortem PD tissue. Autophagy functions to degrade damaged or aged cytosolic proteins and intracellular organelles, such as mitochondria in order to maintain the optimal functioning of the cell under basal conditions, as well as during cellular stress as a means to suppress cell death (Cuervo, 2004). This is especially important in postmitotic cells such as neurons. Moreover, autophagy is an integral mechanism of protein degradation when toxic or aggregate-prone proteins are unable to be removed by the ubiquitin proteosomal system (Ravikumar et al., 2002; Rubinsztein, 2007). Three types of autophagy have been identified. (1) Macroautophagy typically degrades organelles, such as mitochondria and long lasting cytosolic proteins. Proteins to be degraded are enclosed in an

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autophagosome and trafficked to the lysosome where the contents are degraded by hydrolytic enzymes. The fused autophagosome and lysosome are collectively referred to as autophagic vacuoles, which are considered to represent the characteristic morphological component of autophagy (Mortimore et al., 1996). (2) Microautophagy functions in the gradual and continuous turnover of cytosolic proteins. (3) Chaperone-mediated autophagy (CMA) generally degrades cytosolic proteins that contain a specific set of targeting residues that are recognized by the molecular chaperone, heat shock cognate protein of 70 kDa (Hsc70). Binding of Hsc70 traffics the protein to the lysosome where it binds with the lysosomal associated membrane protein 2a (LAMP2a) receptor and is sequestered into the lysosome for breakdown by hydrolases (Cuervo and Dice, 1998). Interestingly, previous research has demonstrated the presence of excitotoxicity-mediated autophagy in various disease models, such as ischemia, traumatic brain injury, and cerebellar degeneration (Bigford et al., 2009; Samara et al., 2008; Yue et al., 2002), thus providing support for a role of glutamatedependent autophagy in PD. Mechanisms underlying autophagy in PD are currently not well understood. One of the proposed links is the degradation and clearance of α-synuclein, whose mutations and increases in intracellular concentrations have been implicated in PD pathogenesis. Chaperone-mediated autophagy is exquisitely involved in the degradation of wildtype α-synuclein (Bandyopadhyay and Cuervo, 2007; Cuervo et al., 2004; Vogiatzi et al., 2008). However, it is unable to degrade any of the mutant forms of α-synuclein. These mutant forms can interact with Hsc70 and be trafficked to the lysosome. Instead of binding with LAMP2a and being internalized for degradation, they block LAMP2a and inhibit the sequestration and subsequent breakdown of other damaged proteins (Cuervo et al., 2004). An earlier report by Meredith et al. (2002) demonstrated lysosomal dysfunction and the presence of aggregated α-synuclein in their chronic MPTP mouse model of PD. In addition to chaperone-mediated autophagy, previous studies have demonstrated a role for macroautophagy in the degradation of wildtype α-synuclein in cell culture models (Vogiatzi et al., 2008; Webb et al., 2003). Thus, it appears that autophagy is an important regulator of α-synuclein and alteration of lysosomal function can increase the potential for accumulation of damaged and aggregate-prone proteins, contributing to neurodegeneration in PD. The detection of autophagic processes suggests that two types of programmed cell death are occurring in the SNpc following exposure to MPTP/probenecid, similar to that seen in PD patients (Anglade et al., 1997; Michel et al., 1994). These results are especially interesting in light of the contribution of glutamate excitotoxicity and the accumulation and degradation of damaged proteins in PD. It should be stressed, however, that autophagy could suggest a neuroprotective mechanism in which the cell is actively trying to degrade proteins that have been damaged. The same process becomes pro-neurodegenerative as the autophagic machinery is overwhelmed or disrupted and begins to degrade functional intracellular components that are vital for cell survival or are unable to clear aggregated, neurotoxic species. It may be that the contribution of autophagy is contingent upon the stage of the disease with early stages associated with a neuroprotective function and neurodegeneration associated with later stages of the disease. Nonetheless, the findings of Meredith et al., (2009) provide a model with which to further tease out the particular contribution of autophagy to cell survival in PD. In summary, the importance of glutamate neurotransmission in the basal ganglia and the functional consequences of its disruption have been well established as major contributors to the motor alterations associated with PD. Furthermore, perturbations in glutamate handling which result in increased concentrations of extracellular glutamate appear to be the predominant initiator of excitotoxic cascades and subsequent neurodegeneration in the SNpc. As the SNpc

receives multiple glutamatergic inputs from various regions within the basal ganglia circuit, the accumulation of synaptic glutamate and cell death could be a direct result of alteration to the normal inhibition and disinhibition of these nuclei. Currently, the dissection of the role of glutamate excitoxicity in either apoptosis, autophagy, or both in neurodegeneration and PD pathogenesis remains to be adequately determined. The recent study by Meredith et al. (2009) makes significant strides in addressing these issues by providing an animal model that recapitulates multiple pathological features of the human parkinsonian phenotype. Most important in the context of the present commentary, the demonstration of increased extracellular glutamate as well as the concomitant presence of both apoptotic and autophagic cell death mechanisms provides an easily accessible, reproducible model that will prove a useful tool for researchers to methodically tease out some of the unanswered questions surrounding excitotoxicity and apoptotic and autophagic cell death in the SNpc. Finally, this model of PD provides an excellent platform to comprehensively evaluate therapeutic interventions aimed at addressing disruption of glutamate homeostasis and either augmenting or attenuating the autophagic pathway in PD. On the other hand, although Meredith et al. (2009) were able to record a significant increase in extracellular glutamate in the SNpc of this model of PD, the authors were only able to speculate as to which pathway may have contributed to the glutamatergic overflow. Furthermore, although a plethora of evidence would suggest that an accumulation of glutamate in the SNpc synapse could generate an excitotoxic response, it is unclear whether this increase in extracellular glutamate is sufficient to elicit excitotoxicity and cell death and could this be attenuated with NMDA or AMPA receptor antagonists. Answers to these questions will likely shed more light on how precisely excitotoxicity contributes to neurodegeneration, dopaminergic or not, in PD. References Albin, R.L., Greenamyre, J.T., 1992. Alternative excitotoxic hypotheses. Neurology 42, 733–738. Albin, R.L., Young, A.B., Penney, J.B., 1989. The functional anatomy of basal ganglia disorders. Trends Neurosci. 12, 366–375. Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M.T., Michel, P.P., Marquez, J., MouattPrigent, A., Ruberg, M., Hirsch, E.C., Agid, Y., 1997. Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease. Histol. Histopathol. 12, 25–31. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., Nicotera, P., 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961–973. Bandyopadhyay, U., Cuervo, A.M., 2007. Chaperone-mediated autophagy in aging and neurodegeneration: lessons from alpha-synuclein. Exp. Gerontol. 42, 120–128. Battaglia, G., Busceti, C.L., Pontarelli, F., Biagioni, F., Fornai, F., Paparelli, A., Bruno, V., Ruggieri, S., Nicoletti, F., 2003. Protective role of group-II metabotropic glutamate receptors against nigro-striatal degeneration induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine in mice. Neuropharmacology 45, 155–166. Battaglia, G., Busceti, C.L., Molinaro, G., Biagioni, F., Storto, M., Fornai, F., Nicoletti, F., Bruno, V., 2004. Endogenous activation of mGlu5 metabotropic glutamate receptors contributes to the development of nigro-striatal damage induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. J. Neurosci. 24, 828–835. Bergman, H., Wichmann, T., DeLong, M.R., 1990. Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science 249, 1436–1438. Bezard, E., Boraud, T., Bioulac, B., Gross, C.E., 1999. Involvement of the subthalamic nucleus in glutamatergic compensatory mechanisms. Eur. J. Neurosci. 11, 2167–2170. Bigford, G.E., Alonso, O.F., Dietrich, D., Keane, R.W., 2009. A novel protein complex in membrane rafts linking the NR2B glutamate receptor and autophagy is disrupted following traumatic brain injury. J. Neurotrauma. 26, 703–720. Braak, H., Del Tredici, K., Rub, U., de Vos, R.A., Jansen Steur, E.N., Braak, E., 2003. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging 24, 197–211. Breysse, N., Baunez, C., Spooren, W., Gasparini, F., Amalric, M., 2002. Chronic but not acute treatment with a metabotropic glutamate 5 receptor antagonist reverses the akinetic deficits in a rat model of parkinsonism. J. Neurosci. 22, 5669–5678. Corti, C., Battaglia, G., Molinaro, G., Riozzi, B., Pittaluga, A., Corsi, M., Mugnaini, M., Nicoletti, F., Bruno, V., 2007. The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection. J. Neurosci. 27, 8297–8308. Cuervo, A.M., 2004. Autophagy: many paths to the same end. Mol. Cell. Biochem. 263, 55–72. Cuervo, A.M., Dice, J.F., 1998. Lysosomes, a meeting point of proteins, chaperones, and proteases. J. Mol. Med. 76, 6–12.

W.M. Caudle, J. Zhang / Experimental Neurology 220 (2009) 230–233 Cuervo, A.M., Stefanis, L., Fredenburg, R., Lansbury, P.T., Sulzer, D., 2004. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 305, 1292–1295. Dauer, W., Przedborski, S., 2003. Parkinson's disease: mechanisms and models. Neuron 39, 889–909. Dingledine, R., Borges, K., Bowie, D., Traynelis, S.F., 1999. The glutamate receptor ion channels. Pharmacol. Rev. 51, 7–61. Farrer, M.J., 2006. Genetics of Parkinson disease: paradigm shifts and future prospects. Nat. Rev. Genet. 7, 306–318. Gobbel, G.T., Chan, P.H., 2001. Neuronal death is an active, caspase-dependent process after moderate but not severe DNA damage. J. Neurochem. 76, 520–531. Greenamyre, J.T., Porter, R.H., 1994. Anatomy and physiology of glutamate in the CNS. Neurology 44, S7–13. Greenamyre, J.T., Eller, R.V., Zhang, Z., Ovadia, A., Kurlan, R., Gash, D.M., 1994. Antiparkinsonian effects of remacemide hydrochloride, a glutamate antagonist, in rodent and primate models of Parkinson's disease. Ann. Neurol. 35, 655–661. Greene, J.G., Greenamyre, J.T., 1996. Bioenergetics and glutamate excitotoxicity. Prog. Neurobiol. 48, 613–634. Hirsch, E.C., Hunot, S., Faucheux, B., Agid, Y., Mizuno, Y., Mochizuki, H., Tatton, W.G., Tatton, N., Olanow, W.C., 1999. Dopaminergic neurons degenerate by apoptosis in Parkinson's disease. Mov. Disord. 14, 383–385. Kingsbury, A.E., Mardsen, C.D., Foster, O.J., 1998. DNA fragmentation in human substantia nigra: apoptosis or perimortem effect? Mov. Disord. 13, 877–884. Klockgether, T., Turski, L., Honore, T., Zhang, Z.M., Gash, D.M., Kurlan, R., Greenamyre, J.T., 1991. The AMPA receptor antagonist NBQX has antiparkinsonian effects in monoamine-depleted rats and MPTP-treated monkeys. Ann. Neurol. 30, 717–723. Kosel, S., Egensperger, R., von Eitzen, U., Mehraein, P., Graeber, M.B., 1997. On the question of apoptosis in the parkinsonian substantia nigra. Acta Neuropathol. 93, 105–108. Lau, Y.S., Trobough, K.L., Crampton, J.M., Wilson, J.A., 1990. Effects of probenecid on striatal dopamine depletion in acute and long-term 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-treated mice. Gen. Pharmacol. 21, 181–187. Levy, O.A., Malagelada, C., Greene, L.A., 2009. Cell death pathways in Parkinson's disease: proximal triggers, distal effectors, and final steps. Apoptosis 14, 478–500. Limousin, P., Krack, P., Pollak, P., Benazzouz, A., Ardouin, C., Hoffmann, D., Benabid, A.L., 1998. Electrical stimulation of the subthalamic nucleus in advanced Parkinson's disease. N. Engl. J. Med. 339, 1105–1111. Liu, W., Liu, R., Chun, J.T., Bi, R., Hoe, W., Schreiber, S.S., Baudry, M., 2001. Kainate excitotoxicity in organotypic hippocampal slice cultures: evidence for multiple apoptotic pathways. Brain Res. 916, 239–248. Meredith, G.E., Totterdell, S., Petroske, E., Santa Cruz, K., Callison Jr., R.C., Lau, Y.S., 2002. Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson's disease. Brain Res. 956, 156–165. Meredith, G.E., Totterdell, S., Potashkin, J.A., Surmeier, D.J., 2008. Modeling PD pathogenesis in mice: advantages of a chronic MPTP protocol. Parkinsonism Relat. Disord. 14 (Suppl 2), S112–115. Meredith, G.E., Totterdell, S., Beales, M., Meshul, C.K., 2009. Impaired glutamate homeostasis and programmed cell death in a chronic MPTP mouse model of Parkinson's disease. Exp Neurol. 219, 334–340. Michel, P.P., Vyas, S., Anglade, P., Ruberg, M., Agid, Y., 1994. Morphological and molecular characterization of the response of differentiated PC12 cells to calcium stress. Eur. J. Neurosci. 6, 577–586.

233

Mortimore, G.E., Miotto, G., Venerando, R., Kadowaki, M., 1996. Autophagy. Subcell Biochem. 27, 93–135. Olney, J.W., 1990. Excitotoxicity: an overview. Can Dis Wkly Rep 16 Suppl 1E, 47–57; discussion 57–48. Ossowska, K., Konieczny, J., Wolfarth, S., Wieronska, J., Pilc, A., 2001. Blockade of the metabotropic glutamate receptor subtype 5 (mGluR5) produces antiparkinsonianlike effects in rats. Neuropharmacology 41, 413–420. Petroske, E., Meredith, G.E., Callen, S., Totterdell, S., Lau, Y.S., 2001. Mouse model of Parkinsonism: a comparison between subacute MPTP and chronic MPTP/ probenecid treatment. Neuroscience 106, 589–601. Ravikumar, B., Duden, R., Rubinsztein, D.C., 2002. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet. 11, 1107–1117. Rodriguez, M.C., Obeso, J.A., Olanow, C.W., 1998. Subthalamic nucleus-mediated excitotoxicity in Parkinson's disease: a target for neuroprotection. Ann. Neurol. 44, S175–188. Rubinsztein, D.C., 2007. Autophagy induction rescues toxicity mediated by proteasome inhibition. Neuron 54, 854–856. Samara, C., Syntichaki, P., Tavernarakis, N., 2008. Autophagy is required for necrotic cell death in Caenorhabditis elegans. Cell Death Differ. 15, 105–112. Silva, R.M., Ries, V., Oo, T.F., Yarygina, O., Jackson-Lewis, V., Ryu, E.J., Lu, P.D., Marciniak, S.J., Ron, D., Przedborski, S., Kholodilov, N., Greene, L.A., Burke, R.E., 2005. CHOP/ GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism. J. Neurochem. 95, 974–986. Spooren, W.P., Gasparini, F., Salt, T.E., Kuhn, R., 2001. Novel allosteric antagonists shed light on mglu(5) receptors and CNS disorders. Trends Pharmacol. Sci. 22, 331–337. Starr, M.S., 1995. Antiparkinsonian actions of glutamate antagonists—alone and with L-DOPA: a review of evidence and suggestions for possible mechanisms. J. Neural. Transm. Park Dis. Dement. Sect. 10, 141–185. Tatton, N.A., Maclean-Fraser, A., Tatton, W.G., Perl, D.P., Olanow, C.W., 1998. A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson's disease. Ann. Neurol. 44, S142–148. Vogiatzi, T., Xilouri, M., Vekrellis, K., Stefanis, L., 2008. Wild type alpha-synuclein is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells. J. Biol. Chem. 283, 23542–23556. Wallace, B.A., Ashkan, K., Heise, C.E., Foote, K.D., Torres, N., Mitrofanis, J., Benabid, A.L., 2007. Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys. Brain 130, 2129–2145. Webb, J.L., Ravikumar, B., Atkins, J., Skepper, J.N., Rubinsztein, D.C., 2003. Alphasynuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 278, 25009–25013. Wichmann, T., DeLong, M.R., 1993. Pathophysiology of parkinsonian motor abnormalities. Adv. Neurol. 60, 53–61. Yue, Z., Horton, A., Bravin, M., DeJager, P.L., Selimi, F., Heintz, N., 2002. A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice. Neuron 35, 921–933. Yu, S.W., Wang, H., Poitras, M.F., Coombs, C., Bowers, W.J., Federoff, H.J., Poirier, G.G., Dawson, T.M., Dawson, V.L., 2002. Mediation of poly(ADP-ribose) polymerase-1dependent cell death by apoptosis-inducing factor. Science 297, 259–263. Yu, S.W., Wang, H., Dawson, T.M., Dawson, V.L., 2003. Poly(ADP-ribose) polymerase-1 and apoptosis inducing factor in neurotoxicity. Neurobiol. Dis. 14, 303–317.