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• W2013503732 abstract "The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DAcyt) in cultured midbrain neurons, we confirm that elevated DAcyt and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DAcyt provide neuroprotection. L-DOPA increased DAcyt in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DAcyt was not altered by α-synuclein deletion, although dopaminergic neurons lacking α-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DAcyt, and α-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets. The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DAcyt) in cultured midbrain neurons, we confirm that elevated DAcyt and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DAcyt provide neuroprotection. L-DOPA increased DAcyt in SN neurons to levels 2- to 3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DAcyt was not altered by α-synuclein deletion, although dopaminergic neurons lacking α-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DAcyt, and α-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets. Parkinson's disease (PD) is characterized by aggregation of alpha-synuclein (α-syn) into Lewy bodies and Lewy neurites, and a progressive loss of specific neuronal populations. In particular, ventral midbrain (VM) dopamine (DA) neurons of the substantia nigra (SN) preferentially degenerate in PD, while neighboring ventral tegmental area (VTA) DA neurons are relatively spared (Dauer and Przedborski, 2003Dauer W. Przedborski S. Parkinson's disease: mechanisms and models.Neuron. 2003; 39: 889-909Abstract Full Text Full Text PDF PubMed Scopus (3649) Google Scholar). A role of α-syn in PD pathogenesis is demonstrated by cases of familial PD that result from mutations or overexpression of α-syn, as well as by the observation that SN neurons in mice with α-syn deletion are protected against the parkinsonian neurotoxins MPTP and 6-OHDA (Alvarez-Fischer et al., 2008Alvarez-Fischer D. Henze C. Strenzke C. Westrich J. Ferger B. Hoglinger G.U. Oertel W.H. Hartmann A. Characterization of the striatal 6-OHDA model of Parkinson's disease in wild type and α-synuclein-deleted mice.Exp. Neurol. 2008; 210: 182-193Crossref PubMed Scopus (113) Google Scholar, Dauer et al., 2002Dauer W. Kholodilov N. Vila M. Trillat A.C. Goodchild R. Larsen K.E. Staal R. Tieu K. Schmitz Y. Yuan C.A. et al.Resistance of α-synuclein null mice to the parkinsonian neurotoxin MPTP.Proc. Natl. Acad. Sci. USA. 2002; 99: 14524-14529Crossref PubMed Scopus (470) Google Scholar). Several hypotheses may explain α-syn-mediated cytotoxicity, including the formation of toxic aggregates that disrupt membrane (Conway et al., 2001Conway K.A. Rochet J.C. Bieganski R.M. Lansbury Jr., P.T. Kinetic stabilization of the α-synuclein protofibril by a dopamine-α-synuclein adduct.Science. 2001; 294: 1346-1349Crossref PubMed Scopus (926) Google Scholar), a blockade of lysosomal protein degradation (Martinez-Vicente et al., 2008Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. et al.Dopamine-modified α-synuclein blocks chaperone-mediated autophagy.J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar), and mitochondrial dysfunction (Ved et al., 2005Ved R. Saha S. Westlund B. Perier C. Burnam L. Sluder A. Hoener M. Rodrigues C.M. Alfonso A. Steer C. et al.Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of α-synuclein, parkin, and DJ-1 in Caenorhabditis elegans.J. Biol. Chem. 2005; 280: 42655-42668Crossref PubMed Scopus (213) Google Scholar). It has also been recently suggested that high Ca2+ levels due to Cav1.3 channel-dependent pacemaking activity (Nedergaard et al., 1993Nedergaard S. Flatman J.A. Engberg I. Nifedipine- and ω-conotoxin-sensitive Ca2+ conductances in guinea-pig substantia nigra pars compacta neurones.J. Physiol. 1993; 466: 727-747PubMed Google Scholar) may contribute to SN susceptibility, as VTA neurons use Na+ channels for pacemaking (Chan et al., 2007Chan C.S. Guzman J.N. Ilijic E. Mercer J.N. Rick C. Tkatch T. Meredith G.E. Surmeier D.J. ‘Rejuvenation’ protects neurons in mouse models of Parkinson's disease.Nature. 2007; 447: 1081-1086Crossref PubMed Scopus (615) Google Scholar). Accordingly, Cav1.3 antagonists block SN cell death in MPTP and other neurotoxin models (Chan et al., 2007Chan C.S. Guzman J.N. Ilijic E. Mercer J.N. Rick C. Tkatch T. Meredith G.E. Surmeier D.J. ‘Rejuvenation’ protects neurons in mouse models of Parkinson's disease.Nature. 2007; 447: 1081-1086Crossref PubMed Scopus (615) Google Scholar). The reason for the preferential death of SN DA neurons is, however, unclear, as both α-syn and Cav1.3 channels are expressed throughout the CNS in neurons that are not lost in PD (Clayton and George, 1998Clayton D.F. George J.M. The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease.Trends Neurosci. 1998; 21: 249-254Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, Rajadhyaksha et al., 2004Rajadhyaksha A. Husson I. Satpute S.S. Kuppenbender K.D. Ren J.Q. Guerriero R.M. Standaert D.G. Kosofsky B.E. L-type Ca2+ channels mediate adaptation of extracellular signal-regulated kinase 1/2 phosphorylation in the ventral tegmental area after chronic amphetamine treatment.J. Neurosci. 2004; 24: 7464-7476Crossref PubMed Scopus (50) Google Scholar, Striessnig et al., 2006Striessnig J. Koschak A. Sinnegger-Brauns M.J. Hetzenauer A. Nguyen N.K. Busquet P. Pelster G. Singewald N. Role of voltage-gated L-type Ca2+ channel isoforms for brain function.Biochem. Soc. Trans. 2006; 34: 903-909Crossref PubMed Scopus (135) Google Scholar). A long-standing hypothesis of neurodegeneration in PD postulates that the buildup of cytosolic DA (DAcyt) with associated oxyradical stress and its possible interaction with α-syn and other PD-related proteins underlie neurotoxicity (Caudle et al., 2008Caudle W.M. Colebrooke R.E. Emson P.C. Miller G.W. Altered vesicular dopamine storage in Parkinson's disease: a premature demise.Trends Neurosci. 2008; 31: 303-308Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Chen et al., 2008Chen L. Ding Y. Cagniard B. Van Laar A.D. Mortimer A. Chi W. Hastings T.G. Kang U.J. Zhuang X. Unregulated cytosolic dopamine causes neurodegeneration associated with oxidative stress in mice.J. Neurosci. 2008; 28: 425-433Crossref PubMed Scopus (161) Google Scholar, Edwards, 1993Edwards R.H. Neural degeneration and the transport of neurotransmitters.Ann. Neurol. 1993; 34: 638-645Crossref PubMed Scopus (51) Google Scholar, Pardo et al., 1995Pardo B. Mena M.A. Casarejos M.J. Paino C.L. De Yebenes J.G. Toxic effects of L-DOPA on mesencephalic cell cultures: protection with antioxidants.Brain Res. 1995; 682: 133-143Crossref PubMed Scopus (128) Google Scholar, Sulzer and Zecca, 2000Sulzer D. Zecca L. Intraneuronal dopamine-quinone synthesis: a review.Neurotox. Res. 2000; 1: 181-195Crossref PubMed Google Scholar). A role for DAcyt in the selectivity of cell death in PD, however, has never been directly studied due to a lack of means to measure DAcyt. Here we use a new electrochemical approach to measure DAcyt in neurons following various pharmacological and genetic interventions. We report that “multiple hits” consisting of high cytoplasmic Ca2+, elevated DAcyt, and α-syn expression are required to evoke selective death of dopaminergic neurons from SN and show that interference with any of these three factors rescues the neurons. We previously introduced intracellular patch electrochemistry (IPE) to study the regulation of cytosolic catecholamine homeostasis in cultured primary murine adrenal chromaffin cells (Mosharov et al., 2003Mosharov E.V. Gong L.W. Khanna B. Sulzer D. Lindau M. Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells.J. Neurosci. 2003; 23: 5835-5845PubMed Google Scholar) and the PC12 cell line (Mosharov et al., 2006Mosharov E.V. Staal R.G. Bove J. Prou D. Hananiya A. Markov D. Poulsen N. Larsen K.E. Moore C.M. Troyer M.D. et al.α-synuclein overexpression increases cytosolic catecholamine concentration.J. Neurosci. 2006; 26: 9304-9311Crossref PubMed Scopus (110) Google Scholar). To extend IPE measurements to DA neurons, we employed VM cultures from mice that express green fluorescent protein under the control of the tyrosine hydroxylase (TH) promoter (TH-GFP; see Figure S1 available online) (Sawamoto et al., 2001Sawamoto K. Nakao N. Kobayashi K. Matsushita N. Takahashi H. Kakishita K. Yamamoto A. Yoshizaki T. Terashima T. Murakami F. et al.Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons.Proc. Natl. Acad. Sci. USA. 2001; 98: 6423-6428Crossref PubMed Scopus (183) Google Scholar). Immunolabeling of fixed 2-week-old cultures of VM neurons for TH showed that approximately 97% of GFP+ cells were TH+ (185 of 191 cells). IPE measurements in cyclic voltammetric (CV) mode that detects DA preferentially over other intracellular metabolites (including L-3,4-dihydroxyphenylalanine [L-DOPA] and dihydroxyphenylacetic acid [DOPAC]) revealed that DAcyt in untreated GFP+ neurons was below the detection limits of the technique (<0.1 μM). This was similar to DAcyt levels in PC12 cells (Mosharov et al., 2006Mosharov E.V. Staal R.G. Bove J. Prou D. Hananiya A. Markov D. Poulsen N. Larsen K.E. Moore C.M. Troyer M.D. et al.α-synuclein overexpression increases cytosolic catecholamine concentration.J. Neurosci. 2006; 26: 9304-9311Crossref PubMed Scopus (110) Google Scholar), but differed from the 10–20 μM cytosolic catecholamine concentrations found in untreated chromaffin cells (Mosharov et al., 2003Mosharov E.V. Gong L.W. Khanna B. Sulzer D. Lindau M. Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells.J. Neurosci. 2003; 23: 5835-5845PubMed Google Scholar). We previously demonstrated that 1 hr pretreatment with 100 μM L-DOPA produces a 2- to 3-fold increase of cytosolic catecholamine concentration in chromaffin cells (Mosharov et al., 2003Mosharov E.V. Gong L.W. Khanna B. Sulzer D. Lindau M. Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells.J. Neurosci. 2003; 23: 5835-5845PubMed Google Scholar). The same dose of L-DOPA increased DAcyt in GFP+ neurons to 17.4 ± 1.7 μM (mean ± SEM; n = 74 cells). To determine the kinetics of DAcyt changes after L-DOPA treatment, we performed IPE at 1, 8, 15, and 24 hr after L-DOPA addition to the media. After 1 hr of 100 μM L-DOPA exposure, DA in the cytosol reached a steady-state level that was maintained for ∼8 hr, followed by a decline to control levels over the succeeding 24 hr of drug treatment (Figure 1A). Interestingly, 500 μM L-DOPA increased DAcyt to the same maximum level, but the elevated steady state was maintained longer, and 24 hr after L-DOPA treatment, DAcyt was still higher than in untreated cells. To study the dependence of DAcyt on extracellular L-DOPA, neuronal cultures were treated with a range of L-DOPA concentrations for 1 hr at 37°C, followed by IPE measurements in the presence of the same L-DOPA concentrations in the bath and in the pipette solutions within the following 30 min at room temperature. The drug response curve was hyperbolic (Figure 1B) with an apparent K0.5 of ∼10 μM L-DOPA. As the steady-state DA concentration in neuronal cytosol is regulated by multiple enzymes and transporters, we attempted to determine which metabolic step limited DAcyt accumulation in L-DOPA-treated cells. High-performance liquid chromatography with electrochemical detection (HPLC-EC) measurements of the total intracellular DA (the majority of which represents vesicular DA; Chien et al., 1990Chien J.B. Wallingford R.A. Ewing A.G. Estimation of free dopamine in the cytoplasm of the giant dopamine cell of Planorbis corneus by voltammetry and capillary electrophoresis.J. Neurochem. 1990; 54: 633-638Crossref PubMed Scopus (140) Google Scholar, Mosharov et al., 2003Mosharov E.V. Gong L.W. Khanna B. Sulzer D. Lindau M. Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells.J. Neurosci. 2003; 23: 5835-5845PubMed Google Scholar) and DOPAC contents showed that these metabolites were elevated to the same degree in neurons exposed to 100 and 500 μM L-DOPA (Figure 1C), indicating that there was no difference in the rates of DA oxidation by monoamine oxidase A (MAO) and vesicular uptake by vesicular monoamine transporter 2 (VMAT2). To establish whether L-DOPA transport into the cell or its conversion to DA by aromatic L-amino acid decarboxylase (AADC) limited the L-DOPA metabolic consumption, we measured total intracellular L-DOPA levels in VM cultures after 1 hr treatment with 100 and 500 μM L-DOPA concentrations in the presence of the AADC inhibitor benserazide to block DA synthesis (Figure 1D). The initial rate of L-DOPA accumulation in the cells was not saturated under these conditions, consistent with previously published data (Sampaio-Maia et al., 2001Sampaio-Maia B. Serrao M.P. Soares-da-Silva P. Regulatory pathways and uptake of L-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells.Am. J. Physiol. Cell Physiol. 2001; 280: C333-C342PubMed Google Scholar) on the kinetics of L-DOPA uptake by cell lines derived from astrocytes and neurons (Km = 50–100 μM). Overall, these data suggest that the activity of AADC limits the steady-state DAcyt concentration following L-DOPA treatment. The data in Figure 1C also provide information about the rate of L-DOPA turnover by the cells. The combined rate of DA and DOPAC synthesis during 1 hr of L-DOPA exposure was ∼40 fmol/μg of total protein. As each culture dish typically contained 50 −100 μg of total protein, the rate of L-DOPA consumption during 10 hr of incubation was 20–40 pmol per culture. This rate, however, is too low to account for the decline in the DAcyt observed after 10 hr of cell incubation with L-DOPA (Figure 1A), as the total amount of available L-DOPA was >3 orders of magnitude higher (200 nmol in 2 ml of 100 μM solution). We therefore examined the availability of extracellular L-DOPA, which is known to auto-oxidize to DOPA-semiquinone and DOPA-quinone derivatives (Sulzer and Zecca, 2000Sulzer D. Zecca L. Intraneuronal dopamine-quinone synthesis: a review.Neurotox. Res. 2000; 1: 181-195Crossref PubMed Google Scholar). HPLC-EC measurements of L-DOPA concentration in cell-free media showed that the drug disappeared with first-order kinetics and a half-life of 4.7 hr (Figure 1E). This suggests that after 100 or 500 μM L-DOPA initial doses, its concentration in the media would reach the K0.5 levels in 16 or 26 hr, respectively (Figure 1F), which is in close agreement with the kinetics of DAcyt changes, as a significant decrease in its initial steady state was observed at approximately these time points (Figure 1A). Together, these data allow us to predict the time dependence of DAcyt changes based on the concentration of L-DOPA added to the media and the initial increase in DAcyt determined by IPE (see Discussion). We next investigated whether exposure to various L-DOPA concentrations, and thereby different durations of sustained elevated DAcyt, correlated with neurotoxicity. We identified an exponential dependence of neurotoxicity on the extracellular L-DOPA concentration (Figure 2A), whereas the time dependence for cell survival in cultures treated with 250 μM L-DOPA demonstrated that the number of TH+ neurons declined linearly after drug exposure, reaching ∼50% of the control levels after 4 days, with no subsequent neuronal loss (Figure 2B). As L-DOPA at this concentration is cleared within ∼22 hr, the data indicate a lag between the end of L-DOPA/DA-mediated stress and the completed course of the catechol-induced toxicity. For further experiments we exposed cultures to 250 μM L-DOPA, which consistently produced ∼50% loss of VM dopaminergic neurons 4 days following drug addition. Extracellular DA released by neuronal activity did not seem to play a significant role in the L-DOPA-induced cell death, as neuronal viability was not affected by the Na+ channel blocker tetrodotoxin (TTX; 1 μM), which inhibits stimulation-dependent transmitter release (data not shown). To further determine which pool of DA was responsible for the observed cell damage, we employed postnatal cultures of cortical and striatal neurons, which do not express TH, DAT, or VMAT. Following L-DOPA treatment of cortical neurons, we detected small amounts of DA measured by HPLC-EC (Figure 1C) and a substantial elevation of DAcyt measured by IPE (Figure 2C). This was accompanied by >60% loss of cells immunoreactive to microtubule-associated protein 2 (MAP2), which was used as a neuronal marker (Figure 2D). L-DOPA neurotoxicity was NSD-1015 sensitive, confirming AADC-mediated DA synthesis in cortical neurons (data not shown). In contrast, almost no DAcyt was detected in L-DOPA-treated striatal neurons, and they were spared following L-DOPA challenge (Figures 2C and 2D). Together, our data suggest DAcyt as the primary source of the L-DOPA-induced neurotoxicity. To study the contribution of individual metabolic pathways in the maintenance of the DAcyt steady state and to determine whether changes in DA level correlate with neurotoxicity, we performed metabolite measurements and toxicity studies on L-DOPA-treated neurons that were preincubated with specific inhibitors of AADC, MAO, and VMAT. Inhibition of the DA-synthesizing enzyme AADC with NSD-1015 (Figures 3A and 3B) or benserazide (not shown) blocked L-DOPA-induced elevation of whole-cell intracellular DA and DOPAC. Benserazide also inhibited the buildup of DAcyt following L-DOPA treatment (Figure 3C), consistent with the idea that the IPE oxidation signal comes from DA synthesized by AADC in the cytosol (note that NSD-1015 alters IPE sensitivity for DA, and therefore was not examined; see Experimental Procedures). Moreover, the blockade of L-DOPA decarboxylation completely prevented drug-induced cell death (Figure 3D), indicating that an L-DOPA metabolite, but not L-DOPA itself, was responsible for the toxicity. (A and B) HPLC-EC measurements of total intracellular DA (A) and DOPAC (B) in rat VM neuronal cultures pretreated for 1 hr with 500 μM NSD-1015, 2 μM reserpine (Res), or 10 μM pargyline (PGL) and then treated with 100 μM L-DOPA for 1 hr (n = 6–10 dishes). N.D., not detected. (C) DAcyt in mouse VM neurons under the same treatments as above except for 2 μM benserazide (Bsrz) to block AADC (n = 22–81 cells). (D) The effect of NSD-1015, benserazide, reserpine, and pargyline on the survival of mouse TH+ neurons treated with 250 μM L-DOPA for 4 days (n = 6–20 dishes). (E) DAcyt concentration in TH-GFP neurons pretreated with 10 μM cocaine for 15 min, then exposed to 100 μM L-DOPA for 1 hr, and then treated with 50 μM METH for 15–30 min (n = 19–46 cells). (F) Cell survival of mouse TH+ neurons pretreated with METH and cocaine as in (E) and exposed to 250 μM L-DOPA for 4 days (n = 3–8 dishes). ∗p < 0.05 versus cells treated with L-DOPA only by one-way ANOVA. Dotted lines and shadowed boxes in (D) and (F) represent mean ± SEM in untreated cells. The contribution of DA catabolism was investigated by treating cells with pargyline, an inhibitor of MAO that almost completely abolished DOPAC synthesis (Figure 3B). Pargyline also produced a several-fold increase in both the total amount of intracellular DA (Figure 3A) and DAcyt (Figure 3C) in L-DOPA-treated neurons, which further correlated with a significantly greater neuronal loss (Figure 3D). The blockade of VMAT-mediated DA uptake into the vesicles depletes vesicular catecholamine storage, as observed by a reduction in the exocytotic quantal size (Colliver et al., 2000Colliver T. Hess E. Pothos E.N. Sulzer D. Ewing A.G. Quantitative and statistical analysis of the shape of amperometric spikes recorded from two populations of cells.J. Neurochem. 2000; 74: 1086-1097Crossref PubMed Scopus (81) Google Scholar). Consistently, pretreatment of VM neurons with reserpine decreased the amount of total intracellular DA synthesized from L-DOPA by ∼2-fold both with and without pargyline (Figure 3A). We observed, however, no effect of reserpine, or another VMAT inhibitor, tetrabenazine (10 μM; data not shown), on either DAcyt or the number of surviving dopaminergic neurons (Figures 3C and 3D). The discrepancy between the reduction of the total DA in reserpine-treated neurons and the lack of the effect of VMAT inhibition on DAcyt might be explained by the distribution of synaptic vesicles between neuronal cell bodies and the neurites. If the majority of vesicles filled with DA are located in synaptic terminals far from the cell bodies, any transmitter redistributed from them would be invisible to IPE (see Experimental Procedures). To examine the effects of other DA-releasing drugs on somatic DA concentration, we exposed L-DOPA-treated neurons to methamphetamine (METH), which disrupts the vesicular proton gradient and redistributes DA from synaptic vesicles to the cytosol (Sulzer et al., 2005Sulzer D. Sonders M.S. Poulsen N.W. Galli A. Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (833) Google Scholar). In contrast to the increased cytosolic catecholamine levels observed in chromaffin cells acutely treated with METH (Mosharov et al., 2003Mosharov E.V. Gong L.W. Khanna B. Sulzer D. Lindau M. Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells.J. Neurosci. 2003; 23: 5835-5845PubMed Google Scholar), neurons exposed to 50 μM METH showed decreased DAcyt (Figure 3E); 5 μM METH produced the same decrease of DAcyt (38% of untreated cells; p < 0.005 by t test). METH-mediated reduction of DAcyt was blocked by the DA uptake transporter (DAT) inhibitor cocaine, suggesting that the effect was due to reverse transport through DAT (Sulzer et al., 2005Sulzer D. Sonders M.S. Poulsen N.W. Galli A. Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (833) Google Scholar). METH-mediated decrease of DAcyt and its blockade by cocaine were also observed in L-DOPA-treated cells in the presence of pargyline (data not shown). Note that DAcyt in L-DOPA-treated neurons was unaffected by DAT inhibitors cocaine (Figure 3E) or nomifensine (5 μM; data not shown), supporting the idea that reverse transport is not induced by the elevated DAcyt alone, but requires additional METH-mediated effects on DAT (Kahlig et al., 2005Kahlig K.M. Binda F. Khoshbouei H. Blakely R.D. McMahon D.G. Javitch J.A. Galli A. Amphetamine induces dopamine efflux through a dopamine transporter channel.Proc. Natl. Acad. Sci. USA. 2005; 102: 3495-3500Crossref PubMed Scopus (206) Google Scholar). Consistent with the IPE results, METH protected neuronal cell bodies from L-DOPA toxicity (Figure 3F), despite the considerable neurite loss that occurs in METH-treated cultured DA neurons (Cubells et al., 1994Cubells J.F. Rayport S. Rajendran G. Sulzer D. Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress.J. Neurosci. 1994; 14: 2260-2271Crossref PubMed Google Scholar, Larsen et al., 2002Larsen K.E. Fon E.A. Hastings T.G. Edwards R.H. Sulzer D. Methamphetamine-induced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis.J. Neurosci. 2002; 22: 8951-8960Crossref PubMed Google Scholar). When applied for 4 days without L-DOPA, neither METH or cocaine nor the combination of the two drugs had a significant effect on the number of surviving neurons (data not shown). We previously developed a recombinant adenovirus that overexpresses VMAT2 (rVMAT2), resulting in reduced neuronal neuromelanin content and enhanced quantal size, and which increased the number of evoked quantal neurotransmitter release events from the terminals of cultured VM dopaminergic neurons (Pothos et al., 2000Pothos E.N. Larsen K.E. Krantz D.E. Liu Y. Haycock J.W. Setlik W. Gershon M.D. Edwards R.H. Sulzer D. Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.J. Neurosci. 2000; 20: 7297-7306Crossref PubMed Google Scholar, Sulzer and Pothos, 2000Sulzer D. Pothos E.N. Regulation of quantal size by presynaptic mechanisms.Rev. Neurosci. 2000; 11: 159-212Crossref PubMed Scopus (154) Google Scholar). To investigate whether enhanced vesicular uptake may decrease DAcyt and rescue neurons from L-DOPA-induced toxicity, we employed an adenoviral construct that resulted in overexpression of the recombinant VMAT2 in 80%–90% of both dopaminergic and nondopaminergic cells (Figure S2). VMAT2 overexpression significantly decreased DAcyt in L-DOPA-treated GFP+ VM neurons (from 17.4 ± 1.7 μM, n = 74 neurons to 3.1 ± 0.8 μM, n = 28; Figure 4A), but had no effects on GFP− neurons from the same culture (2.4 ± 0.8 μM, n = 33 versus 2.3 ± 1.9 μM, n = 7). GFP+ neurons in cultures treated with a virus that did not contain rVMAT2 displayed the same DAcyt levels after L-DOPA treatment as control cells (data not shown). Consistent with the kinetics of L-DOPA metabolic consumption (Figure 1), rVMAT2 lowered DAcyt to the same extent in cells treated with 100 and 500 μM L-DOPA (Figure 4A). Moreover, infection with rVMAT2 effectively protected TH+ neurons from the L-DOPA-mediated neurotoxicity (Figure 4B). It should be noted, however, that these data also suggest that somatic vesicles or other organelles that do not normally sequester substantial levels of DA can do so after transporter overexpression, as has been demonstrated for noncatecholaminergic AtT-20 cells (Pothos et al., 2000Pothos E.N. Larsen K.E. Krantz D.E. Liu Y. Haycock J.W. Setlik W. Gershon M.D. Edwards R.H. Sulzer D. Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.J. Neurosci. 2000; 20: 7297-7306Crossref PubMed Google Scholar) and hippocampal neurons (Li et al., 2005Li H. Waites C.L. Staal R.G. Dobryy Y. Park J. Sulzer D.L. Edwards R.H. Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines.Neuron. 2005; 48: 619-633Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). (A) DAcyt in sister cultures of mouse TH+ neurons treated with L-DOPA for 1 hr; cells were either untransfected or were transfected with rVMAT2 or empty vector (not shown) 1 day before the application of L-DOPA. ∗p < 0.01 versus cells treated with L-DOPA only by one-way ANOVA (n = 15–28 cells). (B) Neuroprotection of TH+ neurons in rat VM cultures infected with rVMAT2 or empty vector and exposed to varying concentrations of L-DOPA on day 1 and assessed for survival on day 7. ∗p < 0.05 versus empty vector by two-way ANOVA. (C) Reduced sensitivity of TH+ neurons from α-syn-deficient mice to L-DOPA-induced neurotoxicity. Neurons from α-syn wild-type, heterozygous, and knockout littermate mice were treated with indicated L-DOPA concentrations for 4 days. ∗p < 0.05 and ∗∗p < 0.001 versus the wild-type by two-way ANOVA with Bonferroni post hoc test (n = 10–29 dishes). (D) Comparison of DAcyt levels in neurons from α-syn knockout, heterozygous, and wild-type mice treated with 100 μM L-DOPA for 1 hr (n = 20–49 cells). To investigate the role of α-syn in mediating DAcyt neurotoxicity, we generated α-syn-deficient mice that express eGFP in dopaminergic neurons (see Experimental Procedures). Whereas VM neurons from α-syn−/− and α-syn+/− mice were more resistant to L-DOPA-induced stress than neurons from their wild-type littermates (Figure 4C), IPE measurements indicated no difference in the DAcyt concentration between the three groups (Figure 4D). These data suggest that the pathogenic effect of α-syn is downstream of DA synthesis (see Discussion), and that the presence of both elevated DAcyt and α-syn is required for L-DOPA-induced neurotoxicity. To address the question of differential susceptibility of neurons from different brain regions, we prepared cultures that were enriched with cells from either SN or VTA, as previously published (Burke et al., 1998Burke R.E. Antonelli M. Sulzer D. Glial cell line-derived neurotrophic growth factor inhibits apoptotic death of postnatal substantia nigra dopamine neurons in primary culture.J. Neurochem. 1998; 71: 517-525Crossref PubMed Scopus (135) Google Scholar). Immunolabeling for calbindin, a protein that is expressed at higher levels in VTA than in SN (Thompson et al., 2005Thompson L. Barraud P. Anderss" @default.
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• W2013503732 date "2009-04-01" @default.
• W2013503732 modified "2023-10-15" @default.
• W2013503732 title "Interplay between Cytosolic Dopamine, Calcium, and α-Synuclein Causes Selective Death of Substantia Nigra Neurons" @default.
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• W2013503732 doi "https://doi.org/10.1016/j.neuron.2009.01.033" @default.
• W2013503732 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2677560" @default.
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