Summary 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 2-3-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 most commonly a sporadic illness, and is characterized by degeneration of substantia nigra dopamine (DA) neurons and abnormal cytoplasmic aggregates of ␣-synuclein. Rarely, PD may be caused by missense mutations in ␣-synuclein. MPTP, a neurotoxin that inhibits mitochondrial complex I, is a prototype for an environmental cause of PD because it produces a pattern of DA neurodegeneration that closely resembles the neuropathology of PD. Here we show that ␣-synuclein null mice display striking resistance to MPTP-induced degeneration of DA neurons and DA release, and this resistance appears to result from an inability of the toxin to inhibit complex I. Contrary to predictions from in vitro data, this resistance is not due to abnormalities of the DA transporter, which appears to function normally in ␣-synuclein null mice. Our results suggest that some genetic and environmental factors that increase susceptibility to PD may interact with a common molecular pathway, and represent the first demonstration that normal ␣-synuclein function may be important to DA neuron viability.T he concept of genetic predisposition to disease suggests that one's genes influence susceptibility to environmental insult. However, the relationship between genetic and environmental factors is poorly understood; most models of disease focus on single genes or toxins. A major challenge of postgenomic biology will be to link the molecular pathways modified by diseaseassociated alleles to the environmental factors implicated in disease susceptibility.There is increasing evidence for genetic susceptibility to Parkinson's disease (PD) (1-3). Additionally, dysfunction of a common molecular pathway has been implicated in the familial and sporadic forms of PD. Mutations in the gene that encodes ␣-synuclein cause a rare form of dominantly inherited PD, and ␣-synuclein is an abundant protein in Lewy bodies, the proteinaceous neuronal inclusions that are the pathological hallmark of sporadic PD (4-6). The ␣-synuclein pathway is also implicated in an autosomal recessive form of PD caused by mutations in the gene encoding parkin (7,8). Epidemiological and twin studies suggest that environmental factors alter susceptibility to PD (9). The fact that exposure of humans to the environmental toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a syndrome that mimics the core neurological symptoms and relatively selective dopamine (DA) neuron degeneration of PD lends support to this concept (10, 11).We asked whether a model neurotoxin for an environmental cause of PD might act on a molecular pathway implicated in genetic and sporadic forms of the disease by generating ␣-synuclein null mice, and testing whether they display altered sensitivity to MPTP-induced degeneration of substantia nigra (SN) DA neurons. MethodsAnimal Generation. A 5.7-kb EcoRV mouse ␣-synuclein fragment (Fig. 1A) was used to generate the targeting construct. A DNA fragment containing, in order, LoxP-phosphoglycerate kinaseNeomycin-transcription blocking '...

Alpha-synuclein mutations have been identified in certain families with Parkinson's disease (PD), and alpha-synuclein is a major component of Lewy bodies. Other genetic data indicate that the ubiquitin-dependent proteolytic system is involved in PD pathogenesis. We have generated stable PC12 cell lines expressing wild-type or A53T mutant human alpha-synuclein. Lines expressing mutant but not wild-type alpha-synuclein show: (1) disruption of the ubiquitin-dependent proteolytic system, manifested by small cytoplasmic ubiquitinated aggregates and by an increase in polyubiquitinated proteins; (2) enhanced baseline nonapoptotic death; (3) marked accumulation of autophagic-vesicular structures; (4) impairment of lysosomal hydrolysis and proteasomal function; and (5) loss of catecholamine-secreting dense core granules and an absence of depolarization-induced dopamine release. Such findings raise the possibility that the primary abnormality in these cells may involve one or more deficits in the lysosomal and/or proteasomal degradation pathways, which in turn lead to loss of dopaminergic capacity and, ultimately, to death. These cells may serve as a model to study the effects of aberrant alpha-synuclein on dopaminergic cell function and survival.

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to L-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells. P arkinson's disease (PD) results from the death of neuromelanin (NM)-containing neurons in the substantia nigra pars compacta (SNC) (1) and the locus coeruleus (2). The presence of NM provides the characteristic pigmented appearance indicated by the names of these brain regions. NM appears in SNC DAergic neurons within 3 years of birth and increases with age (3). Because the neuronal populations that contain NM are those that die in PD, there has long been speculation that NM underlies PD pathogenesis. In one hypothesis, NM binds neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (4) or paraquat (5), providing a pool of toxin within pigmented cells. Similarly, NM binds iron and toxic metals that could promote neurodegeneration (6, 7). Finally, NM could itself produce toxic free radicals (8). However, NM cannot be the sole causal factor in PD pathogenesis because all humans accumulate NM with age.Beyond the suggestions that NM underlies PD, there has been no suggestion of a biological function for this substance. Very little is known about NM biosynthesis, and it is not known where NM is synthesized in the cell, which intracellular or extracellular catecholamine pools are involved, or what triggers its formation (9-12). Although previous studies on NM synthesis have been performed on synthetic polymers arising from sponta...

␣-Synuclein (␣-syn), a protein implicated in Parkinson's disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how ␣-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous ␣-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant ␣-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically "docked" vesicles was apparent in the ␣-synoverexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) ␣-syn, as well as chromaffin cells from control and ␣-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant ␣-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P ␣-syn-overexpressing cells. The ␣-synoverexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that ␣-syn overexpression inhibits a vesicle "priming" step, after secretory vesicle trafficking to "docking" sites but before calcium-dependent vesicle membrane fusion.

Methamphetamine (METH) selectively injures the neurites of dopamine (DA) neurons, generally without inducing cell death. It has been proposed that METH-induced redistribution of DA from the vesicular storage pool to the cytoplasm, where DA can oxidize to produce quinones and additional reactive oxygen species, may account for this selective neurotoxicity. To test this hypothesis, we used mice heterozygous (+/-) or homozygous (-/-) for the brain vesicular monoamine uptake transporter VMAT2, which mediates the accumulation of cytosolic DA into synaptic vesicles. In postnatal ventral midbrain neuronal cultures derived from these mice, METH-induced degeneration of DA neurites and accumulation of oxyradicals, including metabolites of oxidized DA, varied inversely with VMAT2 expression. METH administration also promoted the synthesis of DA via upregulation of tyrosine hydroxylase activity, resulting in an elevation of cytosolic DA even in the absence of vesicular sequestration. Electron microscopy and fluorescent labeling confirmed that METH promoted the formation of autophagic granules, particularly in neuronal varicosities and, ultimately, within cell bodies of dopaminergic neurons. Therefore, we propose that METH neurotoxicity results from the induction of a specific cellular pathway that is activated when DA cannot be effectively sequestered in synaptic vesicles, thereby producing oxyradical stress, autophagy, and neurite degeneration.

Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-t Bu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human a-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our ®ndings suggest that inclusion body formation and cell death may be dissociated from one another.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.