Defective membrane interactions of familial Parkinson's disease mutant A30P alpha-synuclein.

alpha-Synuclein (alpha-Syn) is an abundant presynaptic protein of unknown function, which has been implicated in the pathogenesis of Parkinson's disease. Alpha-Syn has been suggested to play a role in lipid transport and synaptogenesis, and growing evidence suggests that alpha-Syn interactions with cellular membranes are physiologically important. In the current study, we demonstrate that the familial Parkinson's disease-linked A30P mutant alpha-Syn is defective in binding to phospholipid vesicles in vitro as determined by vesicle ultracentrifugation, circular dichroism spectroscopy, and low-angle X-ray diffraction. Interestingly, our data also suggest that alpha-Syn may bind to the lipid vesicles as a dimer, which suggest that this species could be a physiologically relevant and functional entity. In contrast, the naturally occurring murine A53T substitution, which is also linked to Parkinson's disease, displayed a normal membrane-binding activity that was comparable to wild-type alpha-Syn. A double mutant A53T/A30P alpha-Syn showed defective membrane binding similar to the A30P protein, indicating that the proline mutation is dominant in terms of impairing the membrane-binding activity. With these observations, we suggest that the A53T and A30P mutants may have different physiological consequences in vivo and could possibly contribute to early onset Parkinson's disease via unique mechanisms.     

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.     

Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line

Mutations in alpha-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant alpha-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. alpha-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant alpha-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human alpha-synuclein. Lentivirus-induced overexpression of A53T mutant alpha-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant alpha-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant alpha-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in alpha-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.     

Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter

The dopamine transporter (DAT) is a presynaptic plasma membrane protein responsible for the termination of dopaminergic neurotransmission in the central nervous system. While most studies have focused on structure/function analysis, much less information is available regarding the assembly and the trafficking of this protein. To address this problem, we performed a mutational analysis of the DAT protein, combined with biochemical, immunological, and functional approaches. In mammalian cells co-expressing differentially tagged DAT molecules, HA-tagged DAT co-purified with 6His-tagged DAT demonstrating a physical interaction between transporter proteins. Evidence for the functional oligomerization of DAT was obtained using dominant-negative mutants of DAT. Two loss-of-function mutant transporters (Y335A and D79G) that were targeted to the cell surface inhibited wild-type DAT uptake activity without affecting the membrane targeting of the wild-type transporter. Moreover, non-functional amino and carboxyl termini-truncated mutants of DAT inhibited wild-type DAT function by interfering with the normal processing of the wild-type transporter to the cell membrane. Mutations in the leucine repeat of the second transmembrane domain of the transporter could eliminate the dominant-negative effect of all these mutants. In addition, a small fragment comprising the first two transmembrane domains of DAT inhibited wild-type transporter function but not when the leucine repeat motif was mutated. Taken together, our results suggest that the assembly of DAT monomers plays a critical role in the expression and function of the transporter.     

Differential cytotoxicity of human wild type and mutant alpha-synuclein in human neuroblastoma SH-SY5Y cells in the presence of dopamine

Parkinson's disease (PD) involves loss of dopaminergic neurons in the substantia nigra and is characterized by intracellular inclusions, Lewy bodies, consisting primarily of aggregated alpha-synuclein. Two substitution mutations (A53T and A30P) in alpha-synuclein gene have been identified in familial early-onset PD. To understand the biological changes that incur upon alpha-synuclein-induced cytotoxicity in the presence of dopamine, the current studies were undertaken. Human SH-SY5Y neuroblastoma cells coexpressing the human dopamine transporter [hDAT], and either wild type (wt) or mutant alpha-synucleins, were treated with 50 microM dopamine (DA). In cells expressing wt or A30P alpha-synuclein, DA accelerated production of reactive oxygen species and cell death as compared to cells expressing A53T or hDAT alone. The increased sensitivity of such cells to DA was investigated by measuring changes in cellular ionic gradient, by atomic absorption spectrometry, and cell metabolism, by high-resolution nuclear magnetic resonance spectroscopy. Both wt and A30P alpha-synuclein caused rapid decrease in levels of intracellular potassium, followed by mitochondrial damage and cytochrome c leakage, with decreased cellular metabolism as compared to cells expressing A53T or hDAT alone. Collapse of ionic gradient was significantly faster in A30P (t(1/2) = 3.5 h) than in wt (t(1/2) = 6.5 h) cells, and these changes in ionic gradient preceded cytochrome c leakage and depletion of metabolic energy. Neither wt nor mutant alpha-synuclein resulted in significant changes in ionic gradient or cellular metabolism in the absence of intracellular DA. These findings suggest a specific sequence of events triggered by dopamine and differentially exacerbated by alpha-synuclein and the A30P mutant.     

A genetic screen in Caenorhabditis elegans for dopamine neuron insensitivity to 6-hydroxydopamine identifies dopamine transporter mutants impacting transporter biosynthesis and trafficking

The presynaptic dopamine (DA) transporter (DAT) is a major determinant of synaptic DA inactivation, an important target for psychostimulants including cocaine and amphetamine, and a mediator of DA neuron vulnerability to the neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion. To exploit genetic approaches for the study of DATs and neural degeneration, we exploited the visibility of green fluorescent protein (GFP)-tagged DA neurons in transgenic nematodes to implement a forward genetic screen for suppressors of 6-OHDA sensitivity. In our initial effort, we identified three novel dat-1 alleles conferring 6-OHDA resistance. Two of the dat-1 alleles derive from point mutations in conserved glycine residues (G55, G90) in contiguous DAT-1 transmembrane domains (TM1 and TM2, respectively), whereas the third allele results in altered translation of the transporter's COOH terminus. Our studies reveal biosynthetic, trafficking and functional defects in the DAT-1 mutants, exhibited both in vitro and in vivo. These studies validate a forward genetic approach to the isolation of DA neuron-specific toxin suppressors and point to critical contributions of the mutated residues, as well as elements of the DAT-1 COOH terminus, to functional expression of catecholamine transporters in neurons.     

Truncation of human dopamine transporter by protease calpain

It has been shown recently that the N-terminal domain of the dopamine transporter (DAT) plays a role in several transporter functions. Here we provide evidence for a possible cellular mechanism of how the N-terminus of dopamine transporter might be removed in vivo. We isolated a recombinant N-terminal protein region of human dopamine transporter and cleaved it with calpain protease. Peptide fragment analysis revealed the existence of two calpain cleavage sites at positions Thr43/Ser44 and Leu71/Ser72 of the DATN-terminus. We show that calpain activation in rat striatal synaptosomes leads to a rapid decrease of dopamine transporter N-terminal epitopes corresponding to the protein sequences removed by a calpain cleavage at Thr43/Ser44 and that the process is totally blocked by a calpain inhibitor. Calpain truncation of the DATN-terminus abolishes its interaction with the receptor of activated protein kinase C, RACK1 and removes protein sequences previously implicated in amphetamine-induced dopamine release, PKC-dependent endocytosis and the interaction of DAT with the dopamine D2 receptor. The above suggests that cleavage of DAT by calpain may significantly modify dopamine homeostasis under pathological or physiological conditions.     

Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters

The dopamine transporter plays an essential role in the modulation of dopaminergic neurotransmission by mediating the reuptake of dopamine into presynaptic neurons. In cells expressing the dopamine transporter, activation of protein kinase C by phorbol esters results in a significant reduction in dopamine uptake. This phorbol ester-mediated inhibition of dopamine transport is associated with a decrease in V(max), although the apparent affinity of the transporter for dopamine remains unchanged. Using a green fluorescent protein-tagged dopamine transporter stably expressed in Madin-Darby canine kidney cells, we show in live cells that the decrease in transporter activity is caused by the rapid internalization of carriers from the plasma membrane. This redistribution of the transporter is specific to phorbol ester activation and is unaffected by the presence of either substrates or inhibitors of the carrier. Upon the addition of phorbol esters, transporters at the cell surface are rapidly endocytosed through a clathrin-mediated and dynamin-dependent mechanism into early endosomes, where they colocalize with transferrin. The internalized carrier is targeted to the endosomal/lysosomal pathway and is completely degraded within 2 h of protein kinase C activation. Phorbol ester-mediated alterations in the trafficking of the dopamine transporter may serve as a mechanism for controlling extracellular dopamine levels in the central nervous system.     

Cellular localization and trafficking of the human ABCA1 transporter

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.     

Disruption of the interaction of alpha-synuclein with microtubules enhances cell surface recruitment of the dopamine transporter

Mutations in alpha-synuclein have been implicated in the genesis of Parkinson's disease. A probable normative function of alpha-synuclein is the maintenance of dopamine homeostasis, partly through a negative modulation of dopamine transporter (DAT) activity, by reducing its level at the cell surface. To study the possible involvement of the microtubular network in the alpha-synuclein-dependent trafficking of DAT, we treated cotransfected cells and primary mesencephalic neurons with either colchicine, vinblastine, or nocodazole, each of which disrupts microtubules or affects microtubule dynamics. Treatment of both types of cells with vinblastine, colchicine, or nocodazole reversed alpha-synuclein-mediated inhibition of DAT activity, resulting in an increased rate of dopamine uptake and and increased level of extracellular dopamine-induced oxidative stress, with accelerated cell death. Treatment with these agents also reversed the alpha-synuclein-induced decrease in levels of cell surface-associated DAT. This effect of colchicine, vinblastine, or nocodazole was not linked to a disruption of formation of the alpha-synuclein-DAT complex but paradoxically caused an increased level of interaction between these proteins. Both alpha-synuclein and DAT co-immunoprecipitated with both alpha- and beta-tubulins, in both transfected cells and rat primary mesencephalic dopaminergic neurons, suggesting heteromeric complex formation between these various proteins. Treatment with the microtubule depolymerizing agents disrupted the heteromeric protein complex between either alpha-synuclein or the DAT, and alpha- or beta-tubulins. These results indicate a previously unappreciated role of microtubules in the modulation of DAT trafficking, and provide insight into a novel mechanism by which alpha-synuclein regulates DAT activity, by tethering the transporter to the microtubular network.     

Ethanol-sensitive sites on the human dopamine transporter

Previous studies have shown that ethanol enhanced [(3)H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity.     

Lifetime proteomic profiling of an A30P alpha-synuclein Drosophila model of Parkinson's disease

A survey of the proteome changes in an A30P alpha-synuclein Drosophila model of Parkinson's disease (PD) in comparison to age-matched controls is presented for seven different ages across the adult lifespan. The data were acquired by a shotgun proteomic approach that involves multidimensional liquid chromatographies coupled to mass spectrometry and database searching techniques. Semiquantitative analysis to assess relative changes in protein expression between the Drosophila PD model and age-matched controls provides evidence that 28, 19, 12, 5, 7, 23, and 17 proteins are significantly differentially expressed at days 1, 10, 20, 30, 40, 50, and 60, respectively. From the experimental approach employed, it appears that most dysregulated proteins are associated with narrow distributions of ages, such that disease-associated differences change substantially across the lifespan. Previous measurements [J. Proteome Res. 2007, 6, 348] at days 1, 10, and 30 showed dysregulation of actin cytoskeletal proteins at day 1 and mitochondrial proteins at day 10, suggesting that defects in the actin cytoskeleton and the mitochondria are associated with dopaminergic neuron degeneration in PD. Analysis of the day 20, 40, 50, and 60 animals supports the finding that these cytoskeletal and mitochondrial changes predominate in the youngest (pre-symtomatic and early disease stages) animals. Although studies across many time points appear to be important for characterizing disease state, an understanding of molecular changes at the youngest ages should be most important for addressing causation.     

Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

Neurological development and functioning of dopamine (DA) neurotransmission is adversely affected by iron deficiency in early life. Iron-deficient rats demonstrate significant elevations in extracellular DA and a reduction in dopamine transporter (DAT) densities in the caudate putamen and nucleus accumbens. To explore possible mechanisms by which cellular iron concentrations control DAT functioning, endogenous DAT-expressing PC12 cells were used to determine the effect of iron chelation on DAT protein and mRNA expression patterns. In addition, we used human DAT (hDAT)-transfected Neuro2a (N2A) cells to examine DAT degradation and trafficking patterns. A 50 microM treatment for 24 h with the iron chelator, desferrioxamine (DFO), significantly decreased dopamine uptake in a dose-dependent manner, with no apparent change in K(m), in both PC12 and N2A cells. Reduced DA uptake was accompanied by concentration- and time-dependent reductions in total DAT protein levels in both cell lines. Exposure to increasing concentrations of DFO did not significantly alter DAT mRNA in either PC12 or N2A cells. However, DAT degradation rates increased three-fivefold in both cell types exposed to 50 microM DFO for 24 h. Biotinylation studies in N2A cells indicate a more dramatic loss of DAT in the membrane fraction, while OptiPrep fractionation experiments revealed an increase in lysosomal DAT with iron chelation. Inhibition of protein kinase C activation with staurosporin prevented the effect of iron chelation on DAT function, suggesting that in vitro iron chelation affects DAT primarily through the effects on trafficking rather than on synthesis.     

A 40-nucleotide repeat polymorphism in the human dopamine transporter gene

A new polymorphic 40-nucleotide repeat in the human dopamine transporter gene is described. It may underlie individual differences in susceptibility to several neuropsychiatric diseases.     

Effect of familial Parkinson's disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein

Parkinson's disease involves the loss of dopaminergic neurons in the substantia nigra, leading to movement disorders. The pathological hallmark of Parkinson's disease is the presence of Lewy bodies and Lewy neurites, which are intracellular inclusions consisting primarily of alpha-synuclein. Although essentially all cases of sporadic and early-onset Parkinson's disease are of unknown etiology, two point mutations (A53T and A30P) in the alpha-synuclein gene have been identified in familial early-onset Parkinson's disease. Previous reports have shown that mutant alpha-synuclein may form fibrils more rapidly than wild-type protein. To determine the underlying molecular basis for the enhanced fibrillation of the mutants, the structural properties, responses to changes in the environment, and propensity to aggregate of wild-type, A30P, and A53T alpha-synucleins were systematically investigated. A variety of biophysical methods, including far-UV circular dichroism, FTIR, small-angle X-ray scattering, and light scattering, were employed. Neither the natively unfolded nor the partially folded intermediate conformations are affected by the familial Parkinson's disease point mutations. However, both mutants underwent self-association more readily than the wild type (i.e., at much lower protein concentration and more rapidly). We attribute this effect to the increased propensity of their partially folded intermediates to aggregate, rather than to any changes in the monomeric natively unfolded species. This increased propensity of these mutants to aggregate, relative to wild-type alpha-synuclein, would account for the correlation of these mutations with Parkinson's disease.     

Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking,stability, and activity

The function of the human norepinephrine transporter (hNET) depends on its presence at the cell surface. A role for the hNET C-terminus in trafficking the transporter to the surface has been suggested by the report of a bovine NET C-terminal splice variant that accumulates within heterologous host cells, and a human variant homolog has also been reported. We examined the relevance of the C-terminus of hNET to trafficking and function using transfected LLC-PK1 cells. The intracellular and surface expression of NET proteins was evaluated by Western blots, and their functional capacities were assessed using transport assays. We found that the C-terminal residues encoded by hNET 1a enable the efficient maturation and surface expression of hNET and therefore critically impact transporter activity. Alternative splicing causes the retention of immature hNETs within the cell, whereas introduced C-terminal deletions result in significant degradation. The loss of the terminal isoleucine alone (Delta617-hNET) is sufficient to cause the degradation of hNET, an effect that can be mimicked by nonconservative point mutations at the terminal position. The phenotype of Delta617-hNET is recapitulated in neuronal SK-N-MC cells, but is significantly less severe in HEK-293 cells, suggesting a role for host cell factors in enabling the biosynthetic progression of wild-type hNET. Additional proximal residues may act at other steps to affect the expression of the fully mature protein on the cell surface (Q608A) and to more directly affect transporter activity (F609A). Together our studies document a critical contribution of the hNET C-terminus to transporter trafficking, stability, and function.