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Modulation of dopamine transporter function by -synuclein is altered by impairment of cell adhesion and by induction of oxidative stress¹

Department of Pediatrics, Georgetown University, Washington D.C., USA and
^* Institut de Neurobiologie Alfred FESSARD, C.N.R.S., 91198 Gif-sur-Yvette cedex, France

²Correspondence: Laboratory of Molecular Neurochemistry, The Research Building, Room W222, 3970 Reservoir Road NW, Washington, DC 20007, USA. E-mail: sidhua{at}georgetown.edu

SPECIFIC AIMS

-Synuclein, a highly enriched neuronal protein in healthy brain, is a major component of Lewy bodies (LBs) characteristic of idiopathic Parkinson's disease (PD) and other neurodegenerative diseases and is reported to have dual roles of neuroprotection and neurotoxicity, which remain enigmatic. Recent reports that -synuclein regulates synaptic DA (DA) homeostasis prompted us to examine the mechanisms of interactions between -synuclein and the dopamine transporter DAT, a key determinant of synaptic DA reuptake.

PRINCIPAL FINDINGS

1. hDAT and -synuclein were transiently coexpressed in Ltk^- fibroblasts, HEK-293 and neuroblastoma-derived SK-N-MC cells
In all three cell lines, -synuclein significantly (P<0.05) decreased (by 35%) hDAT-mediated cellular uptake of [³H]DA uptake compared with cells transfected with hDAT alone. The attenuation of hDAT function by -synuclein was not due to changes in the affinity (K_m) of the hDAT for DA nor to any changes in protein expression levels. A consequence of reduced hDAT-mediated DA uptake was that reactive oxygen species production generated upon intracellular autoxidation of DA, as indexed by DCF emission fluorescence measurements, was decreased. Decreases in nitrite production and cell death upon exposure of Ltk^- cells to DA were also observed in cotransfected Ltk^- cells compared with cells expressing hDAT alone, showing that -synuclein has a cytoprotective role by virtue of its ability to attenuate DAT activity.

2. Immunocytochemistry and confocal analysis of primary rat mesencephalic neurons and cotransfected Ltk^- cells showed significant coexpression of -synuclein and DAT at both the plasma membrane and intracellular reticular compartments
Such overlap in coexpression suggested the possibility of direct protein:protein interactions between these proteins in endogenously expressing neurons as well as in cotransfected cells.

3. Coimmunoprecipitation (co-IP) studies were conducted using lysates from rat brain tissues coexpressing proteins and lysates of cotransfected Ltk^- cells
In all instances, co-IPs with DAT antibodies revealed the presence of -synuclein and co-IPs with -synuclein antibodies revealed the presence of DAT, in the respective immunopellets. Together, these data confirm that these proteins physically interact with one another to form a stable protein complex.

4. Using cloned truncated cDNAs encoding specific structural domains of -synuclein and hDAT, physical interaction between the proteins occurred through specific binding of the nonamyloid component domain (residues 58-107) of -synuclein to the last 22 residues of the carboxyl-terminal tail of the DAT (residues 598-620)
These studies were also conducted in endogenously expressing rat brain tissues, confirming the physiological relevance of our findings in cotransfected Ltk^- cells. Furthermore, DA uptake measurements performed in cotransfected cells expressing peptides encoded by the truncated cDNAs show that the functional modulation of hDAT activity by -synuclein is mediated by the same structural domains of both proteins.

5. Earlier studies showed that when cells were transfected using a batch transfection method (whereby cells were transfected in a flask, trypsinized, then reseeded into individual dishes), -synuclein failed to attenuate hDAT activity
We speculated that perturbation of cell adhesion may have inadvertently reversed the modulatory properties of -synuclein. To test this, primary mesencephalic neurons or Ltk^- cells cotransfected in individual dishes were subjected to mild trypsinization (0.1% trypsin for 30 s), followed by [³H]DA uptake studies; under these conditions, there was no detachment of cells or changes in the gross morphological features of the neurons or cells. In both Ltk^- cells and neurons, mild trypsinization caused a relief of the -synuclein-mediated inhibition of hDAT function, resulting in increased [³H]DA uptake of 35–40% compared with nontrypsinized cells. Subsequent exposure of cells to DA caused profound cytotoxicity, with accelerated production of free radicals and increased cell death. That mild trypsinization of primary rat mesencephalic neurons also increased DA uptake clearly indicates that in mesencephalic neurons -synuclein also has a negative modulatory action on DAT function.

6. Pretreatment of neurons or Ltk^- cells with MPP⁺ (10–100 nM) or H₂O₂ (1 µM) increased (by 35–40%) DA uptake and DA-induced cytotoxicity (Fig. 1 )
These data underscore the clinical and physiological relevance of our findings whereby PD-linked neurotoxins such as MPP⁺ or oxidative stress can disrupt and convert a neuroprotective role of -synuclein to one that is now neurotoxic.

View larger version (55K): [in this window] [in a new window]   Figure 1. PD-inducing agents reverse -synuclein-mediated modulation of DAT activity and accelerate cytotoxicity. Cotransfected Ltk- cells (A–C) and 6-day-old primary mesencephalic neurons (D–F) were pretreated with indicated concentrations of MPP+ (nM), 1 µM H2O2 (H) or vehicle (0.2% H2O; control; C) for 30 min (48 h after transfection for Ltk- cells) or were subjected (24 h after transfection) to mild trypsinization (0.1%, 30 s; T; A, D). These pretreatments were followed by [3H]DA uptake measurements (A, D) or by a treatment with DA or indatraline (10 µM for Ltk-; 100 nM for neurons) or the antioxidant sodium metabisulfite (200 µM for Ltk-; 20 µM for neurons) for 20 h (B, C, E, F). Studies are the mean ± SE of 4–6 experiments; **P < 0.01 and *P < 0.05, significantly different compared with hDAT-transfected cells.

7. To further investigate the mechanisms by which -synuclein modulates hDAT function, biotinylation of cell surface proteins was analyzed in cotransfected Ltk^- cells
Under normal, nontoxic conditions, -synuclein decreased the presence of hDAT at the plasma membrane in cotransfected Ltk^- cells. In mesencephalic neurons, treatment with either trypsin or MPP⁺ increased biotinylation of DAT, indicating increased presence of DAT at the plasma membrane consistent with increased function of DAT. Together, these data indicate that the mechanism by which -synuclein regulates DAT function is via trafficking of the transporter, thereby regulating the intracellular and synaptic levels of DA.

CONCLUSIONS AND SIGNIFICANCE

Our findings show that a primary function of -synuclein is a neuroprotective one and that this protein has a central role in regulating the synaptic tone of DA through its modulatory effects on DAT activity, which regulates the influx of DA. Disruption of this modulatory effect of -synuclein on DAT function with the known parkinsonian syndrome-inducing agent MPP⁺ or oxidants switches the role of -synuclein to one profoundly neurotoxic, resulting in increased reuptake of DA, increased intracellular autoxidation of DA and enhanced neuronal cell death. These findings are consistent with those of others in transgenic mice that show that absence of -synuclein leads to some pathological features and functional deficits in the nigrostriatal DA system, such as reduced vesicular storage of DA and increased DA release into the synapse, and with the role of -synuclein in plasticity. Yet it is clear that -synuclein can also be neurotoxic; indeed, it has been suggested that -synuclein neurotoxicity is enhanced by cytotoxic insults such as oxidative stress, overexpression of the protein, and transition metals. Our data provide a novel mechanism for the preferential degeneration of mesencephalic dopaminergic neurons in PD, whereby selectivity is conferred through autoxidation of DA. Recent reports showing that oxidized DA stabilizes -synuclein protofibril before its aggregation into insoluble fibrils found in LBs are entirely consistent with and extend the pathophysiological relevance of our findings.

Our data also provide unique insight to the mechanisms by which DAT activity is regulated. A kinase-dependent mechanism of DAT trafficking to and away from the plasma membrane has been suggested, but not definitively demonstrated. The participation of the cytoskeleton in mediating the trafficking process has also been suggested. Our data support the involvement of the cytoskeleton in the trafficking of DAT by -synuclein, since perturbation of cell adhesion, and thus of the anchoring of the cytoskeleton to the plasma membrane or the anchoring of DAT or -synuclein to the cytoskeleton, increases trafficking of DAT to the plasma membrane.

A schematic model summarizing our findings is shown in Fig. 2 . Under nontoxic conditions, -synuclein regulates the activity of DAT by reducing the levels of DAT localized at the plasma membrane, thereby acting in a neuroprotective manner by reducing the amount of DA that enters the neurons. This normative, physiological role of -synuclein can be reversed by impairment of cell adhesion, exposure to neurotoxins such as the PD-linked MPP⁺, or oxidative stress, resulting in increased trafficking of the transporter to the plasma membrane. This triggers a complex chain of events with increased reuptake of DA and increased intracellular autoxidation of DA. Besides its known neurotoxicity, oxidized DA in turn stabilizes -synuclein protofibrils, accelerating its aggregation and accumulation into LBs. Precise elucidation of the molecular partners and events underlying our model is likely to contribute to increased understanding and possibly provide for novel therapeutic approaches in the treatment of PD and other neurodegenerative diseases.

View larger version (34K): [in this window] [in a new window]   Figure 2. Proposed model for interaction between -synuclein and DAT in normal and pathophysiological conditions.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0152fje; doi: 10.1096/fj.03-0152fje

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