Christophe Wersinger

Does α-synuclein modulate dopaminergic synaptic content and tone at the synapse?

α‐Synuclein is a key component of the pathological process of neurodegeneration in Parkinsons disease. Although its contributions to normal physiological conditions remain elusive, converging observations suggest that a primary function of this protein in dopaminergic neurons may be the regulation of dopamine content and synaptic tone at the synapse. We review here cumulative evidence that demonstrates the participation of α‐synuclein in the life cycle of dopamine from its synthesis, storage, release, and reuptake. The regulatory role of α‐synuclein on dopamine metabolism is assessed by discussing the experimental evidence supporting each of these observations in the healthy physiological maintenance of dopaminergic neurons, as well as showing how disruption of these events can initiate the observed neurotoxicity of α‐synuclein and the genesis of the degenerative processes associated with Parkinsons disease.—Sidhu, A., Wersinger, C., Vernier, P. Does α‐synuclein modulate dopaminergic synaptic content and tone at the synapse?

Attenuation of dopamine transporter activity by α-synuclein

Abstract α - synuclein accumulates in Lewy bodies in idiopathic Parkinsons disease. Neither the normal function nor contribution of α-synuclein to the pathophysiology of neurodegeneration is known. Here we show that a normal function of α-synuclein is the negative modulation of human dopamine transporter (hDAT) activity. In cotransfected Ltk − cells, α-synuclein attenuated the reuptake of dopamine by hDAT, in a manner dependent on expression levels of α-synuclein. α-synuclein-mediated inhibition of hDAT activity was independent of expression vectors, cell types and methods of transfection. The α-synuclein-mediated decrease in DAT activity occurred through diminished uptake velocity of dopamine, without changes in the affinity of hDAT for dopamine. Co-immunoprecipitation studies confirmed the formation of a stable complex between α-synuclein and DAT, through direct protein:protein interactions. Thus, under normal (non-toxic) expression conditions, α-synuclein negatively modulates dopamine uptake by DAT.

α-Synuclein regulation of the dopaminergic transporter: a possible role in the pathogenesis of Parkinson's disease

Parkinsons disease (PD) is a slow progressive neurodegenerative disorder. Recent evidence suggests a central role for α‐synuclein, a protein of unknown function, in the genesis of PD. The phenomenon of selective degeneration of dopaminergic neurons in PD may be linked to the potential toxicity of dopamine itself and aberrations in the processes which regulate dopamine content may underlie the pathogenesis of this disease. Here, we review a vital role of α‐synuclein in the modulation of dopamine transporter (DAT) function, and describe how disruption of this modulatory process permits increased re‐uptake of high levels of intracellular dopamine by DAT, causing profound neurotoxicity.

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

Human α‐synuclein accumulates in dopaminergic neurons as intraneuronal inclusions, Lewy bodies, which are characteristic of idiopathic Parkinsons disease (PD). Here, we suggest that modulation of the functional activity of the dopamine transporter (DAT) by α‐synuclein may be a key factor in the preferential degeneration of mesencephalic dopamine (DA)‐synthesizing neurons in PD. In cotransfected Ltk‐, HEK 293, and SK‐N‐MC cells, α‐synuclein induced a 35% decrease in [3H]DA uptake. Biotinylated DAT levels were decreased by 40% in cotransfected cells relative to cells expressing only DAT. DAT was colocalized with α‐synuclein in mesencephalic neurons and cotransfected Ltk‐ cells. Coimmunoprecipitation studies showed the existence of a complex between α‐synuclein and DAT, in specific rat brain regions and cotransfected cells, through specific amino acid motifs of both proteins. The attenuation of DAT function by α‐synuclein was cytoprotective, because DA‐mediated oxidative stress and cell death were reduced in cotransfected cells. The neurotoxin MPP+ (1‐methyl‐4‐phenylpyridinium), oxidative stress, or impairment of cell adhesion ablated the α‐synuclein‐mediated inhibition of DAT activity, which caused increased uptake of DA and increased biotinylated DAT levels, in both mesencephalic neurons and cotransfected cells. These studies suggest a novel normative role for α‐synuclein in regulating DA synaptic availability and homeostasis, which is relevant to the pathophysiology of PD.

The Role of α‐Synuclein in Both Neuroprotection and Neurodegeneration

Abstract: Although α‐synuclein is a central player in the pathophysiology of the dopaminergic neurodegeneration that occurs in Parkinsons disease (PD), emerging results suggest that the fundamental property of the wild‐type form of this protein may be one of neuroprotection, as it can inhibit apoptosis in response to various pro‐apoptotic stimuli. Such properties may be lost by its familial PD‐linked mutations upon alterations in its expression levels or clearance (overexpression of the gene, reduced protein degradation) or following exposure to certain neurotoxins. Moreover, converging observations suggest that a primary function for α‐synuclein in dopaminergic neurons may be the regulation of dopamine content and tone at the synapse. In this paper, we review how, indeed, α‐synuclein regulates both the synthesis of dopamine, its storage into vesicles, its release in the synapse, and its re‐uptake into the dopaminergic neurons. We also show how disruption of these events, and of the neuroprotective effects of α‐synuclein, can initiate the observed neurotoxicity of α‐synuclein in dopaminergic neurons and the genesis of the degenerative processes associated with PD.

Alpha-synuclein induces hyperphosphorylation of Tau in the MPTP model of parkinsonism.

Many neurodegenerative diseases associated with functional Tau dysregulation, including Alzheimers disease (AD) and other tauopathies, also show α‐synuclein (α‐Syn) pathology, a protein associated with Parkinsons disease (PD) pathology. Here we show that treatment of primary mesencephalic neurons (48 h) or subchronic treatment of wild‐type (WT) mice with the Parkinsonism‐inducing neurotoxin MPP+/MPTP, results in selective dose‐dependent hyperphosphorylation of Tau at Ser396/404 (PHF‐1‐reactive Tau, p‐Tau), with no changes in pSer202 but with nonspecific increases in pSer262 levels. The presence of α‐Syn was absolutely mandatory to observe MPP+/MPTP‐induced increases in p‐Tau levels, since no alterations in p‐Tau were seen in transfected cells not expressing α‐Syn or in α‐Syn‐/‐ mice. MPP+/MPTP also induced a significant accumulation of α‐Syn in both mesencephalic neurons and in WT mice striatum. MPTP/MPP+ lead to differential alterations in p‐Tau and α‐Syn levels in a cytoskeleton‐bound, vs. a soluble, cytoskeleton‐free fraction, inducing their coimmunoprecipitation in the cytoskeleton‐free fraction and neuronal soma. Subchronic MPTP exposure increased sarkosyl‐insoluble p‐Tau in striatum of WT but not α‐Syn‐/‐ mice. These studies describe a novel mechanism for MPTP neurotoxicity, namely a MPTP‐inducible, strictly α‐Syn‐dependent, increased formation of PHF‐1‐reactive Tau, suggesting convergent overlapping pathways in the genesis of clinically divergent diseases such as AD and PD. —Duka, T., Rusnak, M., Drolet, R. E., Duka, V., Wersinger, C., Goudreau, J. L., Sidhu, A. Alpha‐synuclein induces hyperphosphorylation of Tau in the MPTP model of Parkinsonism. FASEB J. 20, 2302–2312 (2006)

An Inflammatory Pathomechanism for Parkinsons Disease

Parkinsons disease (PD) is a slowly progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), striatal dopamine deficiency and appearance of Lewy bodies. Inflammatory and immune, or even autoimmune, stigmata, have been described in post-mortem brains of PD patients. Although disputed in humans, a reactive astrocytosis and a lymphocytic infiltration in the SNpc have been observed in animal models of PD, which need further examination. This review summarizes the current knowledge on brain inflammation in humans with PD, and how inflammation and/or (auto)immune reactions within the SNpc could be linked to other pathophysiological mechanisms that have been hypothesized for the etiology of PD, such as oxidative stress, exposure to neurotoxins, and post-infectious or post-traumatic injuries. In particular, we discuss how microglial cells could be activated during the course of PD, and present a new hypothesis that PD-linked protein (alpha-synuclein, in particular) aggregates could be implicated in their activation, to induce a chronic and sustained inflammation involved in the progression, at least, of the disease. The current status of anti-inflammatory agents, either already tried in PD clinical trials or putatively usefull as adjuvant therapies for PD, is also discussed.

Modulation of the trafficking of the human serotonin transporter by human alpha-synuclein.

α‐Synuclein (α‐Syn), a protein primarily localized in the presynaptic compartment of neurons, is known to regulate dopaminergic neurotransmission by negatively modulating dopamine transporter activity and regulating its trafficking to or away from the cell surface. Given the considerable homology between dopamine transporters and the serotonin (5‐HT) transporter (SERT), we examined whether α‐Syn could similarly regulate SERT function. Increasing expression levels of human α‐Syn gradually decreased [3H]5‐HT uptake by human SERT in cotransfected Ltk– cells, by diminishing its Vmax without changing its Km, as compared to cells expressing only SERT. Biotinylation studies to label cell‐surface proteins showed that α‐Syn decreased the levels of SERT present at the plasma membrane. α‐Syn and SERT were able to coimmunoprecipitate (co‐IP), suggesting heteromeric complexes between these two proteins through direct protein–protein interactions. The negative modulation of SERT activity by α‐Syn occurred through the non‐Aβ‐amyloid component (NAC) domain of α‐Syn (aa58–107); DNA constructs encoding this region mimicked the full‐length α‐Syn protein by decreasing [3H]5‐HT uptake by the transporter. Furthermore, only the constructs encoding the NAC domain of α‐Syn prevented the co‐IPs between full‐length α‐Syn and SERT, in both transfected cells and in rat solubilized lysates isolated from the prefrontal cortex. These studies suggest a novel physiological role for α‐Syn in regulating SERT activity and may be of relevance in certain mental illnesses and in depression, in which SERT function is believed to be dysregulated.

Attenuation of the norepinephrine transporter activity and trafficking via interactions with α‐synuclein

Alpha‐synuclein (α‐Syn) has been studied in the context of Parkinsons disease, but its normative role remains elusive. We have shown that α‐Syn regulates the homeostasis of dopaminergic and serotonergic synapses, through trafficking of the dopamine and serotonin transporter, respectively. In the present study we sought to determine if α‐Syn could also modulate noradrenergic signaling, by studying its interactions with the norepinephrine transporter (NET). We co‐transfected Ltk– cells with increasing amounts of α‐Syn DNA and a constant amount of NET DNA, and observed a progressive decrease (68%) in [3H]‐NE uptake in cells co‐transfected with a ratio of 3 : 1 α‐Syn : NET DNA. The Kd of transport did not change, but increasing α‐Syn caused a decrease in the Vmax of the transporter, from 2.27 ± 0.14 to 0.89 ± 0.15 pmol/min/105 cells, with NET expression alone or 4 : 1 ratio of α‐Syn : NET transfection, respectively. Decreases in surface biotinylation and [3H]‐nisoxetine binding kinetics in intact cells revealed that NET cell surface expression was attenuated in correlation to the amount of α‐Syn co‐transfected into cells. The interaction between NET and α‐Syn occurred via the NAC domain of α‐Syn, the region directly responsible for self‐aggregation. These findings are the first to show that α‐Syn has a central role in the homeostasis of noradrenergic neurons. Together with our previous studies on dopamine and serotonin transporters, we propose that a primary physiological role of α‐Syn may be to regulate the homeostasis of monoamines in synapses, through modulatory interactions of the protein with monoaminergic transporters.

The Degeneration of Dopamine Neurons in Parkinson's Disease Insights from Embryology and Evolution of the Mesostriatocortical System

Abstract: Parkinsons disease (PD) is, to a large extent, specific to the human species. Most symptoms are the consequence of the preferential degeneration of the dopamine‐synthesizing cells of the mesostriatal‐mesocortical neuronal pathway. Reasons for that can be traced back to the evolutionary mechanisms that shaped the dopamine neurons in humans. In vertebrates, dopamine‐containing neurons and nuclei do not exhibit homogenous phenotypes. In this respect, mesencephalic dopamine neurons of the substantia nigra and ventral tegmental area are characterized by a molecular combination (tyrosine hydroxylase, aromatic amino acid decarboxylase, monoamine oxidase, vesicular monoamine transporter, dopamine transporter—to name a few), which is not found in other dopamine‐containing neurons of the vertebrate brain. In addition, the size of these mesencephalic DA nuclei is tremendously expanded in humans as compared to other vertebrates. Differentiation of the mesencephalic neurons during development depends on genetic mechanisms, which also differ from those of other dopamine nuclei. In contrast, pathophysiological approaches to PD have highlighted the role of ubiquitously expressed molecules such as a‐synuclein, parkin, and microtubule‐associated proteins. We propose that the peculiar phenotype of the dopamine mesencephalic neurons, which has been selected during vertebrate evolution and reshaped in the human lineage, has also rendered these neurons particularly prone to oxidative stress, and thus, to the fairly specific neurodegeneration of PD. Numerous evidence has been accumulated to demonstrate that perturbed regulation of DAT‐dependent dopamine uptake, DAT‐dependent accumulation of toxins, dysregulation of TH activity as well as high sensitivity of DA mesencephalic neurons to oxidants are key components of the neurodegeneration process of PD. This view points to the contribution of nonspecific mechanisms (α‐synuclein aggregation) in a highly specific cellular environment (the dopamine mesencephalic neurons) and provides a robust framework to develop novel and rational therapeutic schemes in PD.