Muscarinic Receptor Stimulation Induces Translocation of an α-Synuclein Oligomer from Plasma Membrane to a Light Vesicle Fraction in Cytoplasm

Abstract

The close correspondence between the distribution of brain ␣-synuclein and that of muscarinic M 1 and M 3 receptors suggests a role for this protein in cholinergic transmission. We thus examined the effect of muscarinic stimulation on ␣-synuclein in SH-SY5Y, a human dopaminergic cell line that expresses this protein. Under basal conditions, ␣-synuclein was detected in all subcellular compartments isolated as follows: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa ␣-synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer. This finding suggests ␣-synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the ␣-synuclein oligomer in plasma membrane over a 30-min period, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. The carbachol-induced alteration of ␣-synuclein was blocked by atropine. Translocation of the ␣-synuclein oligomer in response to carbachol stimulation corresponds closely with the time course of ligandstimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of the ␣-synuclein oligomer from plasma membrane, and its subsequent association with the endocytotic vesicle fraction may have a role in muscarine receptor endocytosis. We propose that its function may be a transient release of membrane-bound phospholipase D 2 from ␣-synuclein inhibition, thus allowing this lipase to participate in muscarinic receptor endocytosis.The neuronal protein ␣-synuclein, recently implicated in the pathogenesis of Parkinson's disease (1-3), has a brain distribution closely matching that of muscarinic M 1 and M 3 receptors and the muscarine receptor-linked enzymes phospholipase C and protein kinase C (4). The proximity of ␣-synuclein to brain muscarinic receptors and related enzymes suggests that this protein could contribute to cholinergic function. To evaluate this possibility, we investigated the effect of muscarinic receptor stimulation by the cholinergic agonist carbachol on the subcellular distribution of endogenous ␣-synuclein in the human neuroblastoma cell line SH-SY5Y. This dopaminergic cell line shares many properties with dopamine neurons of substantia nigra pars compacta, the major locus of neurodegeneration in Parkinson's disease, including the expression of M 1 and M 3 receptors (5). We now report that carbachol induces a translocation of a 45-kDa ␣-synuclein oligomer from plasma membrane to a light vesicle fraction in cytoplasm, with a time course and subcellular localization matching that of muscarinic receptors during carbachol-stimulated endocytosis. Previously, synucleins have been demonstrated to be endogenous inhibitors of phospholipase D 2 (PLD 2 ) 1 (6). Furthermore, activation of PLD isoforms, including PLD 2 , ...

Use of drugs with anticholinergic effect and impact on cognition in Parkinson's disease: a cohort study

“…There is also evidence that activitation of muscarinic receptors may prevent tau phosphorylation,24 which may explain the findings of increased neurofibrillar tangles after anticholinergic treatment in PD27 and neuroleptic treatment in dementia with Lewy bodies 29. There is also preliminary evidence linking muscarinic receptor activation to the processing of α-synuclein,30 the key protein in the pathogenesis of PD and the main substrate underlying cognitive decline in PD 16. In addition, cholinergic deficits have been reported to be associated with reduced neurogenesis 23…”

Distinct pattern of enteric phospho-alpha-synuclein aggregates and gene expression profiles in patients with Parkinson’s disease

“…Interestingly, M3R activation is linked to α-syn in a human dopaminergic cell line in which muscarinic receptor stimulation leads to translocation of oligomeric α-syn from the plasma membrane to a light vesicle fraction in the cytoplasm [42]. The authors suggest, that α-syn could have a physiological role in the cell, in which its release transiently disinhibits membrane bound phospholipase LD2, freeing this lipase to function in ligand-stimulated manner to muscarinic receptor endocytosis.…”

Membrane Interactions of Oligomeric Alpha-Synuclein: Potential Role in Parkinsons Disease

“…Oligomeric species have been detected in cell cultures expressing S and brain tissue extracts by conformation specific antibodies by using SDS-PAGE [85,[132][133][134]. These oligomeric fractions have been observed to be associated with membrane fractions [129,135]. The structural properties of these oligomeric species have not been studied in detail.…”