Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

Abstract

Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinson's disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our “in vitro” studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, “in vivo” studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the “in situ” visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT.

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons

“…This was associated with a specific rearrangement of synaptic vesicle clusters, a significant reduction of basal and depolarization-dependent dopamine release, and with the onset of an increased locomotor response to the administration of cocaine -a substance known to mobilize synapsin-dependent dopamine-vesicle reserve pools (Venton et al, 2006). In agreement with the results in mice and in cultured neurons, here, we also identify a significant redistribution of synapsin III in the caudate and putamen of individuals with Parkinson's disease, as well as in the striatum of transgenic mice expressing human α-syn, which also show decreased dopamine release and redistribution of synaptic proteins (Bellucci et al, 2011a;Garcia-Reitbock et al, 2010). Finally, by silencing synapsin III in vitro we could prevent α-syn aggregation and the related redistribution of synaptic proteins.…”

“…S1E-G; Bellucci et al, 2008Bellucci et al, , 2011a. Indeed, urea-soluble α-syn aggregates (Bellucci et al, 2008) and thioflavin-S-positive α-syn inclusions (supplementary material Fig.…”

“…We studied synapsin-III distribution in the striatum of 12-month-old control and α-syn-null mice, as well as that of transgenic mice expressing a C-terminally truncated form of human α-syn comprising only residues 1-120 of the protein (termed SYN120) on a mouse-α-syn-null background; we have previously used these mice to demonstrate that the absence or aggregation of α-syn can affect the distribution of synaptic vesicle proteins (Bellucci et al, 2011a;Garcia-Reitbock et al, 2010) (Fig. 2A).…”

“…To test this interaction, we used the in situ proximity ligation assay (PLA) (Fig. 3A) (Bellucci et al, 2011a) and coimmunoprecipitation (Fig. 3B).…”

“…Among the synapsins, synapsin III is believed to be the only isoform that regulates vesicular monoamine transporter 2 (VMAT-2)-positive vesicle pools in the striatum (Bogen et al, 2006). Moreover, previous studies have shown that the protein showing most sequence homology to synaptin III -synaptin I (of which there are two isoforms, synapsin Ia and synapsin Ib encoded by SYN1) -can interact with α-syn (Bellucci et al, 2011a;Maiya et al, 2007) and that the two proteins have similar distribution profiles in hippocampal neurons (Tao-Cheng, 2006). Therefore, we investigated whether synapsin III and α-syn interact, and whether the absence or aggregation of α-syn affects synapsin-III expression and distribution in dopaminergic neurons.…”