Effect of Ser-129 Phosphorylation on Interaction of α-Synuclein with Synaptic and Cellular Membranes

In the healthy brain, less than 5% of α-synuclein (α-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of α-syn is Ser(P)-129. The effects of Ser(P)-129 modification on α-syn distribution and solubility are poorly understood. As α-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 α-syn and analyzed the effects of manipulating Ser(P)-129 levels on α-syn membrane interactions using synaptosomal membranes and neural precursor cells from α-syn-deficient mice or transgenic mice expressing human α-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either α-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT α-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A α-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 α-syn showed a preferential membrane association compared with WT Ser(P)-129 α-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of α-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129.

6-[2-(4-arylpiperazin-1-yl)ethyl]-4-halo-1,3-dihydro-2h-benzimidazole-2-thiones: Synthesis and pharmacological evaluation

Eight new compounds with halogen atom introduced into the benzimidazole- 2-thione dopaminergic pharmacophore of 5-[2-(4-arylpiperazin-1-yl)ethyl]-1,3-dihydro- 2H-benzimidazole-2-thiones with the arylpiperazine part of the molecule being selected according to known structure-affinity requirements, have been synthesized. All the new compounds were evaluated for the in vitro binding affinity at the dopamine (DA) D1 and D2 and serotonin 5-HT1A receptors by the competitive radioassays, performed on synaptosomal membranes prepared from fresh bovine caudate nuclei and hippocampi. All the new compounds were strong competitors for the binding of the radioligands to the D2 and 5-HT1A receptors, with the most active of them having 34 and 170 time higher affinity than non-halogenated congeners in the D2 DA receptor radioassays (compounds 9.1b and 9.2b, respectively). Divergently, these compounds were without significant affinities for the D1 DA receptors. .