In Situ Proximity Ligation Assay Reveals Co-Localization of Alpha-Synuclein and SNARE Proteins in Murine Primary Neurons

Abstract

The aggregation of alpha-synuclein (αSyn) is the pathological hallmark of Parkinson’s disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of αSyn is to promote the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between αSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human αSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human αSyn, a PLA signal indicating close proximity between αSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human αSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of αSyn and the SNARE proteins in primary neuronal cultures.

Identification of Potential Interacting Proteins With the Extracellular Loops of the Neuronal Glycoprotein M6a by TMT/MS

“…Fluorescence immunocytochemistry followed by quantification of protein colocalization is an indirect technique for determining protein-protein interaction. Hence, experiments like in situ proximity ligation assay, PLA (Alam, 2018;Almandoz-Gil et al, 2018), which confirm the direct interaction between two proteins are needed for the final validation of our study. Besides, experiments focused on the functional implication of the potential M6a's interactors are convenient to understand the integral role of M6a in the neuronal development and the neurobiology of the diseases.…”

Dopamine Transporter/α-Synuclein Complexes Are Altered in the Post Mortem Caudate Putamen of Parkinson’s Disease: An In Situ Proximity Ligation Assay Study

“…The in situ PLA allows the detection of protein-protein interactions in situ in intact tissues [ 7 , 27 , 30 , 31 , 32 , 33 , 48 ]. For the fluorescence human in situ PLA studies, we analyzed paraffin embedded brain sections from the caudate putamen of PD patients and age-matched controls by using the Duolink assay kit (O-Link Bioscience, Uppsala, Sweden) with a protocol adapted from the manufacturer’s instructions.…”

Rationally Designed Variants of α-Synuclein Illuminate Its in vivo Structural Properties in Health and Disease

“…Initial reports focused on effects of αS on directly stabilizing curved membrane structures ( Kamp et al, 2010 ; Varkey et al, 2010 ), similar to BAR domain proteins ( DeWitt and Rhoades, 2013 ; Westphal and Chandra, 2013 ). More recent studies described direct effects of αS on membrane clustering Wang et al, 2014 ) or indirect effects of αS on vesicle fusion via the stabilization of vesicle SNARE complexes ( Burré et al, 2010 , Burré et al, 2014 ; Almandoz-Gil et al, 2018 ). Interestingly, both direct and indirect effects have been proposed to be mediated by αS multimerization.…”