Glutamatergic synaptic resilience to overexpressed human alpha-synuclein

Abstract

Alpha synuclein (aSyn) is abundant in the brain and strongly implicated in Parkinson's disease (PD), genetically and through its accumulation in neuronal pathognomonic inclusions. While mutations or increased expression of wild-type aSyn can cause familial PD, it remains unclear whether increased aSyn alone impairs presynaptic function. Here, we overexpressed human aSyn (haSyn) in rodent glutamatergic neurons and analysed presynaptic function. Expression levels mimicked SNCA gene triplications, as seen in certain familial forms of PD. In continental cultures, haSyn overexpression was not toxic nor did it alter the levels of presynaptic SNAP-25 or postsynaptic PSD-95. Analyses of autaptic neurons revealed no significant differences in evoked or spontaneous neurotransmission release, nor in synaptic plasticity. These results indicate that rodent glutamatergic neurons are resilient to aSyn overexpression. Our findings suggest neurotoxicity associated with aSyn overexpression is not universal, and that a deeper understanding of aSyn biology and pathobiology is necessary.

Fig. 1. Overexpression of aSyn is induced at hippocampal neurons by infection with haSyn lentivirus.

A–C Primary hippocampal continental cultures from rat were infected with lentivirus mediating the expression of either eGFP alone or haSyn and eGFP. A Representative immunoblots of total aSyn, and eGFP levels. B 2.2-fold increase in total aSyn levels in haSyn-infected neurons (n = 6 independent cultures). β-actin was used as a loading control. C Representative images of continental hippocampal neurons infected with eGFP or haSyn lentivirus and immunolabelled using anti-MAP2 and corresponding 3D-tracing. Scale bar = 100 μm. D Cell death assessed by ToxiLight assay in rat primary neurons transduced with lentiviral encoding for haSyn for up to DIV21 (n = 7 independent cultures). haSyn transduction alter the total dendritic length (E), apical dendritic length (F) and basal dendritic length (G) at DIV 21 (n = 17–23/3 neurons/independent cultures). H Sholl analysis of aSyn-overexpressed neurons showed reduced number of intersections (n = 17–23/3 neurons/independent cultures). I–P Representative images of immunocytochemistry of continental hippocampal neurons infected with eGFP or haSyn lentivirus and labelled for aSyn (I, M), syntaxin (J, N), and MAP2 (K, O). Overlays are shown (L, P). Scale bar = 10 μm. Quantification of aSyn synaptic mean fluorescence intensity (Q), synaptic area (R), and synaptic volume (S) of neurons infected with haSyn lentivirus compared to neurons infected with eGFP (n = 6–10/1 neurons/independent culture). (T) Quantification of Syntaxin-positive puncta of neurons infected with haSyn lentivirus compared to neurons infected with eGFP (n = 6-10/1 neurons/independent culture). All data are expressed as mean ± SEM; Student’s t-test; * p-value ≤ 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001.

Fig. 2. Lentivirus-mediated overexpression of aSyn at synapses of cultured autaptic neurons.

A–H Representative images of immunocytochemistry of autaptic hippocampal neurons infected with eGFP or haSyn lentivirus and labelled for total aSyn (A, E), VGLUT1 (B, F), and MAP2 (C, G). Overlays are shown (D, H). Scale bar = 10 μm. Column charts showing quantification of aSyn synaptic mean fluorescence intensity (I), synaptic area (J) and synaptic volume (K) for immunolabeled neurons. N = 21 neurons in Control, N = 22 neurons in aSyn condition, from 4 independent cultures. L Quantification of VGLUT1 synaptic mean grey intensity. All data are expressed as mean ± SEM; Student’s t-test; **** p-value ≤ 0.0001.

Fig. 3. Electrophysiological characterization of functional properties in autaptic glutamatergic neurons overexpressing haSyn.

A Representative whole-cell patch clamp recordings traces of evoked EPSC amplitudes stimulated at 0.2 Hz from neurons infected with eGFP (A1) or haSyn lentivirus (A2). B Quantification of eEPSC amplitudes in neurons expressing eGFP alone (n = 69) or haSyn (n = 70). C Quantifications of miniature EPSC amplitudes (C) and frequency (D) recorded in the presence of 300 nM TTX for neurons expressing eGFP (n = 53) or haSyn (n = 52). E–J Normalised eEPSC amplitudes from whole-cell patch clamp recordings of neurons stimulated with trains of either 50 APs at 10 Hz (E) or 100 APs at 40 Hz (H) stimulation. Note that the presynaptic plasticity from neurons expressing eGFP or haSyn appeared highly similar, resulting in depression of the eEPSC before reaching a depressed steady state. F, I Quantification of the paired pulse ratio (PPR) as the ratio of the first two eEPSCs of each stimulation condition. No differences in PPR were observed between neurons expressing eGFP or haSyn when stimulated at 10 Hz (F) or 40 Hz (I). G, J Quantification of the steady-state depression during the stimulation trains, as the average normalised amplitude over the last 10 eEPSCs. For 10 Hz (G), n = 61 for eGFP, n = 67 for haSyn. For 40 Hz (J), n = 59 for eGFP, n = 65 for haSyn. K–N Estimation of readily-releasable pool (RRP) size in autaptic glutamatergic neurons overexpressing haSyn. K Sample traces showing the current elicited by the application of 500 mM sucrose, representing fusion of SVs in the RRP in neurons expressing eGFP (K1) or haSyn (K2). L Quantification of RRP size using the sucrose application method in neurons expressing eGFP or haSyn. M Quantification of the RRP estimation based on extrapolating from cumulative eEPSCs evoked by stimulating neurons at 40 Hz. N Release probability from neurons expressing eGFP or haSyn. n = 45 for eGFP, n = 48 for haSyn. Data collected from 12 independent cultures with three different lentivirus batches. Data are expressed as mean ± SEM; Student’s t-test or Mann-Whitney test, for normally and not-normally distributed data, respectively.