Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice

Abstract

Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.

Keywords: G2019S; LRRK2; LRRK2 mutation; Parkinson disease; cortical culture; electrophysiology; transgenic mice.

Figure 1

Age-dependent LRRK2 expression in vivo and in vitro and generation of LRRK2 G2019S knock-in mice. (A) Representative western blots showing LRRK2 expression at embryonic day 16 (E16) that increases over postnatal days 7-21 (P7-21) in mouse cortex (CTX). Positive control lysate from non-transgenic (NT) P7 CTX was used as a protein standard (STD). A similar pattern of increasing LRRK2 expression is observed in primary neuronal cultures from mouse CTX from 1 to 21 days in vitro (DIV). Semi-quantitative analysis expressed as LRRK2 relative to Grb2 loading control demonstrates significantly increasing LRRK2 levels over time in both CTX tissue and CTX cultures. The increase in vivo at P21 and in vitro at DIV21 occurs to a similar extent, relative to P7 STD (~5-fold). n = 3 Independent cultures ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 by ANOVA and Bonferroni post-test. (B) In lysate from LRRK2 OE mouse CTX, LRRK2 protein is increased relative to NT littermates at 1 month (^*p < 0.05 by ANOVA and Bonferroni post-test) and is absent in knock-out mouse (KO) CTX. In CTX cultures from OE mice LRRK2 expression is ~3-fold increased over NT littermates at DIV14-21. (C) Production of LRRK2 G2019S knock-in mouse model; Exon 41 of the endogenous murine LRRK2 (NT, top) was replaced with G2019S-containing neomycin cassette (middle), prior to cassette excision and retention of the loxP cut site (LRRK2 G2019S KI, bottom). Forward and reverse PCR primers (P1 and P2) were designed to amplify the regions flanking the loxP site, resulting in a 307bp fragment from NT endogenous LRRK2 and a 383bp fragment from each allele of G2019S KI. (D) PCR products reveal clear separation between the predicted band sizes and simple genotyping of NT, heterozygous (HET, herein KI) and homozygous (Homo) KI mice (left). Examples of LRRK2 western blot of lysates from three of the independent paired cultures (pooled KI and NT littermate pups) used in all subsequent experiments. There are no significant differences in the levels of LRRK2 protein in KI CTX cultures at DIV21.

Figure 2

LRRK2 levels subtly alter excitatory transmission and synaptic architecture. (A) Whole-cell patch-clamp recordings of neurons in DIV21 CTX cultures from KO mice. (Top) Example traces of miniature excitatory post-synaptic currents (mEPSCs) mediated by glutamatergic AMPA-type glutamate receptors (AMPARs, left). Quantification of mean mEPSC amplitude and frequency shows no significant difference between genotypes (right). (Bottom) Cumulative probability analysis found no significant differences in mEPSC amplitudes, but did detect a significant interaction between inter-event intervals (IEIs) and genotype (2-way RM-ANOVA ^***p ≤ 0.001), due to generally longer IEIs (indicative of lower frequency) in KO neurons. (B) Example traces of mEPSCs in DIV21 CTX cultures from OE mice (left). Quantification of mean mEPSC amplitude and frequency shows no significant difference between genotypes (right). (Bottom) Cumulative probability analysis found no significant differences in mEPSC amplitues or IEIs in OE neurons. (C) Cultures were stained for neuronal microtubules (MAP2, green) and excitatory pre-synaptic (VGluT1, blue) and post-synaptic (PSD-95, red) markers for neuronal density and synapse (VGluT1+PSD-95 co-clusters) measurements. Left: 40× imaging of MAP staining (bar = 100 um). Right: expanded ROI from 63× images of synaptic markers overlayed with and without MAP2. Co-clusters (white arrow heads) indicative of excitatory synapses are generally located outside of the MAP2 dendritic microtubule scaffold, upon dendritic spines that do not contain microtubules. (D,E) Both KO and OE neuronal densities were similar to those of their respective NT littermate cultures (by MAP2 soma counts) as were their total dendritic areas (not shown). (D) Although cluster intensities were significantly reduced in KO cultures (see text) and they exhibited a trend toward fewer synapses, there were no significant differences in the density or size of VGluT1 clusters, PSD-95 clusters or co-clusters. (E) In OE neurons, there was no significant difference in VGluT1 cluster density, despite a strong trend. There were significantly more PSD95 clusters and synaptic co-clusters in OE neurons ^*p ≤ 0.05 by Student's t-test.

Figure 3

Increased excitatory transmission and altered GABA currents in G2019S KI cortical neurons. (A-C) Whole-cell patch-clamp recordings of neurons in DIV21 CTX cultures from KI mice. (A) Example traces of mEPSCs. (B) Quantification of mean mEPSC amplitude and frequency shows no significant difference in amplitude, but significantly higher frequency of events in KI neurons (^**p ≤ 0.01 by Student's t-test). (C) Cumulative probability analysis found no significant differences in mEPSC amplitudes, but revealed a significant main effect of genotype and interaction between IEI and genotype (2-way RM-ANOVA, ^**p ≤ 0.01, ^****p ≤ 0.0001, values between 40 and 200 ms were also significant by Bonferroni post-test ^***p ≤ 0.001), due to shorter IEIs (indicative of higher frequency) in KI neurons. (D) Cultures were stained (as in Figure 2) for MAP2 (green) and VGluT1 (blue) and PSD-95 (red). Left: 60× 2-times zoom of individual neuron staining. Right: expanded ROI from the 63× image showing synaptic markers overlayed with and without MAP2. Co-clusters are highlighted (white arrow heads). (E) KI neuronal densities were similar to those of NT littermates as were total dendritic areas (not shown), there were no differences (or trends) in the density of VGluT1 clusters, PSD-95 clusters or co-clusters (glutamate synapses). Similarly there were no differences in the density, size or intensity of synapsin 1 (Syn1) clusters, present at all glutamatergic and inhibitory synapses. (F) Example traces of miniature inhibitory post-synaptic currents (mIPSCs). (G) Quantification of mean mIPSC amplitude and frequency shows trends, but no significant differences in event amplitude or frequency of events in KI neurons. (H) Cumulative probability analysis revealed a highly significant interaction (and nearly significant genotype effect) due to increased mIPSC amplitudes in KI neurons (2-way RM-ANOVA, ^****p ≤ 0.0001, values between 25 and 35pA were significant by Bonferroni post-test ^**p ≤ 0.01). There was no significant main effect of genotype on mIPSC IEIs or interaction (despite a trend to higher frequency) in KI neurons.

Figure 4

Reduced Synapsin 1 phosphorylation in KI cortical neurons. Levels of pre-synaptic proteins in DIV21 CTX cultures were assayed by standard western blotting and verified via WES automated capillary-based size sorting system. (A) Representative western blots of EndophilinA (EndoA), vesicle associated membrane protein 1 (VAMP1), vesicle associated membrane protein 2 (VAMP2), dynamin 1, synapsin1 (Syn1), phosphoserine 9 synapsin1 (pS9 Syn1), and phosphoserine 603 synapsin1 (pS603 Syn1). (B) Quantification of synapsin1 levels and associated phosphorylation sites. Synapsin1 levels were similar between NT and KI however the ratio of phosphorylated synapsin1 was significantly reduced at both sites. (C) Standard western blot results were verified using the WES automated capillary-based size sorting system for the S603 phosphorylation site. Representative pseudo-gels (left) and electropherograms (right) exported from the WES compass analysis software. (D) Quantification of synapsin1 and pS603 synapsin1 confirmed significant reductions pS603 synapsin1. Data expressed relative to GAPDH and normalized to NT, ^*p ≤ 0.05 ^**p ≤ 0.01 by paired Student's t-test.