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, 38 (45), 9754-9767

A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes

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A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes

Peter J Teravskis et al. J Neurosci.

Abstract

Abnormalities in α-synuclein are implicated in the pathogenesis of Parkinson's disease (PD). Because α-synuclein is highly concentrated within presynaptic terminals, presynaptic dysfunction has been proposed as a potential pathogenic mechanism. Here, we report novel, tau-dependent, postsynaptic deficits caused by A53T mutant α-synuclein, which is linked to familial PD. We analyzed synaptic activity in hippocampal slices and cultured hippocampal neurons from transgenic mice of either sex expressing human WT, A53T, and A30P α-synuclein. Increased α-synuclein expression leads to decreased spontaneous synaptic vesicle release regardless of genotype. However, only those neurons expressing A53T α-synuclein exhibit postsynaptic dysfunction, including decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. We also found that long-term potentiation and spatial learning were impaired by A53T α-synuclein expression. Mechanistically, postsynaptic dysfunction requires glycogen synthase kinase 3β-mediated tau phosphorylation, tau mislocalization to dendritic spines, and calcineurin-dependent AMPA receptor internalization. Previous studies reveal that human A53T α-synuclein has a high aggregation potential, which may explain the mutation's unique capacity to induce postsynaptic deficits. However, patients with sporadic PD with severe tau pathology are also more likely to have early onset cognitive decline. Our results here show a novel, functional role for tau: mediating the effects of α-synuclein on postsynaptic signaling. Therefore, the unraveled tau-mediated signaling cascade may contribute to the pathogenesis of dementia in A53T α-synuclein-linked familial PD cases, as well as some subgroups of PD cases with extensive tau pathology.SIGNIFICANCE STATEMENT Here, we report mutation-specific postsynaptic deficits that are caused by A53T mutant α-synuclein, which is linked to familial Parkinson's disease (PD). The overexpression of WT, A53T, or A30P human α-synuclein leads to decreased spontaneous synaptic vesicle release. However, only those neurons expressing A53T α-synuclein exhibit tau phosphorylation-dependent postsynaptic dysfunction, which is characterized by decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. The mutation-specific postsynaptic effects caused by human A53T α-synuclein will help us better understand the neurobiological basis of this specific form of familial PD. The differential effects of exogenous human WT, A53T, A30P, and E46K α-synuclein on glutamatergic synaptic responses will help to explain the clinical heterogeneity of sporadic and familial PD.

Keywords: AMPA receptor; Parkinson's disease; frontotemporal dementia; synaptic plasticity; synuclein; tau.

Figures

Figure 1.
Figure 1.
A53T αS causes mutation-specific postsynaptic deficits in AMPAR signaling, whereas overexpression of human αS variants, regardless of genotype, causes presynaptic suppression in acute hippocampal slices. A, List of transgenic mice used in the present study. B, C, Immunoblots and quantification of human αS (HuSyn-1 antibody) and total (mouse and human) αS (BD Biosciences antibody 610787) in hippocampal lysates from 4- to 6-month-old MoPrP-Hu-αS transgenic and TgNg mice. Each lane represents an individual animal. αS levels are normalized to tubulin. I2-2 and H5 have comparable expression levels but have lower expression levels than G2-3 and O2. D, Input–output relationships of EPSCs (TgNg, n = 9; I2-2, n = 10; H5, n = 11; G2-3, n = 9; O2, n = 9; two-way ANOVA, F = 0.29, p = 1.0). E, Paired-pulse ratio induced by two consecutive stimuli delivered at different time intervals (TgNg, n = 15, I2-2, n = 9, H5, n = 11, G2-3, n = 10, O2, n = 11); two-way ANOVA, F = 0.56, p = 0.96. Representative traces are illustrated as insets, scale bars: 20 pA, 30 ms. F, Synaptic fatigue induced by 15 consecutive stimuli at 25 ms interpulse intervals (TgNg, n = 13; I2-2, n = 8; H5, n = 10; G2-3, n = 13; O2, n = 9; two-way ANOVA, F = 0.57, p = 0.99). Representative traces are illustrated as insets. Scale bars, 40 pA, 70 ms. For DF, two-way ANOVA with Fisher LSD post hoc analysis. G, Representative AMPA and NMDA receptor-mediated synaptic response traces and AMPA to NMDA receptor current ratio (TgNg, n = 11; I2-2, n = 7; H5, n = 10; G2-3, n = 8; O2, n = 11). Scale bars, 20 pA, 100 ms. Kruskal–Wallis test with Dunn's method post hoc analysis H = 21.53, df = 4; H5. HJ, Representative traces, mean amplitude, and mean frequency of mEPSCs obtained in the presence of TTX (1 μm) (TgNg, n = 7; I2-2, n = 10; H5, n = 11; G2-3, n = 8; O2, n = 10). Scale bar, 5 pA, 2 s. One-way ANOVA with Fisher LSD post hoc analysis, F = 8.23, p < 0.001 (amplitude); F = 5.54, p = 0.001 (frequency). All data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with TgNg, #p < 0.05 and ##p < 0.01 compared with I2-2. TgNg control was taken from littermates of I2-2 mice. For all, n-values represent neurons, at least 3 3- to 6-month-old mice were used for every experimental condition.
Figure 2.
Figure 2.
A53T αS causes deficits in LTP and spatial learning and memory. A, Top, Representative EPSC traces before (gray) and after (black) a high-frequency stimulation (HFS) of the Schaffer collaterals recorded from TgNg, I2-2, H5, and G2-3 mice. Scale bars, 10 pA, 15 ms. Bottom, EPSC amplitude versus time obtained from TgNg, I2-2, H5, and G2-3 mice (n = 9, n = 9, n = 9, and n = 8, respectively). Arrowhead indicates HFS application. TgNg controls were taken from I2-2 littermates. B, EPSC amplitude before and 45 min after stimulation in the different mouse models. Within-group analysis: two-tailed paired t test: t/df/P = −5.07/8/0.0010; −3.56/0.0074; −0.41/8/0.70; 0.39/6/0.71; for TgNg, I2-2, H5, and G2-3, respectively. Between-group analysis: one-way ANOVA with a Fisher LSD post hoc analysis F = 3.54, p = 0.027. At least 3 3- to 6-month-old mice were used for every experimental condition. n-values represent neurons. C, Diagram of the Barnes circular maze and representative occupancy plots from TgNg and G2-3 probe trials (color gradient bar plot, black: least occupied region, red: highest occupancy). D, Latency time to escape the maze during 4 consecutive training days. Two-way ANOVA, F(3,51) = 0.093. E, Mean distance from target measured on each training day. Two-way ANOVA with Bonferroni post hoc analysis, F(3,51) = 1.056; *p = 0.030, **p = 0.0015. F, Mean time 11- to 12-month-old TgNg and G2-3 animals spent in each quadrant of the maze during the probe trial. Two-way ANOVA with Bonferroni post hoc analysis, F(3,51) = 5.34; ***p = 0.0002. G, Average distance between the animals and the target during the probe trial. G2-3 mice were significantly more distant from the target than their TgNg littermates (TgNg, n = 9; G2-3, n = 10). Analyzed by Student's t test, t = 4.50, df = 17; ***p = 0.0003. All data are expressed as mean ± SEM.
Figure 3.
Figure 3.
A53T αS-induced postsynaptic deficits are independent of expression levels. A, Representative traces of events represented in C. Scale bars, 5 pA, 2 s. B, Relative cumulative frequency of whole-cell mEPSC amplitudes from cultured transgenic mouse hippocampal neurons. Kolmogorov–Smirnov test; D = 0.30, *p = 0.048; D = 0.34, **p = 0.0086; D = 0.43, ***p = 0.0002. C, D, Amplitude and frequency of mEPSCs. One-way ANOVA with Bonferroni post hoc analysis. For C: F(4,47) = 4.48; H5:*p = 0.014; G2-3: *p = 0.026; **p = 0.0036. For D: F(4,47) = 3.54; H5: p = 0.032; G2-3: p = 0.031; O2: p = 0.016. E, Representative images of eGFP-illuminated dendrites and spines from cultured Tg mouse hippocampal neurons. Scale bar, 5 μm. F, Spine density of neurons represented in E. One-way ANOVA, F(4,47) = 2.42. Data are expressed mean ± SEM. n-values are represented parenthetically.
Figure 4.
Figure 4.
A53T αS-induced postsynaptic deficits are cell autonomous. A, Wide-field fluorescence photomicrographs from cultured rat hippocampal DAPI-stained neurons expressing eGFP-tagged WT and A53T αS plasmids via calcium-phosphate transfection. The percentage of untransfected cells was tabulated. B, Photomicrographs of fixed neurons that had been transfected with eGFP, eGFP-WT αS, or eGFP-A53T αS plasmids (left) and subsequently stained with a mouse anti-synaptophysin antibody (middle; with overlay on the right). Axons were visually traced and defined as thin, long neurites emerging from the soma with occasional perpendicular branch points. Arrows represent nonoverlapping synaptophysin clusters; arrowheads point to synaptophysin-filled, eGFP-expressing synaptic boutons. The percentage of synaptophysin clusters free of exogenous αS expression was calculated. C, Whole-cell AMPAR mEPSCs were recorded from cultured rat hippocampal neurons transfected with eGFP alone or eGFP-fused αS species. Scale bar, 10 pA, 100 ms. D, Relative cumulative frequency of mEPSC amplitudes. Kolmogorov–Smirnov test, D = 0.39; ***p < 0.0001. E, F, Mean mEPSC amplitudes and frequencies. One-way ANOVA with Bonferroni post hoc analysis, F(4,32) = 3.65; *p = 0.012. All data are expressed as mean ± SEM.
Figure 5.
Figure 5.
Stable and consistent expression of eGFP-fused human αS across constructs. A, Contour plots of flow cytometry gating parameters from the nontransfected group. B, Contour plots of two populations of cells in the nontransfected group: eGFP-negative, living cells (Q4) and eGFP-negative, dead cells (Q3). CF, Contour plots of neurons transfected with eGFP-fused WT, A30P, E46K, and A53T mutant human αS respectively. A small population of cells emerged that is both living and eGFP-positive (Q1). G, Histogram comparison of fluorescence in eGFP cell population. H, Mean eGFP fluorescence intensity from flow cytometer detection. Data were analyzed by one-way ANOVA, F(3,3638) = 2.38. n-values are eGFP-positive events and shown parenthetically. There was no difference between the cellular distributions of αS variants. I, Deconvoluted example micrographs of an axon and a dendrite of a neuron expressing eGFP-WT αS. Scale bar, 10 μm. J, K, Fifteen-image Z-series of dendrites and axons were analyzed to estimate cellular distribution of αS using linear analysis perpendicular to the shaft. Total area under the curve of dendritic or axonal fluorescence in each image series was averaged and normalized to background fluorescence. One-way ANOVA, F(3,25) = 0.45 (dendrite), F(3,14) = 0.90 (axon); n > 4. All data are expressed as mean ± SEM.
Figure 6.
Figure 6.
A53T αS at two expression levels induces phosphorylation-dependent mislocalization of tau to dendritic spines. Neurons were cultured from TgNg, H5, and G2-3 hippocampi and transfected with DsRed to visualize cellular architecture and eGFP-fused human tau to visualize subcellular location of tau. A, Representative photomicrographs of cultured TgNg, G2-3, and H5 hippocampal neurons expressing WT tau, AP tau (phosphorylation-blocking), or E14 tau (phosphomimetic). Scale bar, 10 μm. B, Quantification of percentage of total dendritic spines containing tau. C, Spine density. For all, TgNg, n = 8; H5, n = 6; G2-3, n = 8; one-way ANOVA with Bonferroni post hoc analysis, F(6,47) = 1.52; ***p < 0.0001. All data are expressed as mean ± SEM.
Figure 7.
Figure 7.
A53T αS induces tau phosphorylation-dependent, cell-autonomous postsynaptic deficits. A, Representative traces of whole-cell mEPSCs recorded from cultured rat hippocampal neurons cotransfected with tau and αS variants. Scale bar, 5 pA, 100 ms. B, Relative cumulative frequency plot of mEPSC amplitude. C, D, Quantification of mean mEPSC amplitude and mEPSC frequency of cotransfected neurons. For all, n = 12; two-way ANOVA with Bonferroni post hoc analysis, F(1,44) = 4.86; **p = 0.0095, ***p < 0.001. All data are expressed as mean ± SEM.
Figure 8.
Figure 8.
GSK3β activation is required for tau mislocalization and synaptic deficits in A53T αS-expressing neurons. A, Representative photomicrographs from cultured TgNg and G2-3 neurons that were either untreated or treated with the GSK3β-specific inhibitor CHIR-99021 (CHIR). Scale bar, 5 μm. B, C, Quantification of spines containing tau and spine density. Two-way ANOVA with Bonferroni post hoc analysis, F(2,42) = 27.27. D, Representative mEPSC traces from untreated (top) and CHIR-treated neurons (bottom) expressing eGFP alone, eGFP-WT αS, and eGFP-A53T αS. Scale bar, 10 pA, 100 ms. E, Relative cumulative frequency of mEPSC amplitudes from neurons represented in D. Kolmogorov–Smirnov comparison with eGFP; D = 0.53 ***p < 0.0001. F, G, Quantification of mEPSC amplitude and frequency. Two-way ANOVA with Bonferroni post hoc analysis. F(2,66) = 3.214; *p = 0.041. For all, n = 12. All data are expressed as mean ± SEM.
Figure 9.
Figure 9.
GluA1 surface expression in dendritic spines is decreased by A53T αS expression in a GSK3β-dependent fashion. A, Photomicrographs of neurons from G2-3 mice and their TgNg littermates without (top two panels) and with treatment of CHIR-99021 (bottom two panels). As described previously (Liao et al., 1999), live neurons were stained for N-GluA1 antibodies (green), fixed, permeablized, and stained for PSD-95 (red). Arrows indicate tightly clustered surface N-GluA1 colocalized with PSD-95, whereas weak, nonspecific N-GluR1 immunoreactivity appeared along the dendritic shafts as diffuse staining rather than distinct clusters in G2-3 neurons (arrowheads). The diffuse staining is likely due to the presence of extrasynaptic AMPA receptors (Newpher and Ehlers, 2008). Treatment with CHIR-99021 restored surface N-GluA1 synaptic localization in G2-3 mice. Scale bar, 10 μm. B, GluA1 surface fluorescence in PSD-95 immunoreactive spines was normalized to dendritic fluorescence. Two-way ANOVA with Bonferroni post hoc analysis, F(1,28) = 5.69; **p = 0.0029. For all, n = 8. All data are expressed as mean ± SEM.
Figure 10.
Figure 10.
Calcineurin activation is required for tau- and A53T αS-induced postsynaptic deficits. A, Representative mEPSC traces recorded from cultured rat hippocampal neurons transfected with WT αS treated with DMSO vehicle or with A53T αS treated with DMSO vehicle or FK506. Scale bar, 10 pA, 100 ms. B, Relative cumulative frequency of mEPSC amplitudes from neurons represented in A. Kolmogorov–Smirnov comparison with vehicle-treated neurons expressing eGFP-WT αS, D = 0.43 ***p < 0.0001. C, D, Quantification of mEPSC amplitude and frequency. One-way ANOVA with Bonferroni post hoc analysis, F(4,41) = 3.84; **p = 0.0025. For all, n = 12. All data are expressed as mean ± SEM. E, Hypothetical pathways for αS-induced changes in neuronal transmission: In pathway #1, A53T αS induces mutation-specific, GSK3β-dependent phosphorylation of tau, leading to tau missorting to dendritic spines. Here, tau leads to calcineurin (CaN)-mediated endocytosis of GluA1-containing AMPA receptors, leading to postsynaptic deficits. However, we cannot rule out tau-mediated inhibition of AMPA receptor insertion into the synaptic membrane. In pathway #2, hyperexpression of WT or mutant αS (A53T, A30P) leads to presynaptic release suppression through an unknown mechanism regardless of genotypes. The differential effects of αS on these two separate pathways may contribute to PD heterogeneity.

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