Alterations in corticostriatal synaptic plasticity in mice overexpressing human alpha-synuclein

Abstract

Most forms of Parkinson's disease (PD) are sporadic in nature, but some have genetic causes as first described for the alpha-synuclein gene. The alpha-synuclein protein also accumulates as insoluble aggregates in Lewy bodies in sporadic PD as well as in most inherited forms of PD. The focus of the present study is the modulation of synaptic plasticity in the corticostriatal pathway of transgenic (Tg) mice that overexpress the human alpha-synuclein protein throughout the brain (ASOTg). Paired-pulse facilitation was detected in vitro by activation of corticostriatal afferents in ASOTg mice, consistent with a presynaptic effect of elevated human alpha-synuclein. However basal synaptic transmission was unchanged in ASOTg, suggesting that human alpha-synuclein could impact paired-pulse facilitation via a presynaptic mechanism not directly related to the probability of neurotransmitter release. Mice lacking alpha-synuclein or those expressing normal and A53T human alpha-synuclein in tyrosine hydroxylase-containing neurons showed, instead, paired-pulse depression. High-frequency stimulation induced a presynaptic form of long-term depression solely in ASOTg striatum. A presynaptic, N-methyl-d-aspartate receptor-independent form of chemical long-term potentiation induced by forskolin (FSK) was enhanced in ASOTg striatum, while FSK-induced cAMP levels were reduced in ASOTg synaptoneurosome fractions. Overall the results suggest that elevated human alpha-synuclein alters presynaptic plasticity in the corticostriatal pathway, possibly reflecting a reduction in glutamate at corticostriatal synapses by modulation of adenylyl cyclase signaling pathways. ASOTg mice may recapitulate an early stage in PD during which overexpressed alpha-synuclein dampens corticostriatal synaptic transmission and reduces movement.

Figure 1. Basal synaptic transmission is unchanged in corticostriatal slices from ASOTg mice and is mainly glutamatergic

A. Western immunoblotting confirmed elevated expression of human α-synuclein in ASOTg forebrain. Density values (mean ± SEM) for α-synuclein were normalized to β-actin in the same lane (N = 4 each, Student t-test, P < 0.05). B. To generate input-output relationships, the amplitudes of field potentials (-mV) were measured in response to a series of increasing intensities in slices. To standardize responses, the % maximum response (where 100% occurred at the highest stimulus intensity) was plotted against stimulus intensity. No significant differences were evident between WT and ASOTg responses (P = 0.588). C. Basal synaptic transmission recorded at 50% maximum stimulus intensity was significantly reduced in both WT and ASOTg slices treated with CNQX (10 μM, 5 min) to inhibit AMPA/kainate glutamate receptors (* P = 0.02). There was no difference in % change in responses for CNQX-treated WT relative to CNQX-treated ASOTg slices (P = 0.41). Responses shown are averaged synaptic field potentials recorded in WT and ASOTg slices either untreated or treated with CNQX (dashed line).

Figure 2. Paired-pulse facilitation is detected in ASOTg striatum

A. PPRs > 1.0 were detected in ASOTg striatum at 25, 50 and 100 msec interstimulus intervals and were significantly different from WT (WT, N = 16 mice; ASOTg, N = 20 mice, Two-Way ANOVA, * P = 0.001). The PPR data demonstrate paired-pulse facilitation in the corticostriatal pathway of ASOTg mice, consistent with a presynaptic effect of over-expressed human α-synuclein. Responses shown are averaged 1^st and 2^nd field potentials recorded at a 50 msec interstimulus interval.

Figure 3. Paired-pulse facilitation is not detected in striatum from Snca^-/- mice

A. Western immunoblotting confirmed loss of expression of endogenous mouse α-synuclein protein in Snca^-/- olfactory bulb compared to Snca^+/+ non-Tg control (N = 3 each, P< 0.05). B. The PPR recorded in corticostriatal slices from Snca^-/- mice was not different from Snca^+/+ controls and did not show paired-pulse facilitation (Snca^+/+, N = 4; Snca^-/-, N = 4,Two-Way ANOVA, P = 0.179). Responses shown are averaged 1^st and 2^nd field potentials recorded at a 50 msec interstimulus interval.

Figure 4. Paired-pulse depression occurs in striatum of THwt Tg mice

A. Westerns blots show similar levels of normal and A53T human α-synuclein in TH-containing olfactory bulb from THwt and THmutA53T Tg mice (P = 0.33). As positive control, human α-synuclein is detected at elevated levels in ASOTg relative to WT olfactory bulb (P = 0.007) resolved on the same blot as the TH Tg samples. B. The PPRs recorded in corticostriatal slices from THwt Tg mice revealed paired-pulse depression (PPR < 1.0) at all interstimulus intervals but was not different from THmutA53T Tg (N = 4 each, Two-Way ANOVA, P = 0.619). Responses shown are averaged 1^st and 2^nd field potentials recorded at a 50 msec interstimulus interval.

Figure 5. HFS-LTD is detected in ASOTg striatum

A. HFS induced significant LTD in ASOTg (* P =0.004) but not in WT striatum (P = 0.13). Responses shown are averaged synaptic field potentials recorded pre-HFS (5 min) and post-HFS (25-30 min). B. Paired-pulse facilitation increases in WT and ASOTg slices after HFS induction of LTD. A 2nd field potential response was recorded at a 50 msec interstimulus interval to obtain a PPR before and after HFS induction. The PPR remained above 1.0 pre-HFS and increased significantly post-HFS, indicative of alterations in paired-pulse facilitation when ASOTg LTD was induced (*P = 0.001).

Figure 6. FSK-induced chemLTP is enhanced in ASOTg striatum

A Slices were perfused with FSK (50 μM) for 10 min to induce chemLTP followed by wash-out with ACSF for 60 min. ChemLTP is enhanced in ASOTg relative to WT striatum (* P = 0.05). Responses shown are averaged synaptic field potentials recorded pre-FSK (5 min) and post-FSK (55-60 min) treatment. Calibrations (0.5mV/5 msec) are shown for each set of traces. B. A 2nd field potential response was recorded at a 50 msec interstimulus interval to obtain PPRs during the time course of chemLTP. The PPR drops significantly below 1.0 after FSK induction of chemLTP only in ASOTg striatum, indicative of paired-pulse depression and presynaptic maintenance of LTP (* P = 0.006). C. Corticostriatal chemLTP is NMDA receptor-independent based on insensitivity to APV post-FSK treatment (25-30 min) but is not different from control (P = 0.51). D. Basal and FSK-induced accumulation of cAMP were measured in SN membrane-enriched subcellular fractions prepared from WT and ASOTg forebrain. The cAMP levels (picomole/μg SN protein) were significantly lower in ASOTg compared to WT SN groups (Two-Way ANOVA, P = 0.048). Pair-wise comparisons showed that FSK-induced cAMP levels in WT SNs to a greater degree than in ASOTg SNs (* Bonferoni t-test, P = 0.015)