Synucleins are developmentally expressed, and alpha-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons

Abstract

alpha-, beta-, and gamma-Synuclein, a novel family of neuronal proteins, has become the focus of research interest because alpha-synuclein has been increasingly implicated in the pathogenesis of Parkinson's and Alzheimer's disease. However, the normal functions of the synucleins are still unknown. For this reason, we characterized alpha-, beta-, and gamma-synuclein expression in primary hippocampal neuronal cultures and showed that the onset of alpha- and beta-synuclein expression was delayed after synaptic development, suggesting that these synucleins may not be essential for synapse formation. In mature cultured primary neurons, alpha- and beta-synuclein colocalized almost exclusively with synaptophysin in the presynaptic terminal, whereas little gamma-synuclein was expressed at all. To assess the function of alpha-synuclein, we suppressed expression of this protein with antisense oligonucleotide technology. Morphometric ultrastructural analysis of the alpha-synuclein antisense oligonucleotide-treated cultures revealed a significant reduction in the distal pool of synaptic vesicles. These data suggest that one function of alpha-synuclein may be to regulate the size of distinct pools of synaptic vesicles in mature neurons.

Fig. 1.

Synuclein characterization by immunofluorescence. 1 (a)-, 2 (b)-, and 3 (c)-week-old cultures stained for α-synuclein (red) and synaptophysin (green). Cytosolic α-synuclein staining is evident in a.d, Double labeling of α-synuclein (green) and GAD (red) show colocalization in an inhibitory neuron and in GABAergic presynaptic terminals (yellow). e, α- and β-synuclein are colocalized (yellow) to the presynaptic terminal. f, γ-synuclein (red) is poorly expressed in mature hippocampal neurons as compared to synaptophysin (green). The nuclei in a–f are labeled by Hoechst (blue) dye. Scale bar, 10 μm.

Fig. 2.

Synuclein protein expression in cultured hippocampal neurons. α- and β-synuclein expression in neurons at 1, 2, 3 weeks in culture was determined by quantitative Western blot analysis from Triton X-100 soluble cell extracts (a). The blots were probed with antibody syn102 (specific for both α- and β-synucleins) and an antibody to NSE.b, Quantitation of α- and β-synuclein expression at 1, 2, and 3 weeks in culture. Closed bars represent α-synuclein expression, and open bars represent β-synuclein expression. All synuclein signals were normalized to the signal for NSE to account for the amount of neuronal protein loaded per lane. Results are presented as the average percentage of expression ± SEM. The expression at 1 week was arbitrarily set at 100%. n = 3; *p < 0.05.

Fig. 3.

A specific AS oligonucleotide decreases α-synuclein expression. Cultured hippocampal neurons were either not treated (a), or treated with AS oligonucleotide (b) or with S oligonucleotide (c) for 6 d and immunostained with antibody SNL-1 (specific for α-synuclein). Scale bar, 10 μm.

Fig. 4.

AS oligonucleotide treatment effectively decreases α-synuclein protein expression. a, Quantitative Western blot analysis of α-synuclein and NSE levels in control (C), antisense (AS)-treated, and sense (S)-treated cultures for 6 d.b, Quantitation of α- and β-synuclein levels in AS-treated cultures compared to control (untreated) cells. No differences were found between control and sense oligonucleotide-treated cultures. The closed barrepresents α-synuclein expression, and the open barrepresents β-synuclein expression. Results are presented as the average percentage of expression of control cells ± SEM.n = 3; *p < 0.05.

Fig. 5.

AS treatment results in an overall decrease of several presynaptic proteins. Left panels (a, c, e, g) are control cultures, right panels(b, d, f, h) are AS oligonucleotide-treated cultures. Synapsin I (a, b), synaptophysin (c, d), VAMP (e, f), and syntaxin (g, h). Although diminished fluorescent puncta were observed inb and d, no changes in the localization of these presynaptic proteins were detected. Scale bar, 10 μm.

Fig. 6.

Effect of α-synuclein AS oligonucleotide treatment on the expression of synaptic proteins. Quantitative Western blot analysis of synaptophysin, VAMP, syntaxin, and synapsin I levels in AS-treated cultures. All protein signals were normalized to the signal for NSE to account for the amount of neuronal protein loaded per lane. The open bar, dotted bar, gray bar, andblack bar represent synaptophysin (SP) expression, VAMP expression, syntaxin expression, and synapsin I expression, respectively. Results are presented as the average percentage of expression of control cells ± SEM.n = 3; *p < 0.05.

Fig. 7.

α-Synuclein AS oligonucleotide treatment decreases the synaptic vesicle pool at the presynapse. Left panels (a, c, e) are representative images of synapses from control hippocampal neurons. Right panels(b, d, f) are images of representative synapses from antisense oligonucleotide-treated hippocampal neurons. Note that synapses from both groups have a similar number of docked vesicles and possess well-defined postsynaptic densities. However, vesicles distal to the docking region are depleted in the AS oligonucleotide-treated cells (compare a, c, e with b, d, f). Scale bar, 200 nm.