The Journal of Neuroscience, May 1, 2000, 20(9):3214–3220 Synucleins Are Developmentally Expressed, and ␣-Synuclein Regulates the Size of the Presynaptic Vesicular Pool in Primary Hippocampal Neurons Diane D. Murphy, Susan M. Rueter, John Q. Trojanowski, and Virginia M.-Y. Lee Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104 ␣-, ␤-, and ␥-Synuclein, a novel family of neuronal proteins, has in the presynaptic terminal, whereas little ␥-synuclein was ex- become the focus of research interest because ␣-synuclein has pressed at all. To assess the function of ␣-synuclein, we sup- been increasingly implicated in the pathogenesis of Parkinson’s pressed expression of this protein with antisense oligonucleo- and Alzheimer’s disease. However, the normal functions of the tide technology. Morphometric ultrastructural analysis of the synucleins are still unknown. For this reason, we characterized ␣-synuclein antisense oligonucleotide-treated cultures revealed ␣-, ␤-, and ␥-synuclein expression in primary hippocampal a significant reduction in the distal pool of synaptic vesicles. neuronal cultures and showed that the onset of ␣- and These data suggest that one function of ␣-synuclein may be to ␤-synuclein expression was delayed after synaptic develop- regulate the size of distinct pools of synaptic vesicles in mature ment, suggesting that these synucleins may not be essential for neurons. synapse formation. In mature cultured primary neurons, ␣- and Key words: Lewy bodies; Parkinson’s disease; primary neu- ␤-synuclein colocalized almost exclusively with synaptophysin rons; synapse; synuclein; vesicle ␣-Synuclein was first isolated in 1988 from synaptic vesicles of sin during CNS development, it has been suggested to play a role Torpedo californica and rat brain (Maroteaux et al., 1988). Sub- in synaptogenesis (Hsu et al., 1998). However, in vitro studies sequently, other members of this family of neuronal proteins have show that the vesicle-associated protein synapsin I appears as been identified: ␤-synuclein, also found mainly in the CNS, early as 24 hr, whereas ␣-synuclein is not detected until day 5 ␥-synuclein, more abundant in the PNS than in the CNS, and (Withers et al., 1997). Thus, it is unclear whether ␣-synuclein is synoretin, present primarily in the retina (Nakajo et al., 1990; needed for the development of synapses or for the maturation and Jakes et al., 1994; Ji et al., 1997; Buchman et al., 1998; Surguchov modulation of previously existing ones. There is also evidence et al., 1999). ␣-Synuclein has been shown to be a primary com- that ␣-synuclein may associate with synaptic vesicles because ponent of Lewy bodies, the neuropathological hallmarks of spo- ␣-synuclein has been detected in vesicular fractions of human radic Parkinson’s disease (PD) (Spillantini et al., 1997; Baba et brain (Irizarry et al., 1996) and binds to both synthetic mem- al., 1998), and pathogenic ␣-synuclein gene mutations have been branes (Davidson et al., 1998) and vesicles isolated from rat brain identified in familial forms of PD (Polymeropoulos et al., 1997; (Jensen et al., 1998). Kruger et al., 1998). Moreover, ␣-synuclein has been detected in The present study assessed ␣-, ␤-, and ␥-synuclein expression in Lewy bodies that are diagnostic of the Lewy body variant of primary hippocampal neurons grown at a substantially increased Alzheimer’s Disease (LBVAD) as well as dementia with Lewy density. These high-density cultures exhibit a robust, rapid devel- bodies (DLB) (Spillantini et al., 1997; Baba et al., 1998; Tro- opment of synaptic contacts and spines (Papa et al., 1995) that janowski et al., 1998). Thus, insight into mechanisms of synuclein corresponds to those seen during postnatal hippocampal devel- pathology may be critical for understanding brain degeneration in opment. Moreover, the synaptic ultrastructure of high-density PD, LBVAD, and DLB (Trojanowski et al., 1998). cultures resembles that found in situ (Bartlett and Banker, 1984). The implication of ␣-synuclein in neurodegenerative disease We used light microscopic immunocytochemistry, electron mi- has stimulated efforts to elucidate the normal distribution and croscopy, and protein biochemistry to characterize the expression functions of ␣-, ␤-, and ␥-synuclein. For example, because of the synuclein family of proteins in hippocampal neurons up to ␣-synuclein is localized to presynaptic terminals throughout the 3 weeks in culture. We also examined changes in synaptic struc- adult mammalian brain and may appear earlier than synaptophy- ture after treatment of the neurons with antisense (AS) oligonu- cleotides to reduce ␣-synuclein expression. Here, we report that Received Nov. 11, 1999; revised Feb. 14, 2000; accepted Feb. 24, 2000. ␣- and ␤-synuclein were expressed after synaptophysin and local- This work was supported by grants from the National Institute on Aging. We acknowledge Drs. N. B. Cole, R.W. Doms, and V. Zhukareva for scientific contri- ized almost exclusively to presynaptic terminals of mature neu- butions, Dr. R. Balice-Gordon for use of the confocal microscope, and N. Shah and rons. In addition, downregulation of ␣-synuclein by AS oligonu- J. Sanzo of the electron microscopy facility for EM preparations and use of the cleotides caused a selective reduction in the size of the electron microscope. ␣ D.M. and S.R. contributed equally to this work. presynaptic vesicular pool. Our data suggest that -synuclein may Correspondence should be addressed to Dr. Virginia M.-Y. Lee, Department of interact with and regulate specific pools of synaptic vesicles, Pathology and Laboratory Medicine, University of Pennsylvania School of Medi- cine, Maloney 3, HUP, 3600 Spruce Street, Philadelphia, PA 19104-4283. E-mail: thereby modulating synaptic functions in the normal brain. These [email protected]. findings have important implications for the role ␣-synuclein may Copyright © 2000 Society for Neuroscience 0270-6474/00/203214-07$15.00/0 play in neurodegenerative disease. Murphy et al. • ␣-Synuclein Regulates Vesicular Pool J. Neurosci., May 1, 2000, 20(9):3214–3220 3215 Figure 1. Synuclein characterization by immunofluorescence. 1 (a)-,2(b)-, and 3 (c)-week-old cultures stained for ␣-synuclein (red) and synaptophysin ( green). Cytosolic ␣-synuclein staining is evident in a. d, Double labeling of ␣-synuclein ( green) and GAD (red) show colocalization in an inhibitory neuron and in GABAergic presynaptic terminals ( yellow). e, ␣- and ␤-synuclein are colocalized ( yellow) to the presynaptic terminal. f, ␥-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 ␮m. MATERIALS AND METHODS 0.1% Triton X-100, 0.5 mM PMSF, and 1 mM DTT, protease inhibitor ϫ Hippocampal cultures. High-density hippocampal neuronal cultures were cocktail). Harvested cells were spun at 100,000 g in a TL100 ultracen- trifuge (Beckman). Protein concentration in the extracts was determined prepared as previously described (Papa et al., 1995). Briefly, 20-d-old ␮ embryos were taken from anesthetized Sprague Dawley rats. The brains by the Bradford assay. Approximately 30 g of the supernatant was were removed and placed in ice-cold (4°C) L-15 medium supplemented fractionated by SDS-PAGE and transferred onto nitrocellulose. The ␮ blots were incubated in blocking solution containing 5% nonfat dry milk with 0.6% glucose and 15 g/ml gentamicin. The hippocampus was ϫ dissected and mechanically disaggregated by gentle trituration using a in 1 Tris-buffered saline (TBS) and then incubated overnight at 4°C Pasteur pipette. Dissociated cells (500,000 cells/well) were plated onto with the various primary antibodies described above diluted in 5% sterile 12 mm glass or Thermanox coverslips that were coated with milk/TBS. As a control, blots were also stained for neuron-specific ␮ ␮ enolase (NSE; Polysciences, Warrington, PA) to normalize the amount collagen (50 g/ml) and poly-L-lysine (15 g/ml). Cells were also pre- ϩ pared on six-well tissue culture dishes for Western blot analysis. Ther- of neuronal protein. The blots were washed in TBS-T (TBS 0.1% Tween 20) and incubated for 2 hr at room temperature with secondary manox coverslips were used for electron microscopy (see below). The 125 plating medium was Eagle’s MEM containing 5% heat-inactivated horse antibodies [ I] conjugated to either anti-mouse IgG or Protein A at a ␮ serum, 5% fetal calf serum, 2 mM glutamine, 0.6% glucose, and 15 ␮g/ml concentration of 1 Ci/ml of PTX buffer (10 mM sodium phosphate, pH 7.3, 1 mM EDTA, 150 mM NaCl, 0.2% Triton X-100, and 4% BSA). The gentamicin. Cells were incubated at 37°C with 5% CO2. The first change of medium, ϳ4–6 d after plating, included 50 ␮g/ml uridine and 20 blots were washed again with TBS-T and exposed in a PhosphorImager ␮g/ml deoxyuridine to prevent glial cell overgrowth. The cultures were cassette (Molecular Dynamics, Sunnyvale, CA) for 24–48 hr. The pro- fed thereafter 1–2 times a week, with Eagle’s MEM as above. tein levels were quantified using ImageQuant software (Molecular Dy- Immunocytochemistry. Cells were fixed in 4% paraformaldehyde in namics), each lane normalized to NSE levels. Ј PBS. After blocking and permeabilization in 5% goat serum with 0.1% Antisense treatments. Oligonucleotides were prepared from the 5 end ␣ saponin, cells were incubated with primary antibodies to the following: