Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse

Abstract

Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKgamma, are major unrecognized components of this synapse type. We demonstrate that MRCKgamma can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.

Figure 1. Tagging the Parallel Fiber/Purkinje Cell Synapse in Transgenic Mice

(A) Southern blot was used to identify transgenic mice having integrated the Pcp2 BAC (a Purkinje cell–specific driver) containing the Venus-tagged GluRδ2 receptor, VGluRδ2. Tg, transgenic; Wt, wild type. (B) VGluRδ2 expression was detected using an anti-GFP antibody on immunoblots from total protein extracts of transgenic (Tg) versus wild-type (Wt) cerebella. An asterisk (*) indicates a nonspecific band. (C) Both VGluRδ2 and GFP were affinity purified using an anti-GFP antibody from 1% Triton X-100 cerebellar extracts from wild-type (Wt), Pcp2/VGluRδ2 (Vδ2), and Pcp2/eGFP control (GFP) mice, as shown by probing the immunoblots with an anti-GFP antibody (left). VGluRδ2 specifically copurified the endogenous GluRδ2, as shown by probing the same blot with an anti-GluRδ2 antibody (right). (D) Immunofluorescence on cerebellar sections using an anti-GFP antibody shows the specific localization of VGluRδ2 in the molecular layer and somata of Purkinje cells of Pcp2/VGluRδ2 mice. Soluble GFP is detected in the molecular layer, dendrites, somata, and axons of Purkinje cells in sections from Pcp2/eGFP mice. Scale bars in the upper panels indicate 200 μm; lower panels, 50 μm.

Figure 2. VGluRδ2 Is Detected in Excitatory Synaptic Fractions Using a New Purification Method

(A) We prepared a crude synaptosome P2 fraction that was solubilized in 0.5% Triton X-100 final concentration. The extract was then separated on a Sephacryl S1000 gel filtration column. Calibration of the column indicated that protein complexes smaller than 669 kDa (arrow in [B]) were resolved after fraction 10. (B) Protein dosage was performed on every fraction collected. (C) Each fraction (0.1% in volume) was run on western blots and assayed for the presence of excitatory postsynaptic markers (GluRδ2, GLUR2, PSD95, and NR2A), presynaptic markers (synapsin I and synaptotagmin), inhibitory synapse markers (GABA(A)Rβ and GABA(A)Rα1), the endoplasmic reticulum marker BiP, and the mitochondrial marker COX. VGluRδ2 was detected using an anti-GFP antibody. The red rectangle outlines the “excitatory synaptic” fractions enriched for synaptic markers and pooled for subsequent affinity-purification of PF/PC PSDs.

Figure 3. Affinity Purification and Protein Profiling of the Parallel Fiber/Purkinje Cell PSDs

(A) Synaptic fractions from Pcp2/VGluRδ2 animals were affinity purified using magnetic beads coated with anti-GFP antibody (VGluRδ2). In parallel, control purifications were performed on preparations from Pcp2/eGFP transgenic mice (GFP); 0.025% of the inputs and flow-throughs (FT), and 25% of the affinity-purified samples (IP) were assayed by western blot using an anti-GFP antibody and showed immunoprecipitation of both VGluRδ2 and GFP, respectively. (B) The same blot was probed for different postsynaptic markers, presynaptic markers (synapsin I and synaptotagmin), the mitochondrial protein COX, and the ER marker BiP, showing specific copurification of postsynaptic markers localized to the PF/PC synapse. (C) Electron microscopy shows the presence of electron-dense structures reminiscent of PSDs on the surface of the magnetic beads used for affinity purification of Pcp2/VGluRδ2 extracts. (D) Proteins from the affinity-purified VGluRδ2 PSDs were separated by SDS-PAGE electrophoresis and stained with Coomassie Blue before MS analysis. (E) MS identified 65 different proteins in the complexes purified from Pcp2/VGluRδ2 mice. These proteins can be classified into 11 functional categories. The number of proteins from each category is indicated in parentheses. Nonshaded areas represent proteins found with high confidence.

Figure 4. Localization of Several Candidate Synaptic Proteins at the Parallel Fiber/Purkinje Cell Synapses

(A) Presence of selected candidates in PF/PC PSDs purified from Pcp2/VGluRδ2 cerebella; 0.025% of the inputs (IN) and flow-throughs (FT), and 25% of the affinity-purified samples (IP) obtained from Pcp2/eGFP control (GFP) and Pcp2/ VGluRδ2 (VGluRδ2) cerebella were assayed by western blot. (B) Localization by immunofluorescence of candidate synaptic proteins. Labeling was performed using antibodies recognizing several candidate proteins identified by MS (green) in conjunction with an anti-calbindin antibody specifically labeling Purkinje cells (red). Scale bars indicate 50 μm.

Figure 5. MRCKγ Modulates Dendritic Spine Morphogenesis and Is Localized to Parallel Fiber/Purkinje Cell Spines

(A) Primary cortical cultures were imaged at DIV14 after transfection with either GFP alone or in conjugation with MRCKγ lacking the kinase domain (DN) or the full-length MRCKγ kinase (FL) (left panel, scale bar indicates 10 μm). Mean protrusion density was not significantly different between the three conditions (middle), but significant changes in mean protrusion length were observed (right panel, asterisk [*] indicates p < 0.05 compared to GFP). (B) Immunofluorescence labeling shows the localization of MRCKγ (green) in Purkinje cell spines (calbindin, red) and its colocalization with VgluT1, a marker of parallel fiber synapses, but not with VgluT2 or GAD65/67, markers of climbing fiber or of inhibitory synapses respectively. Scale bars indicate 5 μm.