Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation

Abstract

Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl), both with regard to localization and function: Cdc42-palm concentrates in dendritic spines and has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.

Figure 1. Global analysis of neuronal protein palmitoylation

a, ABE purification of palmitoyl-proteins (PPs) from cultured rat cortical embryonic neurons. Proteins purified by parallel ABE protocols, with (+) or without (-) hydroxylamine (HA) were subjected to SDS-PAGE and silver-staining. Hashmarks at left mark protein species common to both + and −HA samples, while those at right indicate proteins whose purification is HA-dependent (i.e. presumptive PPs). b, ABE/MuDPIT analysis. The 1643 different proteins identified from MuDPIT analyses of four paired + and −HA samples are each plotted by their associated averaged +HA (x-coordinate) and −HA (y-coordinate) spectral counts. The 58 proteins that were known to be palmitoylated prior to this analysis are shown as red dots. New candidate PPs co-cluster along the x-axis (region indicated) with the known PPs. c, Overlapping identification of known PPs, by the neuronal and synaptosomal proteomic analyses. d, Summary of palmitoylation testing. Results are summarized for the 21 candidate proteins that were individually tested for palmitoylation by either [³H]-palmitate metabolic labeling or by ABE methodologies (see Suppl. Fig. 2). e, Verification of palmitoylation for selected PP candidates. Proteins, ABE-purified from cultured neurons exactly as for proteomic analysis, both in the presence (+) and absence(-) of HA, were analyzed by Western blotting using the indicated specific antibodies. Palmitoylated proteins are expected to show HA-dependent detection. As a control, a portion of the starting protein sample (prior to ABE purification) also was screened (total).

Figure 2. Palmitylation of a brain-specific Cdc42 splice variant

a, C-terminal amino acid sequences of the two alternatively-spliced Cdc42 isoforms. Isoform-1 is the previously characterized prenylated form of Cdc42 (Cdc42-prenyl), while isoform-2 is variant form, shown in panel b to be palmitoylated (Cdc42-palm). The C2S mutation removes the two putative, palmitoyl-accepting cysteines from Cdc42-palm. b, Cdc42 isoform-2 (Cdc42-palm) is palmitoylated. COS-7 cells transfected by the indicated GFP-Cdc42 constructs were metabolically labeled with [³H]-palmitic acid. GFP-Cdc42 proteins were immunoprecipitated then subjected either to autoradiography to assess palmitate incorporation or Western analysis. c, Cdc42-palm expression is limited to the brain. Tissue specific expression of the two Cdc42 isoforms was analyzed by RT-PCR. RNA was analyzed from the indicated organs, brain regions [cerebellum (CB); hippocampus (HIP); cortex (CX)], as well as from developing cultured embryonic cortical neurons.

Figure 3. Cdc42-palm role in dendritic spine induction

a, Different dendritic localizations for Cdc42-palm and Cdc42-prenyl. Hippocampal neurons transfected with the indicated GFP-Cdc42 constructs on DIV 7 were analyzed on DIV 14 by anti-GFP immunofluorescent analysis. Relative distribution to dendritic shaft versus spine was analyzed with the Spine Targeting Index (STI; see Suppl. Methods). A STI of 1 indicates passive distribution; a STI of >1 indicates accumulation of the GFP-Cdc42 construct within spines. (n=10 cells). b, Differential spine induction activity for Cdc42-palm and Cdc42-prenyl. Constitutively-active (CA; G12V mutation) versions of the GFP-Cdc42 constructs were co-transfected with a DsRED expression plasmid into hippocampal neurons on DIV 7 with spine density being assessed on DIV 14. Parallel cultures were treated with 100 μM 2BP treatment for 5 h on DIV 14 to assess effects of palmitoylation inhibition. Spine numbers per 100 μm dendritc length are reported (n=14-24 cells). The inhibition of spine induction by Cdc42(CA)-C2S relative to the vector control is significant, suggesting a dominant-negative action for this mislocalized mutant. c, Cdc42-palm isoform is required for spine development. pSUPER/GFP-based siRNA expression plasmids, targeting sequences specific to either the Cdc42-prenyl or Cdc42-palm mRNAs, were transfected into hippocampal neurons on DIV 9, with spine densities assessed on DIV 14. Results for six different knockdown constructs are reported: a prenyl siRNA construct, targeting the Cdc42-prenyl isoform (41 cells analyzed); two different palm siRNA constructs (#1 and #2, 25 and 10 cells analyzed, respectively), targeting the Cdc42-palm isoform; a pan siRNA construct, targeting a sequence common to both isoforms (12 cells); a scrambled siRNA, a scrambling of a pan siRNA target sequence (31 cells); empty pSUPER/GFP vector (56 cells). Spine numbers per 100 μm dendritc length are reported. COS-7 cell testing of knockdown efficacy showed that the four knockdown constructs reduced expression of their target isoform by 65-70% (Suppl. Fig. 6). Statistical significance levels for panel a-c quantitative analysis: * P<0.05, ** P<0.01, *** P<0.001, scale bar, 5 μm. All error bars are mean ± s.e.m.

Fig. 4. Broad modulation of palmitoylation levels in neuronal activity paradigms

ABE/Western analysis was used to follow palmitoylation changes within selected panels of neuronal PPs in response to three treatment regimens: a,b, 5-h treatment of cortical neurons with 100 μM 2BP to assess constitutive palmitoyl-turnover; c, 5-min treatment of cortical neurons with a 50 μM glutamate excitatory stimulus; d, following kainic acid-induced seizures (10mg/kg kainic acid injected intraperitoneally with brain harvested within 30 min of the onset of seizure activity). a, Example Western blots are shown for the 2BP treatment regimen. Total PPs, ABE-purified from the 2BP-treated and parallel, untreated neuronal cultures were blotted with the indicated specific antibodies. To control for 2BP effects on test protein expression levels, the initial, unpurified protein extracts also were blotted (total). Western blot data for glutamate- and seizure-induced changes is provided in Suppl. Fig. 10. b-d, Quantification of the palmitoylation changes induced by the three treatment protocols. Protein levels measured from the purified PP samples (Palm) were normalized to levels measured from the corresponding unpurified extracts (Total). (See Suppl. Methods for the details.) Data is presented from three independent experiments as means ± s.e.m.. Significance levels are * P<0.05,** P<0.01,*** P<0.001. Note, some antibodies detect multiple paralogues and/or isoforms: the Cdc42 antibody recognizes both Cdc42-palm and Cdc42-prenyl isoforms; the Ras antibody recognizes H-, N-, and K-Ras (only H- and N-Ras are palmitoylated); the Rho A/B antibody recognizes both Rho A and Rho B (only Rho B is palmitoylated).