Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex

Abstract

Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP downregulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this data set identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating.

Figure 1

A) Schematic of the experimental design. Unlabeled ¹⁴N rats were injected with saline or PCP then either underwent PPI analysis or mock PPI analysis. The brains of the ¹⁴N rats were dissected then mixed with ¹⁵N rat brain. The ¹⁴N/¹⁵N mixtures were digested and then phosphopeptides were enriched for analysis. B) PCP (1.25 mg/kg) significantly disrupted PPI and non-significantly increased startle magnitude (inset). * main effect of drug on PPI, [F(1,12)=14.6, p<0.01].C) The total number of phosphopeptides (y-axis) identified and quantified. X-axis represents each rat in the study. D) The percentage of phosphorylation sites per quantified peptide.

Figure 2

A) The distribution of the functions of the quantified phosphoproteins. B) Distribution of kinases. The red bars represents the percentage of kinases family members quantified by phosphopeptides and the black bars represent the percentage of each genes in each kinase family of the known 514 kinases. For example, AGC family represents 12% of all the known kinases but represents 24% of all the kinases quantified by phosphopeptides in this study. C) Two examples of multistate phosphorylation in the dataset. The peptide NQSDLDDQHDYDSVASDEDTDQEPLPSAGATR from GIT1 (red) was quantified over 100 times with one phosphorylation event, but much less with two or three phosphorylation events. The peptide GEGAQDEEEGGASSDATEGHDEDDEIYEGEYQGIPR from SVA2 (blue) was rarely quantified with one phosphorylation event, but more often was quantified with two and three phosphorylation events. D) The peptide ASGQAFELILpSPR from stathmin was significantly increased upon PCP treatment. * p value < 0.05

Figure 3

Phosphoproteins down-regulated in PCP and PCPPPI compared to Sal and SalPPI, respectively, which were significantly enriched in LTP signaling pathway. The blue phosphoproteins were down-regulated in PCPPPI, the red phosphoproteins were down-regulated in PCP, and the green phosphoproteins were down-regulated in both PCP and PCPPPI rats. Uncolored proteins were not quantified in our study. The solid lines represent direct relationship have been reported and the dashed lines represent relationships inferred from the literature based on the relationships of proteins from the same family.

Figure 4

A) The peptide ADSpFSEGDDLSQGHLAEPCFLR of Begain was significantly down-regulated after PCP treatment. B) The peptide LPpSVGDQEPPGHEK of xCT was significantly up-regulated after PCPPPI. The same trend was trend observed for the PCP and Sal comparison but not enough measurements were collected for statistical analysis. C) Abolishing the phosphorylation site in B) decreases ³H-glutamate uptake through xCT in HT22 cells. Cells were transfected with WT, D26, or A26 xCT cDNA and glutamate uptake assays was performed. The data represents three independent experiments. The y-axis is the ³H-glutamate measurements of D26 and A26 as a percentage of the WT xCT glutamate uptake measurements. D) Phosphopeptides significantly altered in the PFC between rats that underwent PPI or mock PPI (control). Paralemmin (SETMVNAQQpTPLGpTPK), myosin-11(VIENTDGpSEEEMDAR), JunD (LAALKDEPQTVPDVPSFGDpSPPLpSPIDMDTQER). * p value < 0.05, ** p value< 0.01.