NMDA receptor hypofunction induces dysfunctions of energy metabolism and semaphorin signaling in rats: a synaptic proteome study

Abstract

There is considerable evidence to suggest that aberrations of synapse connectivity contribute to the pathophysiology of schizophrenia and that N-methyl-D-aspartate (NMDA) receptor-mediated glutamate transmission is especially important. Administration of MK-801 ([+]-5-methyl-10, 11-dihydro-5H-dibenzo-[a, d]-cycloheptene-5, 10-iminehydrogenmaleate) induces hypofunction of NMDA receptors in rats, which are widely used as a model for schizophrenia. We investigated synaptosomal proteome expression profiling of the cerebral cortex of MK-801-treated Sprague-Dawley rats using the 2-dimensional difference gel electrophoresis method, and 49 differentially expression proteins were successfully identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight/Time-of-Flight mass spectrometry. We carried out a literature search for further confirmation of subsynaptic locations and to explore the relevance to the diseases of differentially expressed proteins. Ingenuity Pathways Analysis (IPA) was used to further examine the underlying relationship between the changed proteins. The network encompassing "cell morphology, cell-to-cell signaling and interaction, nervous system development and function" was found to be significantly altered in the MK-801-treated rats. "Energy metabolism" and "semaphorin signaling in neurons" are the most significant IPA canonical pathways to be affected by MK-801 treatment. Using western blots, we confirmed the differential expression of Camk2a, Crmp2, Crmp5, Dnm1, and Ndufs3 in both synaptosome proteins and total proteins in the cerebral cortex of the rats. Our study identified the change and/or response of the central nervous transmission system under the stress of NMDA hypofunction, underlining the importance of the synaptic function in schizophrenia.

Fig. 1.

Enrichment Profile of 4 Representative Proteins Monitored by Western Blot. H1, H2: cerebral cortex homogenate from 2 randomly selected control rats; Sy1, Sy2: the cerebral cortex synaptosomal fraction of the 2 rats; GluR1: a PSD protein, copurification with synaptosomes; Synaptophysin 1: a synaptic vesicle protein, copurification with synaptosomes; Syntaxin 1: ubiquitous distribution on subcellular membranes; TPI1: protein that is lost during the purification.

Fig. 2.

The Representative Difference Gel Electrophoresis of Rat Synaptosome Samples. One hundred fifty micrograms protein mixture of 50 μg Cy3-labeled, 50 μg Cy5-labeled, and 50 μg Cy2-labeled synaptosome proteins were separated on a 24-cm immobilized pH gradient strip (pH 3–10, nonlinear) as the first dimension and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis as the second dimension.

Fig. 3.

The Silver-Stained Gel of Rat Synaptosome Samples for MS Identification. Proteins (400 μg) were separated on a 24-cm immobilized pH gradient strip (pH 3–10 nonlinear) as the first dimension and 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis as the second dimension.

Fig. 4.

Top Networks Associated With MK-801 Treatment Identified by Ingenuity Pathway Analysis. Protein symbols with red were upregulated while green were downregulated. Hub proteins not significantly altered in this study are shown as clear. (A) The merged network of 2 top networks associated with 7-day MK-801 treatment. (B) The top network associated with 21-day MK-801 treatment.

Fig. 5.

Western Blot Analysis Confirming Results of 2-Dimensional Difference Gel Electrophoresis. (A) Western blot results of Camk2a, Crmp2, Crmp5, Dnm1, and Ndufs3 in cerebral synaptosome proteins and total cerebral proteins of the control, 7-day MK-801–treated, and 21-day MK-801–treated groups. (B) The fold changes and P values of western blot results. FC, fold change. #, not consistent with 2-dimensional difference gel electrophoresis result. *P < .05; **P < .01; ***P < .001.