The application of selective reaction monitoring confirms dysregulation of glycolysis in a preclinical model of schizophrenia

Abstract

Background: Establishing preclinical models is essential for novel drug discovery in schizophrenia. Most existing models are characterized by abnormalities in behavioral readouts, which are informative, but do not necessarily translate to the symptoms of the human disease. Therefore, there is a necessity of characterizing the preclinical models from a molecular point of view. Selective reaction monitoring (SRM) has already shown promise in preclinical and clinical studies for multiplex measurement of diagnostic, prognostic and treatment-related biomarkers.

Methods: We have established an SRM assay for multiplex analysis of 7 enzymes of the glycolysis pathway which is already known to be affected in human schizophrenia and in the widely-used acute PCP rat model of schizophrenia. The selected enzymes were hexokinase 1 (Hk1), aldolase C (Aldoc), triosephosphate isomerase (Tpi1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), phosphoglycerate mutase 1 (Pgam1), phosphoglycerate kinase 1 (Pgk1) and enolase 2 (Eno2). The levels of these enzymes were analyzed using SRM in frontal cortex from brain tissue of PCP treated rats.

Results: Univariate analyses showed statistically significant altered levels of Tpi1 and alteration of Hk1, Aldoc, Pgam1 and Gapdh with borderline significance in PCP rats compared to controls. Most interestingly, multivariate analysis which considered the levels of all 7 enzymes simultaneously resulted in generation of a bi-dimensional chart that can distinguish the PCP rats from the controls.

Conclusions: This study not only supports PCP treated rats as a useful preclinical model of schizophrenia, but it also establishes that SRM mass spectrometry could be used in the development of multiplex classification tools for complex psychiatric disorders such as schizophrenia.

Figure 1

Glycolysis metabolic pathway. The differentially expressed enzymes and metabolites revealed by proteomics in post-mortem human brains from schizophrenia patients are contrasted in black (by KEGG - http://www.genome.jp/kegg).

Figure 2

Univariate analysis of glycolysis enzymes. Mass spectrometry intensities are normalized relative to the spiked enolase peptides.

Figure 3

Multivariate analyses of glycolysis enzymes analyzed by SRM. a) Partial least squares discriminant analysis (PLS-DA) scores plot shows the distribution of the different samples in two dimensions, it should be noted that both PCP groups were treated as one class. Good separation was achieved between the model and the control. Also, the plot shows little difference between the samples in the 1 mg and 2.5 mg of PCP models. b) Variable influence on projection (VIP) bar chart shows that Tpi is most important to the separation, as shown in the scores plot. c) The loading plot also identifies Tpi as an important analyte for separation and shows that it is reproducible across samples in a group. Gapdh, Hk, Aldoc and Eno (inside red circle) cluster tightly together.