Cortical N-methyl-d-aspartate receptor in schizophrenia: Characterising molecular changes at the cellular and sub-cellular level

Abstract

Schizophrenia is a mental disorder that affects approximately 1% of the world’s population. The N-methyl-d-aspartate receptor (NMDAR) seems likely to play a prominent role in schizophrenia; as when NMDAR antagonists are introduced, psychosis occurs in individuals with no mental disorders. This thesis explores how NMDAR may be endogenously changed in the brains of people with schizophrenia. By using tissue fractionation, discussed in chapter 3, to examine the post-synaptic density (PSD) specifically one can specifically examine the NMDAR protein at the synapse of the neuron. Also, one can examine the NMDAR mRNA level in the brains of people with schizophrenia compared to controls. My Masters’ thesis research covers four broad areas, namely subcellular tissue fractionation of human cerebral cortex, the determination of the NMDAR subunit NR1 protein levels specifically in the PSD, analysis of NR1 mRNA transcript across cortical lamina, and the quantification of NMDAR NR1 mRNA at the cellular level. It is hypothesized that the NMDAR NR1 protein will be decreased in the PSD and mRNA will be decreased in neurons in schizophrenia patients. There are four main aims of this study. The first aim is to extract the postsynaptic density fraction from human brains in the BA10 region. Using the New South Wales Tissue Resource Centre Cohort (37 schizophrenia patients and 37 controls). Tissue fractionation, discussed in chapter two, was completed in order to obtain total homogenate and PSD-enriched fractions from the PFC [Brodmann’s Area (BA) 10]) from individuals with schizophrenia and controls. The second aim was to measure NMDAR NR1 protein levels in schizophrenia compared to controls in the total homogenate and PSD associated homogenate from the BA10 region. The amounts of NMDAR NR1 protein were determined using Western blotting techniques, described in chapter four. The third aim was to examine examining the laminar expression of NMDAR NR1 mRNA in BA46 in schizophrenia compared to controls using a riboprobe GRIN1 (human genome designation for the NMDAR NR1 subunit). Film from in situ hybridization was used to determine the amount of mRNA from NMDAR NR1 in each cortical layer in the BA46 in patients with schizophrenia and controls, discussed in chapter five. The fourth aim was to measure the cellular expression of NMDAR NR1 mRNA in BA46 in schizophrenia compared to controls in three laminar layers. In addition, silver grain analysis (corresponding to the amount of NMDAR NR1 mRNA/cells) of individual large and small neurons was analysed to determine if changes in mRNA could be localised to specific cell types within cortical layer V. mRNA for the NR1 subunit was studied in the PFC using in situ hybridization, discussed in chapter six. Through the process of Western blotting, it was concluded that our fractionation samples containing the synapse were indeed enriched for proteins (including NR1) known to be enriched in the PSD. From there, analysis of the PSD fractions revealed a statistically significant decreased NR1 protein (39%) in the PSD in people with schizophrenia compared to controls. There were no significant diagnostic differences in NR1 mRNA expression when analysing the individual layers of the cortex from the autoradiographic films. However, when delving deeper and looking at individual neurons, I found that pyramidal neurons were significantly lower in NR1 mRNA expression in people with schizophrenia, and thus may play a role in the hypoglutamatergia in schizophrenia. Future work required would be to determine which neurons are responsible for the NR1 protein changes at the PSD. A comprehensive examination of NR1 mRNA expression across more cortical layers at a microscopic level in BA46 is warranted.