Accumulating evidence suggests dysfunction of the glutamate signaling system in major depressive disorder. Previously, we observed elevated levels of NR2C subunit of N-methyl-D-aspartate receptor (NMDAR) and lower levels of neuronal nitric oxide synthase (nNOS), intracellular mediator of NMDAR activation, in the noradrenergic locus coeruleus in depressed subjects (Karolewicz et al. 2004, 2005). The aim of the present study was to investigate potential abnormalities in the NMDAR signaling molecules in the amygdala and hippocampus from the same depressed subjects. Tissue samples containing the lateral nucleus of amygdala, and hippocampal dentate gyrus were obtained from 10 -11 matched pairs of depressed subjects and healthy controls. Subjects were matched for age, sex, brain pH, and postmortem interval. Changes in concentration of NMDAR subunits NR1 and NR2 as well as intracellular associated proteins, neuronal nitric oxide synthase (nNOS) and postsynaptic density protein (PSD-95), that might occur in depression were assessed by immunoblotting. NR1 subunit immunoreactivity appeared as a prominent band at 120 kDa; NR2A as a band at 180 kDa; PSD-95 and nNOS immunoreactivities appeared as bands at 95 kDa and 155 kDa, respectively. The overall amount of NR1 and nNOS was normal in subjects diagnosed with depression as compared to controls. Amounts of PSD-95 were significantly higher in the amygdala (+115%, p<0.05) and hippocampus (+34%, p<0.05), respectively. The level of NR2A subunit was elevated in the amygdala (+51%) in depressed subjects as compared to controls. Our data indicate that glutamatergic signaling is abnormal in depression. Higher levels of NMDA receptor subunits and its associated protein may represent an adaptive response to decreased synaptic release of glutamate. This hypothesis is in agreement with postmortem and neuroimaging findings of altered glutamatergic transmission in depression. Further studies of the glutamatergic signaling system may lead to the development of novel therapeutics for the treatment of depressive disorder.

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.