Title

Expression of Protocadherin-gammaC5 in the rat brain and interaction with GABAA receptors

Date of Completion

January 2010

Keywords

Biology, Neuroscience|Biology, Cell|Biology, Neurobiology

Degree

Ph.D.

Abstract

GABAA receptors (GABAARs) play a central role in mediating most of the neuronal inhibition in central nervous system. For efficient synaptic neurotransmission, it is essential that GABAARs are recruited and clustered at the synapses. However, the mechanism for this process is still unclear. Previous work done in our laboratory indicated that in a yeast-two-hybrid assay (Y2H) there is an interaction between the intracellular domain of protocadherin-γC5 (Pcdh-γC5) and the large intracellular loop of the GABAAR γ2 subunit (γ2IL). I have now shown that this interaction occurs in cultured neurons and in the brain. I have also studied the expression and localization of Pcdh-γC5 in the rat brain. For these studies, we have developed several antibodies to Pcdh-γC5. With a novel anti-Pcdh-γC5 specific antibody, I have found that in the entire rat brain, Pcdh-γC5 is highly expressed in the olfactory bulb, corpus striatum, dentate gyms, CA1 region of the hippocampus, cerebral cortex and the molecular layer of the cerebellum. The expression of Pcdh-γC5 started after the second postnatal week, coinciding with the time frame of synapse development. A significant percentage of synapses, more GABAergic than glutamatergic, have associated Pcdh-γC5 clusters. Nevertheless there is also non-synaptic Pcdh-γC5. Pcdh-γC5 is expressed in both neurons and astrocytes. In the intact rat brain, a significant number of Pcdh-γC5 clusters are located at non-synaptic contact points between neurons and astrocytes. ^ An anti-Pcdh-γC5 antiserum coprecipitated assembled GABAARs from brain extracts. An anti-γ2 or α1 GABA AR subunit antiserum coprecipitated Pcdh-γC5. In cultured hippocampal neurons, an anti-γ2 or an anti-Pcdh-γC5 antibody induced capping increasing both the γ2 and Pcdh-γC5 cluster densities, indicating that there is interaction between γ2 and Pcdh-γC5 in cultured neurons. ^ In cultured HEK293 cells, overexpression of Pcdh-γC5 helped the trafficking of GABAARs to the cell surface. Overexpression of Pcdh-γC5 increases the number of γ2 clusters in hippocampal neurons. Knocking down the Pcdh-γC5 expression level in hippocampal neurons with Pcdh-γC5 shRNAs, both the postsynaptic γ2 cluster density and the presynaptic GAD innervation were dramatically reduced, indicating that Pcdh-γC5 is essential for the stability of GABAergic synapses. In contrast, the glutamatergic synapses were not affected by the shRNAs. These results indicate that Pcdh-γC5 (i) plays synaptic and non-synaptic function; (ii) interacts with GABA ARs in cultured hippocampal neurons and in brain; (iii) helps GABA ARs trafficking to the cell surface via direct interaction with γ2 subunit; (iv) this interaction plays role in GABAergic synapses formation or maintenance. ^^