Previous studies from this laboratory have suggested that the glutamatergic dysfunction in schizophrenia may be associated with changes in the expression levels of certain  molecules that modulate glutamatergic function. For example, higher expression of the transcripts encoding the glutamate transporter subtypes EAAT1 and EAAT2 have been detected in the thalamus of individuals with schizophrenia and decreased expression of EAAT3 has been detected in the striatum of individuals with schizophrenia.

Several proteins have been identified in humans that may interact with distinct subtypes of glutamate transporters and may modulate their activity. One of them, ARHGEFII, is highly homologous with the rat glutamate transporter-4-assciated protein 48 (GTRAP48) that has been shown to specifically interact with the intracellular carboxy-teminal domain of EAAT4 and modulate its glutamate transport activity. Another protein, KIAA0302, is highly homologous to rat GTRAP41 that has been shown to interact specifically with the carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity. The proteins GTRAP41 and GTRA48 have been found to be expressed predominantly in cerebellum, with low levels in the striatum, hippocampus and thalamus. The protein JWA is encoded by a vitamin-A-responsive gene and is the human homologue of the rat protein GTRAP3-18. GTRAP3-18 interacts with the C-terminal intracellular domain of EAAC1/EAAT3 and modulates its activity. For example, increasing the expression of GTRAP3-18 in cells reduces EAAC1-mediated glutamate transport by lowering substrate affinity. GTRAP3-18 mRNA has been found to be widely expressed in brain (cerebellum, cortex, brainstem) in a pattern consistent with the distribution of EAAC1. Immunohistochemical analysis has shown that GTRAP3-18 protein is localized primarily to neurons such as cerebellar Purkinje cells.

Ajuba is a cytoplamic LIM protein that has been reported to interact with the N-terminus of GLT-1/EAAT2. Ajuba and GLT-1 have been shown to localize in the same cells in the CNS (glia in cerebellum and hippocampus, neurons in retina) but Ajuba has been found to have a wider distribution. Analysis of GLT-1 expressed in COS cells in the presence and absence of Ajuba has not revealed an effect of Ajuba on glutamate transport. It is possible that Ajuba influences the EAAT2 in several different ways. For example, similarly to the EAAT4 interacting protein GTRAP41, Ajuba may link GLT-1 to the cytoskeleton or modulate trafficking of transporters to the surface membrane.

The overall goal of this project will be to characterize and quantitate the expression of the mRNAs encoding for the molecules ARHGEFII, JWA, KIAA0302, Ajuba that are believed to modulate one of the several subtypes of the glutamate transporters. The mRNA expression levels will be initially studied in monkey brain tissue, followed by studies in human postmortem brain tissue from schizophrenic and control psychiatrically-healthy individuals from the Mount Sinai Medical Center Brain Bank. In situ hybridization on frozen tissue sections using 35S radiolabelled riboprobes will be performed according to an established method in this laboratory. Riboprobes for the glutamate transporter modulator proteins ARHGEFII, JWA and KIAA0302 have been already obtained  by subcloning an unique part of each mRNA into the Zero Blunt TOPO vector using a kit from Invitrogen and sequence-specific primers in order to amplify by PCR the desired sequence from human brain cDNA library. A similar approach will be used in order to obtain a riboprobe for Ajuba. For data analysis, images will be aquired from digitized X-ray films a analysed with an imaging system.

Characterization of the mRNA expression levels of these molecules in human brain will increase our understanding of the molecular changes in schizophrenia. It is interesting that schizophrenia is believed to have a neurodevelopmental component and glutamatergic abnormalities and changes in glutamate-transporter interacting proteins might have role in organization of the neuronal cytoskeleton and possibly might participate in regulating neuronal migration, neurite outgrowth, synaptogenesis and neuronal "pruning."

Ibone Huerta