During the current grant period, we established that GABA synthesis depends on two forms of glutamate decarboxylase (GAD65 and GAD67), which differ in size, sequence, intracellular location, and in their interactions with the obligate cofactor, pyridoxal phosphate. Using a retroviral vector, we have engineered a number of cell lines to produce either or both GADs and shown that these cells produce GABA both in culture and after transplantation into lesioned rats. The proposed studies will extend this work to problems specifically related to epilepsy. The rationale of the proposed approach is the finding from several other laboratories that increased GABA levels in the substantia nigra suppresses chemically and electrically induced seizures as well as the development of amygdaloid kindling.
The Specific Aims for the next grant period are: 1. to use GADs produced in engineered bacteria and eukaryotic cells to study the regulation of GAD enzymatic activity both in vitro and in cells; 2. to use cell lines engineered to produce GAD to understand (a) differences between the two GADs in intracellular targeting and (b) the contributions of each GAD to vesicular and non-vesicular GABA release; 3. to transplant into the substantia nigra cells engineered to produce GABA (either constitutively or in an inducible manner) in order to study the effect of in situ GABA release on the propagation of seizures evoked by electroshock; and 4. to study the effects of grafted GABA-producing cells on the expression of GABA- and glutamate-related genes and on the development of amygdaloid kindling. The proposed experiments will test the following hypotheses: (1) that the cellular regulation of GAD activity involves changes in the relative rates of decarboxylation and transamination of the GAD-PLP-glutamate complex, with transamination leading to the formation of inactive apoGAD; (2) that the two GADs are responsible for the production of distinct GABA pools, with GAD67 contributing primarily to the production of non-vesicular GABA and GAD65 to the production of vesicular GABA; (3) that GABA produced by transplanted cells can alter gene expression of GABA- and glutamate-related genes and can suppress seizure propagation; and (4) that changes in the expression of genes and proteins associated with the production and action of GABA and glutamate accompany the development of kindled seizures.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS022256-10
Application #
2264446
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1985-04-01
Project End
2000-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
10
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Zanjani, Hadi; Lemaigre-Dubreuil, Y; Tillakaratne, Niranjala J K et al. (2004) Cerebellar Purkinje cell loss in aging Hu-Bcl-2 transgenic mice. J Comp Neurol 475:481-92
Carlson, B B; Behrstock, S; Tobin, A J et al. (2003) Brain implantations of engineered GABA-releasing cells suppress tremor in an animal model of Parkinsonism. Neuroscience 119:927-32
Tillakaratne, Niranjala J K; de Leon, Ray D; Hoang, Thao X et al. (2002) Use-dependent modulation of inhibitory capacity in the feline lumbar spinal cord. J Neurosci 22:3130-43
Tillakaratne, N J; Mouria, M; Ziv, N B et al. (2000) Increased expression of glutamate decarboxylase (GAD(67)) in feline lumbar spinal cord after complete thoracic spinal cord transection. J Neurosci Res 60:219-30
Thompson, K; Anantharam, V; Behrstock, S et al. (2000) Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures. Exp Neurol 161:481-9
Behrstock, S P; Anantharam, V; Thompson, K W et al. (2000) Conditionally-immortalized astrocytic cell line expresses GAD and secretes GABA under tetracycline regulation. J Neurosci Res 60:302-10
Srinivasan, S; Sapp, D W; Tobin, A J et al. (1999) Biphasic modulation of GABA(A) receptor binding by steroids suggests functional correlates. Neurochem Res 24:1363-72
Mi, J; Chatterjee, S; Wong, K K et al. (1999) Recombinant adeno-associated virus (AAV) drives constitutive production of glutamate decarboxylase in neural cell lines. J Neurosci Res 57:137-48
Srinivasan, S; Nichols, C J; Lawless, G M et al. (1999) Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly. J Biol Chem 274:26633-8
Banerjee, P K; Olsen, R W; Tillakaratne, N J et al. (1998) Absence seizures decrease steroid modulation of t-[35S]butylbicyclophosphorothionate binding in thalamic relay nuclei. J Pharmacol Exp Ther 287:766-72

Showing the most recent 10 out of 44 publications