Cleavage of brain glutamic acid decarboxylase 65 by calpain under pathological conditions

Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica