Abstract
Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2–3 times 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 physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA 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 as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission.
Highlights
The brain constantly strives to maintain a balance between the excitatory and inhibitory networks, whose key players are the neurotransmitters L-glutamic acid and c-amino butyric acid (GABA) respectively
We reported the presence of truncated glutamic acid decarboxylase 65 (GAD65) during protein purification of human recombinant full length GAD65 from a bacterial expression system in vitro [20]. fGAD65 could be readily cleaved by mild trypsin treatment or incubation with Factor Xa to yield a 58 KDa tGAD65, confirming an earlier purported report that a proteolytic hot-spot is contained within the first 100 amino acids of GAD65 [21]
The presence of the cleaved product was demonstrated by immunoblotting using a monoclonal antibody GAD6, whose epitopes are directed against the C terminus of the protein. tGAD65 was essentially absent at physiologically low concentrations of K+ (10 mM K+; Fig. 1A, lane 2) and in the control sample (Fig. 1A, lane 1), respectively
Summary
The brain constantly strives to maintain a balance between the excitatory and inhibitory networks, whose key players are the neurotransmitters L-glutamic acid and c-amino butyric acid (GABA) respectively. Such an alignment in close proximity between these two molecules, licenses VGAT to preferentially load the newly synthesized GABA generated by the transiently activated membrane - associated GAD65 into the SVs, over the pre-existing GABA constitutively generated by GAD67 in the soma This key phenomenon is disrupted in GAD652/2 mice. Mice deficient in GAD65 appear normal but are highly susceptible to epilepsy and general anxiety disorder which could be spontaneously precipitated by fear or mild stress [18,19] These findings underscore the pivotal role played by membrane associated GAD65 to regulate GABA for extra-synaptic tonic inhibition
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