Cleavage of L‐glutamate decarboxylase 65 is associated with brain injury

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 have showed that formation of tGAD65 progressively increases with increase of stimulus concentration both in rat brain synaptosomes and primary rat neuronal cultures. More importantly, direct cleavage of SV‐associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme detached from the SVs. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SV. Furthermore, we also observed that the level of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to elevation of local glutamate as measured by microdialysis. Moreover, the level of tGAD65 is 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 due to decrease of activity of SV‐associated GAD65 resulting in decrease of GABA synthesis/packaging coupling process leading to decrease in GABA transmission. Supported in part by James & Esther King Grant # 09KW‐11, DOH, State of Florida.