Calpain Cleavage of Brain Glutamic Acid Decarboxylase 65 Is Pathological and Impairs GABA Neurotransmission

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.

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…One of the calpain targets are the glutamic acid decarboxylase isoforms (see also section 3.1.3.2), which synthesize GABA from glutamate. GAD65 and GAD67 were shown to be cleaved after transient MCAO in rats, both in the core and penumbra regions (Buddhala et al, 2012), but the mechanisms involved were not elucidated. A role for calpain in GAD cleavage was first suggested based on studies showing i) a Ca 2+ -dependent cleavage of both GAD isoforms in rat synaptosomes incubated with the Ca 2+ ionophore ionomycin, and ii) a truncation of GAD65 following depolarization of the synaptosomes with KCl (Sha et al, 2008;Wei et al, 2006).…”

“…After truncation by calpains, a cleavage product containing the active site of GAD65 is detached from synaptic vesicles, thereby reducing the vesicular uptake of the newly synthesized GABA (Buddhala et al, 2012). Furthermore, cleavage of GAD65 in hippocampal neurons subjected to excitotoxic conditions disrupts the characteristic punctate distribution of the enzyme along neurites, suggesting an impairment in synaptic targeting (Baptista et al, 2010).…”

Epigenetic profiling of human brain differential DNA methylation networks in schizophrenia

“…It also significantly contributes to platelet activity and thrombosis [53], which may contribute to the increased risk of thrombogenesis in schizophrenic patients [2]. From the interaction aspect of neurotransmission, calpain-mediated break down of fGAD65 results in decreased level of the GABA synthesis which leads to reduced GABA neurotransmission [54]. The mTOR has a role mediated by Wnt signaling pathway in the neuropathology of schizophrenia [55].…”

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

“…The calpain sensitivity of KCC2, VIAAT, and GABA synthesizing enzyme glutamic acid decarboxylase (Wei et al, 2006; Sha Buddhala et al, 2012), as well as of the glycine transporters GlyT1 and GlyT2 (Baliova et al, 2004;Baliova and Jursky, 2005), suggests a more general calpain-mediated regulation of GABAergic and glycinergic neurotransmission. This idea gains further support from the finding that gephyrin, a key protein for clustering of GABA receptors (Kneussel and Betz, 2000) that colocalizes with KCC2 (Hübner et al, 2001), is also a calpain substrate (Tyagarajan et al, 2011).…”