Role of the Proteasome in Excitotoxicity-Induced Cleavage of Glutamic Acid Decarboxylase in Cultured Hippocampal Neurons

Márcio S Baptista,Carlos V Melo,Mário Bengtson,Diogo O Pimentel,Dennis Herrmann,Ramiro D Almeida,Ben A Bahr,Carlos B Duarte,Mário Armelão,Graciano Leal,Margarida V Caldeira,Rafael Linden

doi:10.1371/journal.pone.0010139

Abstract

Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms—GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.

Highlights

In traumatic brain injury, epilepsy, and following episodes of hypoxia-ischemia the excessive release of glutamate and the consequent overactivation of glutamate receptors leads to cell death by excitotoxicity [1,2,3,4]
Previous studies have shown the cleavage of the glutamic acid decarboxylase isoforms GAD65 and GAD67 under excitotoxic conditions [23,25], and pointed out a key role for calpains in this process [22,23,24]
In this work we show that the activity of E1 ubiquitin ligase and the proteasome are required for glutamate-evoked cleavage of GAD67, no clear evidences were obtained showing a direct ubiquitination of the enzyme

Summary

Introduction

Epilepsy, and following episodes of hypoxia-ischemia the excessive release of glutamate and the consequent overactivation of glutamate receptors leads to cell death by excitotoxicity [1,2,3,4]. Brain ischemia has a strong impact in GABAergic neurotransmission. The Ca2+-dependent exocytotic release of GABA appears to account for the initial phase of neurotransmitter release at the onset of ischemia, while the reversal of the plasma membrane transporters is responsible for much of the subsequent efflux [5,6]. Following transient focal ischemia there is a decrease in the expression of the vesicular GABA transporter, which may have a delayed impact on the exocytotic release of the neurotransmitter [8]. The plasma membrane GABA transporter GAT1 is a calpain substrate [9], and calpain activation in the postischemic brain [10] may contribute to the deregulation of the transporter

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Results

Conclusion