Editors' ChoiceNeuroscience

Good and Bad Products of Calpain Cleavage

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Science's STKE  06 Feb 2007:
Vol. 2007, Issue 372, pp. tw43
DOI: 10.1126/stke.3722007tw43

Calpain is a calcium-dependent protease that is activated in response to stimuli that activate N-methyl-D-aspartate (NMDA)-type glutamate receptors, leading to an increase in intracellular calcium. Two groups provide evidence for substrates of calpain in neurons. Abe and Takeichi show that in hippocampal cultures, glutamate stimulation triggers an NMDA-mediated (based on pharmacological properties) cleavage of a small proportion (~5%) of β-catenin. Cleavage was blocked by treatment of the neurons with a calpain inhibitor, and in vitro purified calpain cleaved immunoprecipitated β-catenin or β-catenin as part of N-cadherin-catenin complexes. The sites of calpain cleavage were mapped, and fragments representing the cleavage products transfected into L cells were not phosphorylated and were resistant to degradation. The cleavage sites are either very close to the sites of glycogen synthase kinase 3β (GSK-3β) phosphorylation sites or eliminate these phosphorylation sites in the β-catenin fragments; GSK-3β phosphorylation is required for constitutive degradation of β-catenin. Cultured pyramidal neurons treated with glutamate (acting through NMDA receptors) exhibited nuclear accumulation of β-catenin. Wnt antagonists did not alter this redistribution; however, calpain inhibitors blocked it. The nuclear β-catenin was a cleaved form, as antibodies to the N terminus did not recognize it. Treatment of neurons transfected with a β-catenin reporter construct with glutamate increased reporter gene expression, and calpain or NMDA receptor inhibition blocked this response. Transfection of the shorter of the two cleaved forms stimulated the reporter constitutively in the absence of glutamate stimulation. Most intriguing was evidence that mice placed into a new stimulating environment produced β-catenin fragments in hippocampal and cortical regions and that these fragments were not produced if the mice were treated with calpain inhibitor before exposure to the novel exploration environment. Furthermore, enhanced expression of β-catenin-dependent reporter was also observed in mice in response to the novel environment, and this was also dependent on calpain.

The second group, Xu et al., determined that the metabotropic glutamate receptor 1α (mGluR1α) was cleaved by calpain in response to neurotoxic concentrations of glutamate and that this response required activation of NMDA receptors. Cortical neurons treated with glutamate showed the formation of a cleaved form of the mGluR1α that was blocked by NMDA antagonists or calpain inhibitors. The site of cleavage was mapped, and an inhibitory peptide was created that selectively blocked NMDA-stimulated calpain cleavage of mGluR1α. Coupling of the mGluR1α was measured in human embryonic kidney (HEK) 293 cells transfected with the full-length protein or the cleaved form, and activation of either form stimulated an increase in intracellular calcium, presumably through the known coupling mechanism of phosphoinositide hydrolysis, inositol trisphosphate production, and release of calcium from intracellular stores. However, the cleaved form did not stimulate whole-cell currents when cotransfected with various channels, whereas the full-length mGluR1α did. In neurons, activation of mGluR1α with a specific agonist stimulated the production of phosphorylated Akt, indicating activation of the survival pathway mediated by phosphatidylinositol 3-kinase. Pretreatment of the neurons with NMDA reduced basal phospho-Akt and completely blocked any increase by activation of mGluR1α. Immunofluorescence analysis of tagged forms of the full-length mGluR1α and the cleaved form transfected into neurons suggested that cleavage disrupts the dendritic localization of mGluR1α. To assess the roles of full-length and cleaved mGluR1α in neurotoxicity, the authors applied various combinations of pharmacological agents to activate mGluR1α before and after excitotoxic activation of NMDA receptors. If the mGluR1α was activated before NMDA activation, then enhanced survival was observed compared with NMDA alone, whereas if mGluR1α was activated after NMDA activation, then the percent of dead cells was greater than with NMDA alone. A protective effect was observed by application of the mGluR1α cleavage inhibitory peptide. In vivo cleavage of mGluR1α in the hippocampus was observed following glutamate receptor (kainate receptor)-induced seizures. Cleavage appeared to be limited to particular regions. Pretreatment of the mice with the mGluR1α cleavage inhibitory peptide reduced seizure-induced damage, especially in the CA1 region.

Together these two articles suggest a physiological and pathological role for calpain-mediated cleavage in neurons. Under conditions that produce physiological glutamate signaling, NMDA receptors activate calpain, which can cleave β-catenin producing transcriptionally active fragments through a pathway independent of Wnt. Under pathological conditions, glutamate signaling stimulates calpain-mediated cleavage of the metabotropic mGluR1α, apparently uncoupling the receptor from the survival pathway without loss of coupling to calcium signaling, thus contributing to excitotoxicity. Lynch and Gleichman discuss these results and suggest that crucial unanswered questions remain regarding how calpain activity is regulated and substrate selection is achieved in neurons.

K. Abe, M. Takeichi, NMDA-receptor activation induces calpain-mediated β-catenin cleavages for triggering gene expression. Neuron 53, 387-397 (2007). [Online Journal]

W. Xu, T. P. Wong, N. Chery, T. Gaertner, Y. T. Wang, M. Baudry, Calpain-mediated mGluR1α truncation: A key step in excitotoxicity. Neuron 53, 399-412 (2007). [Online Journal]

D. R. Lynch, A. J. Gleichman, Picking up the pieces: The roles of functional remnants of calpain-mediated proteolysis. Neuron 53, 317-319 (2007). [Online Journal]

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