No abstract

Calpain is a calcium-dependent protease that plays a significant role in synaptic plasticity, cell motility, and neurodegeneration. Two major calpain isoforms are present in brain, with -calpain (calpain1) requiring micromolar calcium concentrations for activation and m-calpain (calpain2) needing millimolar concentrations. Recent studies in fibroblasts indicate that epidermal growth factor (EGF) can activate m-calpain independently of calcium via mitogen-activated protein kinase (MAPK)-mediated phosphorylation. In neurons, MAPK is activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate m-calpain by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells, both of which express BDNF and EGF receptors. Calpain activation was monitored by quantitative analysis of spectrin degradation and by a fluorescence resonance energy transfer (FRET)-based assay, which assessed the truncation of a calpain-specific peptide flanked by the FRET fluorophore pair DABCYL and EDANS. In both cell types, BDNF and EGF rapidly elicited calpain activation, which was completely blocked by MAPK and calpain inhibitors. BDNF stimulated m-calpain but not -calpain serine phosphorylation, an effect also blocked by MAPK inhibitors. Remarkably, BDNF-and EGF-induced calpain activation was preferentially localized in dendrites and dendritic spines of hippocampal neurons and was associated with actin polymerization, which was prevented by calpain inhibition. Our results indicate that, in cultured neurons, both BDNF and EGF activate m-calpain by MAPK-mediated phosphorylation. These results strongly support a role for calpain in synaptic plasticity and may explain why m-calpain, although widely expressed in CNS, requires nonphysiological calcium levels for activation.

17-␤-Estradiol (E2) is a steroid hormone involved in numerous brainfunctions. E2 regulates synaptic plasticity in part by enhancing NMDA receptor function and spine density in the hippocampus, resulting in increased long-term potentiation and facilitation of learning and memory. As the calcium-dependent neutral protease, calpain, is also involved in these processes, we tested whether E2 could activate calpain and examined the functional consequences of E2-mediated calpain activation in hippocampus. Calpain activity was analyzed by a fluorescence resonance energy transfer (FRET)-based assay that allows both quantitative determination and spatial resolution. E2 rapidly activated calpain in cultured cortical and hippocampal neurons, prominently in dendrites and dendritic spines. E2-induced calpain activation was mediated through mitogen-activated protein kinase (MAPK), as it was completely blocked by MEK inhibitors. It was also calcium-independent, as it was still evident in presence of the calcium chelator, BAPTA-AM. Activation of ER␣ and ER␤ receptors by specific agonists stimulated calpain activity. Finally, the rapid E2-mediated increase in excitability in acute hippocampal slices was prevented by a membrane-permeable calpain inhibitor. Furthermore, E2 treatment of acute hippocampal slices resulted in increased actin polymerization and membrane levels of GluR1 but not GluR2/3 subunits of AMPA receptors; both effects were also blocked by a calpain inhibitor. Our results indicate that E2 rapidly stimulates calpain activity through MAP kinase-mediated phosphorylation, resulting in increased membrane levels of AMPA receptors. These effects could be responsible for E2-mediated increase in neuronal excitability and facilitation of cognitive processes.FRET ͉ hippocampus ͉ synaptic plasticity C alpains are intracellular calcium-dependent neutral cysteine proteases that have been implicated in several brain processes, including synaptic plasticity [e.g., long term potentiation (LTP)] and learning and memory formation (1, 2). To account for the role of calpain in these processes, calpain activity has been proposed to be intricately involved in activity-dependent changes in dendritic spine morphology via truncation of a variety of cytoskeletal proteins, including spectrin, as well as of enzymes participating in regulation of spine morphology and function (3-6). Several studies have indicated that m-calpain could be rapidly activated by MAP kinase-mediated phosphorylation in various cell types (7-9), and we recently reported that a similar process occurs in neurons (10). Moreover, we showed that brain-derived neurotrophic factor (BDNF)-elicited increase in actin polymerization in cultured cortical neurons was blocked by a calpain inhibitor (10). These findings provided a link between several elements proposed to play a critical role in LTP, namely BDNF, calpain, MAP kinase, and actin polymerization.17-␤-Estradiol (E2) is a steroid hormone that is critical for the functions of multiple organs, including the brain. In partic...

The transcription factor p53 suppresses tumorgenesis by regulating cell proliferation and migration. We investigated whether p53 could also control cell motility in postmitotic neurons. p53 isoforms recognized by phospho-p53-specific (at Ser-15) or "mutant" conformation-specific antibodies were highly and specifically expressed in axons and axonal growth cones in primary hippocampal neurons. Inhibition of p53 function by inhibitors, small interfering RNAs, or by dominant-negative forms, induced axonal growth cone collapse, whereas p53 overexpression led to larger growth cones. Furthermore, deletion of the p53 nuclear export signal blocked its axonal distribution and induced growth cone collapse. p53 inhibition-induced axonal growth cone collapse was significantly reduced by the Rho kinase (

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG–polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin–proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.