CaMKII phosphorylates collapsin response mediator protein 2 and modulates axonal damage during glutamate excitotoxicity

Abstract

In the context of stroke-induced brain damage, the molecular and biochemical mechanisms involving retraction and collapse of the axonal network remain unclear. One of the early morphological changes accompanying excitotoxicity-induced neuronal death in cultured neurons is the retraction/collapse of the neurite network, which indicates that axonal damage occurs before the emergence of typical morphological hallmarks of neuronal death (Deckwerth and Johnson 1994;Raff et al. 2002). Typically, axonal degeneration is manifested by irregular blebbing of axons with thinning and fragmentation, followed by retraction and collapse of the axonal network. While axonal damage was regarded as an outcome of the death process occurring within the cell body, more importantly, it Address correspondence and reprint requests to Sheng T. Hou, Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, ON, Canada, K1A 0R6. E-mail: sheng.hou@nrc-cnrc.gc.caAbbreviations used: AIPII, autocamtide-2-related inhibitory peptide; AKT, protein kinase B; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; CRMP, collapsin response mediator protein; DAPI, 4¢,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GSK, glycogen synthase kinase; MAP2, microtubule-associated protein 2; MAPK, mitogen-activated protein kinase; MCAO, middle cerebral artery occlusion; MK801, dizocilpine; PI3K, phosphoinositide 3-kinase; PSD, post-synaptic density; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. AbstractIntracellular calcium influx through NMDA receptors triggers a cascade of deleterious signaling events which lead to neuronal death in neurological conditions such as stroke. However, it is not clear as to the molecular mechanism underlying early damage response from axons and dendrites which are important in maintaining a network essential for the survival of neurons. Here, we examined changes of axons treated with glutamate and showed the appearance of bIII-tubulin positive varicosities on axons before the appearance of neuronal death. Dizocilpine blocked the occurrence of varicosities on axons suggesting that these microstructures were mediated by NMDA receptor activities. Despite early increased expression of pCaMKII and pMAPK after just 10 min of glutamate treatment, only inhibitors to Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and calpain prevented the occurrence of axonal varicosities. In contrast, inhibitors to Rho kinase, mitogen-activated protein kinase and phosphoinositide 3-kinase were not effective, nor were they able to rescue neurons from death, suggesting CaMKII and calpain are important in axon survival. Activated CaMKII directly phosphorylates collapsin response mediator protein (CRMP) 2 which is independent of calpain-mediated cleavage of CRMP2. Over-expression of CRMP2, but not the phosphorylation-resistant mutant CRMP2-T555A, increased axonal resistance to glutamate toxicity with reduced numbers of varicosities. The levels of both pCRMP2 ...

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

“…Interestingly, overexpression of CRMP-2 increases axonal resistance to glutamate-induced varicosity formation (Hou et al, 2009). Like cypin, CRMP-2 promotes microtubule assembly (Fukata et al, 2002), and, together, this report and our data suggest that promotion of microtubule assembly moderates dendritic swelling.…”

“…What does varicosity formation mean for the neuron? A number of studies show that a reduction of beaded neurons or number of varicosities promotes functional recovery (Inoue and Okada, 2007;Meller et al, 2008;Hou et al, 2009). Others have reported the opposite and indicate that varicosity production is a self-protective response against excitotoxicity and that prevention of focal swelling increases the level of cell death (Ikegaya et al, 2001).…”

Cdk5‐mediated phosphorylation of CRMP‐2 enhances its interaction with CaV2.2

“…Precedence for this is exemplified in CRMP-2's interaction with tubulin; phosphorylation of CRMP-2 by cyclin dependent kinase 5 (Cdk5), glycogen synthase kinase 3 beta (GSK-3b) and/or Rho-dependent kinase (RhoK) lowers the ability of CRMP-2 to interact with tubulin which leads to arrest of axonal growth and collapse of cones [22][23][24]. Phosphorylation, by glycogen synthase kinase-3b (GSK-3b), cyclin dependent kinase 5 (Cdk5), and Rho kinase [10], as well as calmodulin dependent protein kinase II (CaMKII) [25] and the src family kinase Fyn [26] and Yes [27] have been reported, identifying phosphorylation as a fulcrum in CRMP-2 signaling. Based upon the established important role of phosphorylation in modulating CRMP-2's activity and interaction with partners proteins, we hypothesized that phosphorylation may also affect its modulation of CaV2.2.…”

Phaseic Acid, an Endogenous and Reversible Inhibitor of Glutamate Receptors in Mouse Brain

“…C57B/6 mice (20 -23 g) were obtained from Charles River and bred locally. Under temporary isofluorane anesthesia, MCAO was induced by the intraluminal insertion of a silicon-coated nylon filament (Re L910 PK5, Doccol Corporation) through the common carotid artery into the internal carotid artery and left in place for 60 min as we previously described (20)(21)(22)(23)(24)(25)(26). Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry using a probe located in the ipsilateral parietal bone (1-2 mm posterior to bregma), and a >90% reduction in CSF was considered to indicate successful occlusion.…”

“…Indirect immunofluorescence staining and confocal microscopy-The procedures for indirect immunofluorescence immunocytochemistry were exactly as described previously (20,22). Mouse monoclonal antibody to PA was a kind gift from Prof E.W.…”