Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of alpha-synuclein (alpha-Syn), and although it is now recognized that alpha-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of alpha-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a beta-sheet structure. To determine whether calpain-cleavage of alpha-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to alpha-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved alpha-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved alpha-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved alpha-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of alpha-Syn in various alpha-synucleinopathies.

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 ...

CRMP proteins play critical regulatory roles during semaphorin-mediated neurite outgrowth, neuronal differentiation and death. Albeit having a high degree of structure and sequence resemblance to that of liver dihydropyrimidinase, purified rodent brain CRMPs do not hydrolyze dihydropyrimidinase substrates. Here we found that mouse CRMP3 has robust histone H4 deacetylase activity. During excitotoxicity-induced mouse neuronal death, calpain-cleaved, N-terminally truncated CRMP3 undergoes nuclear translocation to cause nuclear condensation through deacetylation of histone H4. CRMP3-mediated deacetylation of H4 leads to de-repression of the E2F1 gene transcription and E2F1-dependent neuronal death. These studies revealed a novel mechanism of CRMP3 in neuronal death. Together with previous well established bodies of literature that inhibition of histone deacetylase activity provides neuroprotection, we envisage that inhibition of CRMP3 may represent a novel therapeutic approach towards excitotoxicity-induced neuronal death.