Gangliosides, a member of the glycosphingolipid family, are heterogeneously expressed in biological membranes and are particularly enriched within the central nervous system. Gangliosides consist of mono- or poly-sialylated oligosaccharide chains of variable lengths attached to a ceramide unit and are found to be intimately involved in brain disease development. The purpose of this study is to examine the spatial profile of ganglioside species using matrix-assisted laser desorption/ionization (MALDI) imaging (IMS) following middle cerebral artery occlusion (MCAO) reperfusion injury in the mouse. IMS is a powerful method to not only discriminate gangliosides by their oligosaccharide components, but also by their carbon length within their sphingosine base. Mice were subjected to a 30 min unilateral MCAO followed by long-term survival (up to 28 days of reperfusion). Brain sections were sprayed with the matrix 5-Chloro-2-mercaptobenzothiazole, scanned and analyzed for a series of ganglioside molecules using an Applied Biosystems 4800 MALDI TOF/TOF. Traditional histological and immunofluorescence techniques were performed to assess brain tissue damage and verification of the expression of gangliosides of interest. Results revealed a unique anatomical profile of GM1, GD1 and GT1b (d18∶1, d20∶1 as well as other members of the glycosphingolipid family). There was marked variability in the ratio of expression between ipsilateral and contralateral cortices for the various detected ganglioside species following MCAO-reperfusion injury. Most interestingly, MCAO resulted in the transient induction of both GM2 and GM3 signals within the ipsilateral hemisphere; at the border of the infarcted tissue. Taken together, the data suggest that brain region specific expression of gangliosides, particularly with respect to hydrocarbon length, may play a role in neuronal responses to injury.

Semaphorin 3A (Sema3A) increased significantly in mouse brain following cerebral ischemia. However, the role of Sema3A in stroke brain remains unknown. Our aim was to determine wether Sema3A functions as a vascular permeability factor and contributes to ischemic brain damage. Recombinant Sema3A injected intradermally to mouse skin, or stereotactically into the cerebral cortex, caused dose- and time-dependent increases in vascular permeability, with a degree comparable to that caused by injection of a known vascular permeability factor vascular endothelial growth factor receptors (VEGF). Application of Sema3A to cultured endothelial cells caused disorganization of F-actin stress fibre bundles and increased endothelial monolayer permeability, confirming Sema3A as a permeability factor. Sema3A-mediated F-actin changes in endothelial cells were through binding to the neuropilin2/VEGFR1 receptor complex, which in turn directly activates Mical2, a F-actin modulator. Down-regulation of Mical2, using specific siRNA, alleviated Sema3A-induced F-actin disorganization, cellular morphology changes and endothelial permeability. Importantly, ablation of Sema3A expression, cerebrovascular permeability and brain damage were significantly reduced in response to transient middle cerebral artery occlusion (tMCAO) and in a mouse model of cerebral ischemia/haemorrhagic transformation. Together, these studies demonstrated that Sema3A is a key mediator of cerebrovascular permeability and contributes to brain damage caused by cerebral ischemia.

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 ...

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J. Neurochem. (2012) 122, 764–774. Abstract Microglia are the ‘immune cells’ of the brain and their activation plays a vital role in the pathogenesis of many neurodegenerative diseases. Activated microglia produce high levels of pro‐inflammatory factors, such as TNFα, causing neurotoxicity. Here we show that vimentin played a key role in controlling microglia activation and neurotoxicity during cerebral ischemia. Deletion of vimentin expression significantly impaired microglia activation in response to LPS in vitro and transient focal cerebral ischemia in vivo. Reintroduction of the functional vimentin gene back into vimentin knockout microglia restored their response to LPS. More importantly, impairment of microglia activation significantly protected brain from cerebral ischemia‐induced neurotoxicity. Collectively, we demonstrate a previously unknown function of vimentin in controlling microglia activation.