Fractalkine is a recently identified chemokine that exhibits cell adhesion and chemoattractive properties. It represents a unique member of the chemokine superfamily because it is located predominantly in the brain in which it is expressed constitutively on specific subsets of neurons. To elucidate the possible role of neuronally expressed fractalkine in the inflammatory response to neuronal injury, we have analyzed the regulation of fractalkine mRNA expression and protein cleavage under conditions of neurotoxicity. We observed that mRNA encoding fractalkine is unaffected by experimental ischemic stroke (permanent middle cerebral artery occlusion) in the rat. Similarly, in vitro, levels of fractalkine mRNA were unaffected by ensuing excitotoxicity. However, when analyzed at the protein level, we found that fractalkine is rapidly cleaved from cultured neurons in response to an excitotoxic stimulus. More specifically, fractalkine cleavage preceded actual neuronal death by 2-3 hr, and, when evaluated functionally, fractalkine represented the principal chemokine released from the neurons into the culture medium upon an excitotoxic stimulus to promote chemotaxis of primary microglial and monocytic cells. We further demonstrate that cleavage of neuron-derived, chemoattractive fractalkine can be prevented by inhibition of matrix metalloproteases. These data strongly suggest that dynamic proteolytic cleavage of fractalkine from neuronal membranes in response to a neurotoxic insult, and subsequent chemoattraction of reactive immune cells, may represent an early event in the inflammatory response to neuronal injury.

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAGresponsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.Expression of myelin-associated glycoprotein (MAG; siglec 4a), 1 is restricted to myelinating glial cells on myelin membrane adjacent to the axon and is required for maintenance of myelin integrity (1-4). In vitro, MAG inhibits outgrowth of postnatal neurons (5-8), involving activation of Rho GTPase, a key signaling step for the inhibitory effect of myelin on regeneration of neurons in vivo (9). MAG is therefore thought to contribute to the inhibitory properties of myelin, which is in part responsible for the lack of regenerative capacity of the central nervous system after injury or disease (10, 11).Like other siglecs, MAG binds to sialic acid residues at the termini of glycans on opposing cells through a sialic acid binding site located in the N-terminal V-set Ig domain (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). MAG binds specifically to terminal sialic acid residues in ␣2-3 linkage to galactose, which occurs in glycans linked ␤1-3 to GalNAc or GlcNAc or ␤1-4 to GlcNAc (12,[23][24][25]. Use of sialic acid analogues has identified specific groups on sialic acid essential for interaction with MAG (26), consistent with interactions seen between sialic acid and conserved amino acids in the siglec 1 crystal structure (21). It is also thought that the core glycan structure on which the terminal sialic acid is presented plays a role in recognition by MAG (23,26,27). The ability of MAG to bind specific gangliosides bearing terminal ␣2-3-linked sialic acid has been well documented. Gangliosides bind to MAG with the relative potencies GQ1b␣ Ͼ GT1a␣, GD1␣ Ͼ GD1a, GT1b Ͼ Ͼ GM3, GM4, whereas GM1, GD1b, GD3, and GQ1b do not support adhesion (23,27,28).Although the binding of MAG to sialylated glycans and gangliosides is well characterized, the functional importance of these intera...

Spreading depression (SD) has long been associated with the underlying pathophysiology of migraine. Evidence that the Nmethyl-D-aspartate (NMDA) glutamate receptor (NMDA-R) is implicated in the generation and propagation of SD has itself been available for more than 15 years. However, to date, there are no reports of NMDA-R antagonists being developed for migraine therapy. In this study, an uncompetitive, pan-NMDA-R blocker, memantine, approved for clinical use, and two antagonists with selectivity for NMDA-R containing the NR2B subunit, (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606) and (Ϯ)-(R*,S*)-␣-(4-hydroxyphenyl)-␤-methyl-4-(phenylmethyl)-1-piperidine propanol (Ro 25-6981), were investigated to assess their protective effects against SD in the rat. Under isoflurane anesthesia, d.c. potential and the related cortical blood flow and partial pressure of O 2 (pO 2 ) were recorded simultaneously at separate cortical sites. Drugs (1, 3, and 10 mg/kg i.p.) were given 1 h or 30 min before KCl application to the brain surface. Core temperature and arterial pCO 2 , pO 2 , and pH measurements confirmed physiological stability. KCl induced 7.7 Ϯ 1.8 (mean Ϯ S.D.) SD events with d.c. amplitude of 14.9 Ϯ 2.8 mV.Memantine and CP-101,606 dose-dependently decreased SD event number (to 2.0 Ϯ 1.8 and 2.3 Ϯ 2.9, respectively) and SD amplitude at doses relevant for therapeutic use. Ro 25-6981 also decreased SD events significantly, but less effectively (to 4.5 Ϯ 1.6), without affecting amplitude. These results indicate that NR2B-containing NMDA receptors are key mediators of SD, and as such, memantine-and NR2B-selective antagonists may be useful new therapeutic agents for the treatment of migraine and other SD-related disorders (e.g., stroke and brain injury). Whether chronic, rather than acute, treatment may improve their efficacy remains to be determined.Spreading depression (SD) is a slowly (2-6 mm/min) propagating wave of transiently increased cerebral blood flow, suppressed cortical activity, and loss of membrane potential accompanied by major metabolic disturbance (for review, see Smith et al., 2006). SD can be induced in the brains of all animal species investigated, including human, using a variety of mechanical and chemical methods. Typically, in the anesthetized rat, the brain is surgically exposed using a small craniotomy, and SD is evoked by topical administration of KCl. SD can be detected via a reduction in d.c. amplitude using electrodes placed on the brain surface distant from the SD induction site. Associated changes in extracellular ion concentrations and cerebral blood flow can also be detected with appropriate probes. Changes in cerebral tissue water, This study was supported by Marie Curie Industry Host Fellowship QLG5-CT-2001-60018 and the Fonds Spécial de Recherche de l'Université Catholique de Louvain, Belgium) (to M.P.).

The mechanisms by which neurons die after cerebral ischemia and related conditions in vivo are unclear, but they are thought to involve voltage-dependent Na ϩ channels, glutamate receptors, and nitric oxide (NO) formation because selective inhibition of each provides neuroprotection. It is not known precisely what their roles are, nor whether they interact within a single cascade or in parallel pathways. These questions were investigated using an in vitro primary cell culture model in which striatal neurons undergo a gradual and delayed neurodegeneration after a brief (5 min) challenge with the glutamate receptor agonist NMDA. Unexpectedly, NO was generated continuously by the cultures for up to 16 hr after the NMDA exposure. Neuronal death followed the same general time course except that its start was delayed by ϳ4 hr. Application of the NO synthase inhibitor nitroarginine after, but not during, the NMDA exposure inhibited NO formation and protected against delayed neuronal death. Blockade of NMDA receptors or of voltage-sensitive Na ϩ channels [with tetrodotoxin (TTX)] during the postexposure period also inhibited both NO formation and cell death. The NMDA exposure resulted in a selective accumulation of glutamate in the culture medium during the period preceding cell death. This glutamate release could be inhibited by NMDA antagonism or by TTX, but not by nitroarginine. These data suggest that Na ϩ channels, glutamate receptors, and NO operate interdependently and sequentially to cause neurodegeneration. At the core of the mechanism is a vicious cycle in which NMDA receptor stimulation causes activation of TTX-sensitive Na ϩ channels, leading to glutamate release and further NMDA receptor stimulation. The output of the cycle is an enduring production of NO from neuronal sources, and this is responsible for delayed neuronal death. The same neurons, however, could be induced to undergo more rapid NMDA receptor-dependent death that required neither TTX-sensitive Na ϩ channels nor NO.

Certain cytokines have been reported to exert neurotrophic actions in vivo and in vitro. In the present study, we investigated the possible neuroprotective actions of the cytokine human recombinant interleukin-1 beta (hrIL-1 beta) against excitatory amino acid (EAA)-induced neurodegeneration in cultured primary cortical neurons. Brief (15 min) exposure of cultures to submaximal concentrations of glutamate, NMDA, AMPA, or kainate caused extensive neuronal death (approximately 70% of all neurons). Neuronal damage induced by the EAAs was significantly reduced (up to 70%) by pretreatment with 500 ng/ml (6.5 x 10(3) U/ml) hrIL-1 beta for 24 hr. The neuroprotective effect of hrIL-1 beta was reversed by coapplication of an IL-1 receptor antagonist (IL-1ra, 50 micrograms/ml). Neuroprotective actions of hrIL-1 beta were also reduced by administration of a neutralizing monoclonal antibody to NGF (65% inhibition). In concordance, the neurotoxic actions of EAAs were significantly reduced (by 40%) after pretreatment with NGF (100 ng/ml for 48 hr). Furthermore, an additive neuroprotective effect of approximately 75% was observed when cultures were exposed to a combination of hrIL-1 beta and NGF. In contrast, exposure of cultures to high concentrations hrIL-1 beta alone (100 micrograms/ml, 1.3 x 10(6) U/ml) for periods up to 72 hr resulted in neurotoxicity, which was reversed by IL-1ra (1 mg/ml). These findings suggest that hrIL-1 beta can limit EAA-induced neuronal damage. These effects appear to be may be mediated, at least in part, via NGF. These findings may be relevant to the understanding of neurodegenerative diseases.