Mechanism of glial activation by S100B: involvement of the transcription factor NFkappaB.

Compelling evidence links chronic activation of glia and the subsequent cycle of neuroinflammation and neuronal dysfunction to the progression of neurodegeneration in disorders such as Alzheimer's disease (AD). S100B, a glial-derived cytokine, is significantly elevated in the brains of AD patients and high concentrations of S100B are believed to be detrimental to brain function. As a first step toward elucidating the mechanisms by which S100B might be serving this detrimental role, we examined the mechanisms by which S100B stimulates glial inducible nitric oxide synthase (iNOS), an oxidative stress related enzyme that has been linked to neuropathology through the production of neurotoxic peroxynitrite. We report here that S100B stimulates iNOS in rat primary cortical astrocytes through a signal transduction pathway that involves activation of the transcription factor NFkappaB. NFkappaB activation was demonstrated by nuclear translocation of the p65 NFkappaB subunit, stimulation of NFkappaB-specific DNA binding activity, and stimulation of NFkappaB-dependent transcriptional activity. Furthermore, S100B-induced iNOS promoter activation was inhibited upon mutation of the NFkappaB response element in the promoter, and transfection of cells with an NFkappaB inhibitor blocked S100B-induced iNOS promoter activation and nitric oxide production. These studies define a signal transduction pathway by which S100B activation of glia could participate in the generation of oxidative stress in the brain.

S100B proteins that lack one or both cysteine residues can induce inflammatory responses in astrocytes and microglia.

The astrocytic protein S100B stimulates neurite outgrowth and neuronal survival during CNS development. S100B can also stimulate glial activation, leading to induction of pro-inflammatory molecules like interleukin-1 beta (IL-1 beta) and inducible nitric oxide synthase (iNOS). Although it is known that S100B's neurotrophic activity requires a disulfide-linked dimeric form of the protein, the structural features of S100B that are important for glial activation have not been defined. As an initial step towards understanding the structural features of S100B required for its action on glia and to determine if these features are different from those required for its action on neurons, we tested two mutants of S100B for their ability to activate glia. The C68VC84S mutant lacks S100B's two cysteine residues (cys68, cys84) and lacks neurotrophic activity (Winningham-Major et al., 1989, J. Cell Biol. 109 3063-3071), and the truncation mutant S100B83stop lacks the C-terminal nine residues (including cys84) that have been shown to be important for some S100B:target protein interactions. We report here that both C68VC84S and S100B83stop stimulate glial activation, as determined by induction of iNOS and IL-1 beta in rat primary astrocyte and microglial cultures. C68VC84S showed activation profiles similar to those of wild-type S100B, demonstrating that a disulfide-linked dimer is not required for glial activation. S100B83stop also stimulated both iNOS and IL-1 beta, although S100B83stop was significantly less effective than wild-type S100B in inducing iNOS. These results indicate that the C-terminal region of S100B is not required for glial activation; however, its presence may influence the degree of activation by the protein. Altogether, these studies demonstrate that the structural features required for S100B's neurotrophic activity are distinct from those affecting its glial activation activity.

Group II selective metabotropic glutamate receptor agonists and local cerebral glucose use in the rat.

The novel mGluR agonist LY354740 and a related analogue LY379268 are selective for mGluR2/3 receptors and are centrally active after systemic administration. In this study, rates of local cerebral glucose use were measured using the [14C]2-deoxyglucose autoradiographic technique to examine the functional consequences of their systemic administration in the conscious rat. Both LY354740 (0.3, 3.0, 30 mg/kg) and LY379268 (0.1, 1.0, 10 mg/kg) produced dose-dependent changes in glucose use. After LY354740 (3.0mg/kg), 4 of the 42 regions measured showed statistically significant changes from vehicle-treated controls: red nuclei (-16%), mammillary body (-25%), anterior thalamus (-29%), and the superficial layer of the superior colliculus (+50%). An additional 15 regions displayed significant reductions in function-related glucose use (P < .05) in animals treated with LY354740 (30 mg/ kg). LY379268 (0.1, 1.0, 10 mg/kg) produced changes in glucose metabolism in 20% of the brain regions analyzed. Significant increases (P < .05) in glucose use were evident in the following: the superficial layer of the superior colliculus (+81%), locus coeruleus (+57%), genu of the corpus callosum (+31%), cochlear nucleus (+26%), inferior colliculus (+20%), and the molecular layer of the hippocampus (+14%). Three regions displayed significant decreases: mammillary body (-34%), anteroventral thalamic nucleus (-28%), and the lateral habenular nucleus (-24%). These results show the important functional involvement of the limbic system together with the participation of components of different sensory systems in response to the activation of mGluR2 and mGluR3 with LY354740 and LY379268.

Reversible neural inactivation reveals hippocampal participation in several memory processes.

Studies of patients and animals with brain lesions have implicated the hippocampal formation in spatial, declarative/relational and episodic types of memory. These and other types of memory consist of a series of interdependent but potentially dissociable memory processes-encoding, storage, consolidation and retrieval. To identify whether hippocampal activity contributes to these processes independently, we used a novel method of inactivating synaptic transmission using a water-soluble antagonist of AMPA/kainate glutamate receptors. Once calibrated using electrophysiological and two-deoxyglucose techniques in vivo, drug or vehicle was infused chronically or acutely into the dorsal hippocampus of rats at appropriate times during or after training in a water maze. Our findings indicate that hippocampal neural activity is necessary for both encoding and retrieval of spatial memory and for either trace consolidation or long-term storage.

Effects of the selective metabotropic glutamate agonist LY354740 in a rat model of permanent ischaemia.

The neuroprotective effects of a systemically active, potent, group II selective metabotropic glutamate receptor agonist, LY354740, was assessed in the middle cerebral artery occlusion model of focal ischaemia in rats. LY354740 (0.3, 3.0 or 30.0 mg/kg) was administered subcutaneously (s.c.) 30 min prior to and 3 hours after the induction of ischaemia. Twenty four hours after the ischaemic insult, the brains were processed for the evaluation of infarct volumes. No significant reduction in infarct volumes were observed in treated animals at any of the doses investigated. These data provide no support for the view that group II metabotropic glutamate receptors have a major influence on ischaemic damage in this model.

Local cerebral glucose utilization in the AS/AGU rat: a mutant with movement disorders.

The AS/AGU mutant rat is characterized by a wide staggering gait and a movement disorder of the hindlimbs. Local cerebral glucose utilization in the brain was investigated using the [14C]2-deoxyglucose autoradiographic technique to map any functional alterations in the mutant AS/AGU (agu/agu) compared with Albino Swiss controls (+/+). Locomotor tests were also performed to confirm the phenotypic assignment of the animals. Statistically significant reductions in glucose utilization were apparent in 12 of the 44 regions examined in the AS/AGU animals. The regions showing the most significant differences (P < 0.01) from the control AS strain were the substantia nigra pars compacta (-23%) and medial geniculate body (-17%). Statistically significant decreases (P < 0.05 and P < 0.02) in glucose utilization ranging from -15 to -26% were also displayed in the superior colliculus superficial layer, auditory cortex, ventroposterior nucleus of the thalamus, molecular layer of the hippocampus, dentate gyrus, medial amygdaloid nucleus, median raphe nucleus, subthalamic nucleus, medial preoptic area of the hypothalamus and anterior hypothalamus. In no region studied was the mean value of glucose use in the AS/AGU rat greater than in the control animals. The results of this study complement previous behavioural and neurochemical characterization studies of this mutant, confirm that the disorder involves functional disturbances of the basal ganglia, and demonstrate the involvement of the limbic system and some sensory systems.