Laura Facci

Selective small‐molecule inhibitors of glycogen synthase kinase‐3 activity protect primary neurones from death

The phosphatidylinositol 3‐kinase (PI 3‐kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase‐3 (GSK‐3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK‐3 in response to insulin and growth factors, which inhibits GSK‐3 activity and leads to the modulation of multiple GSK‐3 regulated cellular processes. We show that the novel potent and selective small‐molecule inhibitors of GSK‐3; SB‐415286 and SB‐216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3‐kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK‐3 activity and modulation of GSK‐3 substrates tau and β‐catenin. Thus, in addition to the previously assigned roles of GSK‐3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK‐3 activity in primary neurones is sufficient to prevent death, implicating GSK‐3 as a physiologically relevant principal regulatory target of the PI 3‐kinase/PKB neuronal survival pathway.

Activation of (Na+, K+)‐ATPase by Nanomolar Concentrations of GM1 Ganglioside

Abstract: GM1 ganglioside binding to the crude mitochondrial fraction of rat brain and its effect on (Na+, K+)‐ATPase were studied, the following results being obtained: (a) the binding process followed a biphasic kinetics with a break at 50 nM‐GM1; GM1 at concentrations below the break was stably associated, while over the break it was loosely associated; (b) stably bound GM1 activated (Na+, K+)‐ATPase up to a maximum of 43%; (c) the activation was dependent upon the amount of bound GM1 and was highest at the critical concentration of 20 pmol bound GM1× mg protein‐1; (d) loosely bound GM1 suppressed the activating effect on (Na+, K+)‐ATPase elicited by firmly bound GM1; (e) GM1‐activated (Na+, K+)‐ATPase had the same pH optimum and apparent Km (for ATP) as normal (Na+, K+)‐ATPase but a greater apparent Vmax; (f) under identical binding conditions (2 h, 37°C, with 40 nM substance) all tested gangliosides (GM1, GD1a, GD1b, GT1b) activated (Na+, K+)‐ATPase (from 26–43%); NeuNAc, sodium dodecylsulphate, sulphatide and cerebroside had only a very slight effect. It is suggested that the ganglioside activation of (Na+ ‐K+)‐ATPase is a specific phenomenon not related to the amphiphilic and ionic properties of gangliosides, but due to modifications of the membrane lipid environment surrounding the enzyme.

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Abstract: Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro‐2a clone, in monolayer culture. GM1 bound to neuro‐2a cells in three distinct forms, one removable by treatment with serum‐containing solutions, one serum‐resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell‐associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10−6M); the trypsin‐labile and ‐stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro‐2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite‐bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10−4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite‐bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin‐labile and ‐stable forms of cell‐associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, <1:20,000–1:50,000. Therefore the neuritogenic effect can be attributed to ganglioside itself. The results obtained suggest that under the experimental conditions used the stimulation of neuro‐2a cell differentiation by GM1 is related to changes of the plasma membrane properties following association of exogenous GM1 molecules. This would facilitate the spontaneous process of differentiation, or enhance cell responsiveness to differentiating factors present in the serum.

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway.

Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum‐free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation‐induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation‐induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high‐K+ to low‐K+/serum‐free medium decreased viability by >50% when measured after 30 h. Treatment of low‐K+ granule neurons at DIV 4 with nerve growth factor, brain‐derived neurotrophic factor (BDNF), neurotrophin‐3, or neurotrophin‐4/5 (NT‐4/5) demonstrated concentration‐dependent (1–100 ng/ml) protective effects only for BDNF and NT‐4/5. Between DIV 10 and 20, K+‐deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT‐4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin‐3. BDNF and NT‐4/5 also circumvented glutamate‐induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate‐triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin‐protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3‐kinase or the mitogen‐activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival‐promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.

Myelin-associated Glycoprotein Interacts with Ganglioside GT1b A MECHANISM FOR NEURITE OUTGROWTH INHIBITION

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 MAG-responsive 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.

Microglia and mast cells: two tracks on the road to neuroinflammation

One of the more important recent advances in neuroscience research is the understanding that there is extensive communication between the immune system and the central nervous system (CNS). Proinflammatory cytokines play a key role in this communication. The emerging realization is that glia and microglia, in particular, (which are the brains resident macrophages), constitute an important source of inflammatory mediators and may have fundamental roles in CNS disorders from neuropathic pain and epilepsy to neurodegenerative diseases. Microglia respond also to proinflammatory signals released from other non‐neuronal cells, principally those of immune origin. Mast cells are of particular relevance in this context. These immunity‐related cells, while resident in the CNS, are capable of migrating across the blood‐spinal cord and bloodbrain barriers in situations where the barrier is compromised as a result of CNS pathology. Emerging evidence suggests the possibility of mast cell‐glia communications and opens exciting new perspectives for designing therapies to target neuroinflammation by differentially modulating the activation of non‐neuronal cells normally controlling neuronal sensitization, both peripherally and centrally. This review aims to provide an overview of recent progress relating to the pathobiology of neuroinflammation, the role of microglia, neuroimmune interactions involving mast cells, in particular, and the possibility that mast cell‐microglia crosstalk may contribute to the exacerbation of acute symptoms of chronic neurodegenerative disease and accelerate disease progression, as well as promote pain transmission pathways. We conclude by considering the therapeutic potential of treating systemic inflammation or blockade of signaling pathways from the periphery to the brain in such settings.—Skaper, S. D., Giusti, P., Facci, L. Microglia and mast cells: two tracks on the road to neuroinflammation. FASEB J. 26, 3103–3117 (2012). www.fasebj.org

MAPK-activated Protein Kinase 2 Deficiency in Microglia Inhibits Pro-inflammatory Mediator Release and Resultant Neurotoxicity RELEVANCE TO NEUROINFLAMMATION IN A TRANSGENIC MOUSE MODEL OF ALZHEIMER DISEASE

MAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon γ-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2–/–) mice demonstrated significant inhibition in release of tumor necrosis factor α, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1α on stimulation with LPS + interferon γ or amyloid-β peptide (1–42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon γ-stimulated or amyloid-β peptide (1–42)-stimulated MK2–/– microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with β-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor α, macrophage inflammatory protein 1α, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented.

Excitotoxicity, Oxidative Stress, and the Neuroprotective Potential of Melatonin

The Brain Consumes Large Quantities of Oxygen Relative to its Contribution to total body mass. This, together with its paucity of oxidative defense mechanisms, places this organ at risk for damage mediated by reactive oxygen species. The pineal secretory product melatonin possesses broad‐spectrum free radical scavenging and antioxidant activities, and prevents kainic acid‐induced neuronal lesions, glutathione depletion, and reactive oxygen species‐mediated apoptotic nerve cell death. Melatonins action is thought to involve electron donation to directly detoxify free radicals such as the highly toxic hydroxyl radical, which is a probable end‐product of the reaction between NO· and peroxynitrite. Moreover, melatonin limits NO·‐induced lipid peroxidation, inhibits cerebellar NO· synthase, scavenges peroxynitrite, and alters the activities of enzymes that improve the total antioxidative defense capacity of the organism. Melatonin function as a free radical scavenger and antioxidant is likely facilitated by the ease with which it crosses morphophysiological barriers, e.g., the blood‐brain barrier, and enters cells and subcellular compartments. Pinealectomy, which eliminates the nighttime rise in circulating and tissue melatonin levels, worsens both reactive oxygen species‐mediated tissue damage and brain damage after focal cerebral ischemia and excitotoxic seizures. That melatonin protects against hippocampal neurodegeneration linked to excitatory synaptic transmission is fully consistent with the last study. Conceivably, the decreased melatonin secretion that is documented to accompany the aging process may be exaggerated in populations with dementia.

Inflammatory Mediator Stimulation of Astrocytes and Meningeal Fibroblasts Induces Neuronal Degeneration via the Nitridergic Pathway

Abstract: The role of inflammatory cytokines in the pathogenesis of neurological disorders is not entirely clear. The neurotoxic effects of cytokines, and perhaps indirectly bacterial endotoxins, could be mediated by the stimulation of immunocompetent cells in the brain to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. NO is a short‐lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. Both constitutive and inducible NO synthase has been described in astrocytes in vitro. Here we demonstrate that newborn mouse cortical astrocytes, when coincubated with neonatal mouse cerebellar granule cells or hippocampal neurons, induced neurotoxicity upon stimulation with endotoxin (lipopolysaccharide) (ED50 30 ng/ml). Astrocytes were unresponsive to the cytokines tumor necrosis factor‐α or interleukin‐1β individually, but exhibited a marked synergistic stimulation in their combined presence. Moreover, meningeal fibroblasts treated with tumor necrosis factor‐α, but not interleukin‐1β or lipopolysaccharide, elaborated neurotoxicity for cocultured granule cells (ED50 30 U/ml). In cocultures of immunostimulated astrocytes or meningeal fibroblasts, neurotoxicity was blocked by the NO synthase inhibitors Nω‐nitro‐l‐arginine and Nω‐nitro‐d‐arginine methyl ester, and by oxyhemoglobin, which inactivates NO. Astroglial‐induced neurotoxicity was not affected by N‐methyl‐d‐aspartate receptor antagonists. Superoxide dismutase, which degrades superoxide anion, attenuated astrocyte‐ and fibroblast‐mediated neurotoxicity, indicating that endogenous superoxide anion may react with NO to form toxic peroxynitrite and its breakdown products. These findings suggest a potentially important role for glial‐ and meningeal fibroblast‐induced NO synthase in the pathophysiology of CNS disease states of immune or inflammatory origin.

Mast cells, glia and neuroinflammation: partners in crime?

Glia and microglia in particular elaborate pro‐inflammatory molecules that play key roles in central nervous system (CNS) disorders from neuropathic pain and epilepsy to neurodegenerative diseases. Microglia respond also to pro‐inflammatory signals released from other non‐neuronal cells, mainly those of immune origin such as mast cells. The latter are found in most tissues, are CNS resident, and traverse the blood–spinal cord and blood–brain barriers when barrier compromise results from CNS pathology. Growing evidence of mast cell–glia communication opens new perspectives for the development of therapies targeting neuroinflammation by differentially modulating activation of non‐neuronal cells that normally control neuronal sensitization – both peripherally and centrally. Mast cells and glia possess endogenous homeostatic mechanisms/molecules that can be up‐regulated as a result of tissue damage or stimulation of inflammatory responses. Such molecules include the N‐acylethanolamine family. One such member, N‐palmitoylethanolamine is proposed to have a key role in maintenance of cellular homeostasis in the face of external stressors provoking, for example, inflammation. N‐Palmitoylethanolamine has proven efficacious in mast‐cell‐mediated experimental models of acute and neurogenic inflammation. This review will provide an overview of recent progress relating to the pathobiology of neuroinflammation, the role of microglia, neuroimmune interactions involving mast cells and the possibility that mast cell–microglia cross‐talk contributes to the exacerbation of acute symptoms of chronic neurodegenerative disease and accelerates disease progression, as well as promoting pain transmission pathways. We will conclude by considering the therapeutic potential of treating systemic inflammation or blockade of signalling pathways from the periphery to the brain in such settings.