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Interleukin-1β (IL-1β) is a crucial mediator in the pathogenesis of inflammatory diseases at the periphery and in the central nervous system (CNS). Produced as an unprocessed and inactive pro-form which accumulates intracellularly, release of the processed cytokine is strongly promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Microglia are central to the inflammatory process and a major source of IL-1β when activated. Here we show that purified (>99%) microglia cultured from rat cortex, spinal cord and cerebellum respond robustly to ATP-dependent IL-1β release, upon priming with a number of TLR isoform ligands (zymosan and Pam3CSK4 for TLR2, poly(I:C) for TLR3). Cytokine release was prevented by a P2X7R antagonist and inhibitors of stress-activated protein kinases. Enriched astrocytes (≤ 5% microglia) from these CNS regions displayed responses qualitatively similar to microglia but became unresponsive upon eradication of residual microglia with the lysosomotropic agent Leu-Leu-OMe. Activation of multiple TLR isoforms in nervous system pathology, coupled with elevated extracellular ATP levels and subsequent P2X7R activation may represent an important route for microglia-derived IL-1β. This phenomenon may have important consequences for neuroinflammation and its position to the common pathology of CNS diseases.

Summary Report of the International Association of Neurorestoratology VII Conference : Regulations, Ethics, Science, and the Need of Patients Care in Neurorestoratology

The role of the Wnt signaling pathway during synaptic development has been well established. In the adult brain, different components of Wnt signaling are expressed, but little is known about its role in mature synapses. Emerging in vitro studies have implicated Wnt signaling in synaptic plasticity. Furthermore, activation of Wnt signaling has shown to protect against amyloid-β-induced synaptic impairment. The present study provides the first evidence that in vivo activation of Wnt signaling improves episodic memory, increases excitatory synaptic transmission, and enhances long-term potentiation in adult wild-type mice. Moreover, the activation of Wnt signaling also rescues memory loss and improves synaptic dysfunction in APP/PS1-transgenic mice that model the amyloid pathology of Alzheimer's diseases. These findings indicate that Wnt signaling modulates cognitive function in the adult brain and could be a novel promising target for Alzheimer's disease therapy.

Inflammatory and neuroinflammatory processes are increasingly recognized as critical pathophysiologic steps in the development of multiple chronic diseases and in the etiology of persistent pain and depression. Mast cells are immune cells now viewed as cellular sensors in inflammation and immunity. When stimulated, mast cells release an array of mediators to orchestrate an inflammatory response. These mediators can directly initiate tissue responses on resident cells, and may also regulate the activity of other immune cells, including central microglia. New evidence supports the involvement of peripheral and central mast cells in the development of pain processes as well as in the transition from acute, to chronic and neuropathic pain. That behavioral and endocrine states can increase the number and activation of peripheral and brain mast cells suggests that mast cells represent the immune cells that peripherally and centrally coordinate inflammatory processes in neuropsychiatric diseases such as depression and anxiety which are associated with chronic pelvic pain. Given that increasing evidence supports the activated mast cell as a director of common inflammatory pathways/mechanisms contributing to chronic and neuropathic pelvic pain and comorbid neuropsychiatric diseases, mast cells may be considered a viable target for the multifactorial management of both pain and depression.

During development of the nervous system, neurons extend axons over considerable distances in a highly stereospecific fashion in order to innervate their targets in an appropriate manner. This involves the recognition, by the axonal growth cone, of guidance cues that determine the pathway taken by the axons. These guidance cues can act to promote and/or repel growth cone advance, and they can act either locally or at a distance from their place of synthesis. The directed growth of axons is partly governed by cell adhesion molecules (CAMs) on the neuronal growth cone that bind to CAMs on the surface of other axons or non-neuronal cells. In vitro assays have established the importance of the CAMs (N-CAM, N-cadherin and the L1 glycoprotein) in promoting axonal growth over cells, such as Schwann cells, astrocytes and muscle cells. Strong evidence now exists implicating the fibroblast growth factor receptor tyrosine kinase as the primary signal transduction molecule in the CAM pathway. Cell adhesion molecules are important constituents of synapses, and CAMs appear to play important and diverse roles in regulating synaptic plasticity associated with learning and memory. Negative extracellular signals which physically direct neurite growth have also been described. The latter include the neuronal growth inhibitory proteins Nogo and myelin-associated glycoprotein, as well as the growth cone collapsing Semaphorins/neuropilins. Although less well characterised, evidence is now beginning to emerge describing a role for Rho kinase-mediated signalling in inhibition of neurite outgrowth. This review focuses on some of the major themes and ideas associated with this fast-moving field of neuroscience.

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.

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 beta-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.

Cerebellar granule neurons can be maintained in culture in a medium containing high serum and depolarising levels of KCl. When serum is removed and the KCl levels lowered from 25 to 5 mM, the cells undergo apoptosis. Apoptosis can be prevented by inhibitors of transcription or translation, suggesting a need for macromolecular synthesis in the apoptotic process. Using quantitative reverse transcription-polymerase chain reaction the levels of mRNA for a range of genes postulated to be important in apoptosis have been examined. Elevated levels of caspase 3, c-Jun, and Fas ligand were found, in addition to a corresponding increase in c-Jun protein and activation of caspase-3. These results suggest that cerebellar granule neurons upregulate components of both death receptor-mediated and the mitochondrial-mediated death pathways.

Nerve growth factor (NGF) is widely recognized as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurons and basal forebrain cholinergic nuclei during development and maturation. Other NGF-responsive cells are now known to belong to the hemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells themselves are capable of producing and responding to NGF, suggesting that alterations in mast cell behavior may trigger maladaptive neuroimmune tissue responses, including those of an autoimmune nature. Moreover, NGF exerts a modulatory role on sensory nociceptive nerve physiology in the adult, and appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. NGF can thus be viewed as a multifactorial modulator of neuroimmune-endocrine functions.

Unilateral injection into the right substantia nigra of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) produces extensive loss of dopaminergic cells ('hemi-parkinsonian rat'). The pineal hormone melatonin, which is a potent antioxidant against different reactive oxygen species and has been reported to be neuroprotective in vivo and in vitro, was evaluated for potential anti-Parkinson effects in this model. Imbalance in dopaminergic innervation between the striata produced by intranigral administration of 6-OHDA results in a postural asymmetry causing rotation away from the nonlesioned side. Melatonin given systemically prevented apomorphine-induced circling behavior in 6-OHDA-lesioned rats. Reduced activity of mitochondrial oxidative phosphorylation enzymes has been suggested in some neurodegenerative diseases; in particular, selective decrease in complex I activity is observed in the substantia nigra of Parkinson's disease patients. Analysis of mitochondrial oxidative phosphorylation enzyme activities in nigral tissue from 6-OHDA-lesioned rats by a novel BN-PAGE histochemical procedure revealed a clear loss of complex I activity, which was protected against in melatonin-treated animals. A good correlation between behavioral parameters and enzymatic (complex I) analysis was observed independent of melatonin administration. A deficit in mitochondrial complex I could conceivably contribute to cell death in parkinsonism via free radical mechanisms, both directly via reactive oxygen species production and by decreased ATP synthesis and energy failure. Melatonin may have potential utility in the treatment of neurodegenerative disorders where oxidative stress is a participant.

Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at/=2.5 mM (P0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.

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. Melatonin's 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.

gp120 induction of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was studied in cultures of purified astrocytes. Incubation of pure mouse cortical astrocytes with gp120 IIIB induced the expression of TNF-alpha mRNA, assessed by in situ hybridization. Anti- TNF-alpha immunocytochemical staining of gp120 IIIB stimulated astrocytes indicated the presence of TNF-alpha. gp120 IIIB treatment also stimulated secretion of bioactive TNF-alpha from astrocytes, which was prevented by inhibitors of transcription and translation. Hippocampal and cerebellar astrocytes displayed similar behaviors. Further, gp120 displayed cytotoxicity for astrocytes that depended on macromolecular synthesis. The data are the first to show gp120 IIIB induction of de novo TNF-alpha production by pure astrocytes. Because TNF-alpha exerts a wide array of effects in the brain of infected individuals and has HIV-1 inducing activity as well, induction of this cytokine by gp120 IIIB in astrocytes may contribute importantly to the pathogenesis of AIDS dementia complex. Since TNF-alpha can stimulate astrocyte reactivity and proliferation by an autocrine mechanism, the extent of the gp120 effect could conceivably increase with HIV-1 disease progression in a self-amplifying loop, involving other cell types, thus favoring both virus persistence and a chronic disease state.

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.

Depression is often associated with local and/or systemic diseases. Chronic inflammation, pain and depression are a clinical triad increasingly recognized as co-morbid. Peripheral and central inflammation can alter neuronal activity, thereby contributing to the psych emotional and somatic symptoms of depression. Preliminary evidence suggests that mast cells direct the immune pathways mediating this triad. Chronic pelvic pain in women could be a paradigm of the triad, and of considerable clinical relevance. Mast cells are immune cells which contribute to and modulate inflammation and immunity. Upon stimulation mast cells release an array of mediators, cytokines, and growth factors to orchestrate an inflammatory response. These mediators can directly initiate tissue responses on resident cells, and may also regulate the activity of other immune cell functions, including central nervous system immune cells like microglia. Mast cell mediators can interact also with neurons, either alone or in concert with other immune cells. New evidence supports the involvement of peripheral and central mast cells in the development of pain processes, in the transition from acute, nociceptive to chronic and neuropathic pain and in the depressive states associated with them. A significant increase of pro-inflammatory cytokines is evident in depression associated with chronic diseases. Marked increases of tissue mast cells and pro-inflammatory cytokines have been documented in disorders associated with chronic pelvic pain presenting as well with significant affective comorbidities (anxiety and depression). Mast cells could be thus considered as a target of therapeutic approaches which aim to reduce inflammation and the chronicization of pain. The present review analyzes current evidence on co-morbidity between depression with peripheral and central inflammation associated with chronic pelvic pain in women. The analysis will briefly focus on key mechanisms of inflammation and role of mast cells as a common denominator of depression and chronic pain. The goal is to offer mental health professionals a biologicallyoriented contribution in the pathophysiologic reading of co-morbidities between depression and chronic pelvic pain in women. Results underline the increasing evidence supporting a role of inflammatory pathways/mechanisms, orchestrated by activated mast cells, as common denominators contributing to co-morbidity between depression and chronic pelvic pain in women.

The accumulation and aggregation of misfolded proteins can be highly cytotoxic and may underlie several human degenerative diseases characterized by neuronal inclusions such as Alzheimer's, Parkinson's, prion-like and polyglutamine repeat diseases. In this context small heat shock proteins, molecular chaperones known to be induced by cell stress, play a fundamental role by facilitating folding of nascent polypeptides, preventing aggregation of misfolded proteins and enhancing their degradation. A recently identified member of the small heat shock protein family, HspB8, is of particular interest in the field of neurological diseases since mutations in its sequence correlate with development of distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 expression has been detected in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington disease and spinocerebellar ataxia type 3. In the latter, HspB8 appears to be involved in protecting the cell from accumulation of insoluble aggregates either by preventing aggregation or by promoting degradation of improperly folded proteins. These data propose that HspB8 may be a major player in the neuroprotective response and a promising target for the development of therapeutic strategies.

Persistent pain affects nearly half of all people seeking medical care in the US alone, and accounts for at least $80 billion worth of lost productivity each year. Among all types of chronic pain, neuropathic pain stands out: this is pain resulting from damage or disease of the somatosensory nervous system, and remains largely untreatable. With few available treatment options, neuropathic pain represents an area of significant and growing unmet medical need. Current treatment of peripheral neuropathic pain involves several drug classes, including opioids, gabapentinoids, antidepressants, antiepileptic drugs, local anesthetics and capsaicin. Even so, less than half of patients achieve partial relief. This review discusses a novel approach to neuropathic pain management, based on knowledge of: the role of glia and mast cells in pain and neuroinflammation; the body's innate mechanisms to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation. The discovery that palmitoylethanolamide, a member of the N-acylethanolamine family which is produced from the lipid bilayer on-demand, is capable of exerting anti-allodynic and anti-hyperalgesic effects by down-modulating both microglial and mast cell activity has led to the application of this fatty acid amide in several clinical studies of neuropathic pain, with beneficial outcome and no indication of adverse effects at pharmacological doses. Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain.

The in vitro and in vivo actions of ciliary neurotrophic factor (CNTF) suggest that endogenous CNTF plays a role in nervous system development and maintenance. CNTF produces most, possibly all, of its effects by binding to a protein referred to as CNTF receptor alpha (CNTFRalpha). Information on CNTFRalpha tissue expression and dynamics would be advanced by the availability of reagents suitable for studying the subcellular localization and trafficking of CNTFRalpha. This paper describes the genetic construction, synthesis, purification and properties of a chimeric protein in which a highly fluorescent form of the green fluorescent protein (GFP) has been fused to human CNTF. The fusion protein, termed GFP-CNTF, was expressed in Escherichia coli. Histidine tagging of GFP-CNTF permitted ready purification by means of immobilized Ni(II) chromatography. Under non-reducing conditions GFP-CNTF migrated on SDS-PAGE with an apparent molecular mass of 50 kDa, although under reducing conditions it behaved electrophoretically as a 67 kDa species. Despite these discrepancies, the molecular mass of GFP-CNTF determined by mass spectrometry (54755) agreed well with its deduced relative molecular mass of 54536. Importantly, the absorbance profile of the GFP chromophore in GFP-CNTF was not modified by the presence of the CNTF domain. Moreover, the fluorescence emission spectrum of GFP-CNTF overlapped that of GFP, showing neither a change in absorbance shift nor a difference in the fluorescence quantum yield. Circular dichroism spectroscopy confirmed that the CNTF and GFP domains of GFP-CNTF folded independently of each other. GFP-tagged CNTF was equipotent to human CNTF in supporting the survival of cultured embryonic chicken sensory and ciliary ganglion neurons. GFP-CNTF, but not GFP, bound to immobilized CNTFRalpha and was displaced by an excess of human CNTF. GFP-CNTF specifically labeled the Purkinje cell layer in cerebellar slices from adult rat. This report is the first to describe a GFP chimera with a neurotrophic factor as the fusion partner. GFP-CNTF should provide a valuable tool for elucidating the role of CNTFRalpha in nervous system function.

Oxidation reactions are essential biological reactions necessary for the formation of high-energy compounds used to fuel metabolic processes, but can be injurious to cells when produced in excess. Cutaneous tissue is especially susceptible to damage mediated by reactive oxygen species and low-density lipoprotein oxidation, triggered by dysmetabolic diseases, inflammation, environmental factors, or aging. Here we have examined the ability of the flavonoid quercetin to protect cutaneous tissue-associated cell types from injury induced by oxidative stress, and possible cooperative effects of ascorbic acid. Human skin fibroblasts, keratinocytes, and endothelial cells were cultured in the presence of buthionine sulfoximine (BSO), an irreversible inhibitor of glutathione (GSH) synthesis. Depletion of intracellular levels of GSH leads to an accumulation of cellular peroxides and eventual cell death. Quercetin concentration-dependently (EC50: 30-40 microM) reduced oxidative injury of BSO to all cell types, and was also effective when first added after BSO washout. BSO caused marked decreases in the intracellular level of GSH, which remained depressed in quercetin-protected cells. Ascorbic acid, while by itself not cytoprotective synergized with quercetin, lowered the quercetin EC50 and prolonged the window for cytoprotection. The related flavonoids rutin and dihydroquercetin also decreased BSO-induced injury to dermal fibroblasts, albeit less efficaciously so than quercetin. The cytoprotective effect of rutin, but not that of dihydroquercetin, was enhanced in the presence of ascorbic acid. Further, quercetin rescued sensory ganglion neurons from death provoked by GSH depletion. Direct oxidative injury to this last cell type has not been previously demonstrated. The results show that flavonoids are broadly protective for cutaneous tissue-type cell populations subjected to a chronic intracellular form of oxidative stress. Quercetin in particular, paired with ascorbic acid, may be of therapeutic benefit in protecting neurovasculature structures in skin from oxidative damage.

Glia are key players in a number of nervous system disorders. Besides releasing glial and neuronal signaling molecules directed to cellular homeostasis, glia respond also to pro-inflammatory signals released from immune-related cells, with the mast cell being of particular interest. A proposed mast cell–glia communication may open new perspectives for designing therapies to target neuroinflammation by differentially modulating activation of non-neuronal cells normally controlling neuronal sensitization—both peripherally and centrally. Mast cells and glia possess endogenous homeostatic mechanisms/molecules that can be upregulated as a result of tissue damage or stimulation of inflammatory responses. Such molecules include the N-acylethanolamines, whose principal family members are the endocannabinoid N-arachidonoylethanolamine (anandamide), and its congeners N-stearoylethanolamine, N-oleoylethanolamine, and N-palmitoylethanolamine (PEA). A key role of PEA may be to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation: PEA is produced and hydrolyzed by microglia, it downmodulates mast cell activation, it increases in glutamate-treated neocortical neurons ex vivo and in injured cortex, and PEA levels increase in the spinal cord of mice with chronic relapsing experimental allergic encephalomyelitis. Applied exogenously, PEA has proven efficacious in mast cell-mediated experimental models of acute and neurogenic inflammation. This fatty acid amide possesses also neuroprotective effects, for example, in a model of spinal cord trauma, in a delayed post-glutamate paradigm of excitotoxic death, and against amyloid β-peptide-induced learning and memory impairment in mice. These actions may be mediated by PEA acting through “receptor pleiotropism,” i.e., both direct and indirect interactions of PEA with different receptor targets, e.g., cannabinoid CB2 and peroxisome proliferator-activated receptor-alpha.

Nerve growth factor (NGF) is largely known as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurones and basal forebrain cholinergic nuclei during development and maturation. However, NGF also exerts a modulatory role on sensory, nociceptive nerve physiology during adulthood that appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other NGF-responsive cells are now recognized as belonging to the haemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with an increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general 'alert' molecule capable of recruiting and priming tissue defence processes following insult as well as systemic defensive mechanisms. Moreover, mast cells themselves produce NGF, suggesting that alterations in normal mast cell behaviours can provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states. This review discusses recent discoveries involving novel and diverse biological activities of this fascinating molecule.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Nerve growth factor (NGF), initially characterized for its survival and differentiating actions on embryonic sensory and sympathetic neurons, is now known to display a greatly extended spectrum of biological functions. NGF exerts a profound modulatory role on sensory nociceptive nerve physiology during adulthood which appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other newly detected NGF-responsive cells belong to the hematopoietic-immune and neuroendocrine systems. In particular, mast cells and NGF both appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general "alert" molecule capable of recruiting and priming both local tissue and systemic defense processes following stressful events. NGF can thus be viewed as a multifactorial mediator modulating neuroimmune-endocrine functions of vital importance to the regulation of homeostatic interactions, with potential involvement in pathological processes deriving from dysregulation of either local or systemic homeostatic balances.

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. This study describes the genetic construction, expression, purification and properties of a diphtheria toxin-related CNTF fusion gene in which the native receptor binding domain of diphtheria toxin was genetically replaced with a synthetic gene encoding human CNTF. The fusion protein expressed from the chimeric tox gene was designated DAB389-CNTF. This fusion toxin has a deduced molecular weight of 67 440 and is formed by the fusion of the first 389 amino acids of diphtheria toxin to amino acids 15-200 of mature human CNTF (Cys17-->Ser), using a bridge of 34 additional amino acids including six consecutive histidine residues. This latter span allows for a single-step purification of the fusion protein by Ni(2+)-immobilized metal ion affinity chromatography, and provides a degree of flexibility which facilitates polypeptide refolding. DAB389-CNTF was selectively cytotoxic for clonal cells bearing CNTF receptors and for CNTF-responsive spinal sensory ganglion neurons in primary culture. The cytotoxic action of DAB389-CNTF, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The delivery of the catalytic domain to the target cell cytosol results in inhibition of protein synthesis and cell death. This latter point was confirmed by the observation that both CNTF and DAB389-CNTF increased c-fos mRNA expression, but only CNTF induced Fos protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The psychoactive component of the cannabis resin and flowers, delta9-tetrahydrocannabinol (THC), was first isolated in 1964, and at least 70 other structurally related ‘phytocannabinoid’ compounds have since been identified. The serendipitous identification of a G-protein-coupled cannabinoid receptor at which THC is active in the brain heralded an explosion in cannabinoid research. Elements of the endocannabinoid system (ECS) comprise the cannabinoid receptors, a family of nascent lipid ligands, the ‘endocannabinoids’ and the machinery for their biosynthesis and metabolism. The function of the ECS is thus defined by modulation of these receptors, in particular, by two of the best-described ligands, 2-arachidonoyl glycerol and anandamide (arachidonylethanolamide). Research on the ECS has recently aroused enormous interest not only for the physiological functions, but also for the promising therapeutic potentials of drugs interfering with the activity of cannabinoid receptors. Many of the former relate to stress-recovery systems and to the maintenance of homeostatic balance. Among other functions, the ECS is involved in neuroprotection, modulation of nociception, regulation of motor activity, neurogenesis, synaptic plasticity and the control of certain phases of memory processing. In addition, the ECS acts to modulate the immune and inflammatory responses and to maintain a positive energy balance. This theme issue aims to provide the reader with an overview of ECS pharmacology, followed by discussions on the pivotal role of this system in the modulation of neurogenesis in the developing and adult organism, memory processes and synaptic plasticity, as well as in pathological pain and brain ageing. The volume will conclude with discussions that address the proposed therapeutic applications of targeting the ECS for the treatment of neurodegeneration, pain and mental illness.

Communication between the immune and nervous systems depends a great deal on pro-inflammatory cytokines. Both astroglia and microglia, in particular, constitute an important source of inflammatory mediators and may have fundamental roles in central nervous system (CNS) disorders from neuropathic pain and epilepsy to neurodegenerative diseases. Glial cells respond also to pro-inflammatory signals released from cells of immune origin. In this context, mast cells are of particular relevance. These immune-related cells, while resident in the CNS, are able to cross a compromised blood-spinal cord and blood-brain barrier in cases of CNS pathology. Emerging evidence suggests the possibility of mast cell–glia communication, 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 glia, neuro-immune interactions involving mast cells and the possibility that glia–mast cell interactions contribute to exacerbation of acute symptoms of chronic neurodegenerative disease and accelerated disease progression, as well as promotion of pain transmission pathways. Using this background as a starting point for discussion, we will consider the therapeutic potential of naturally occurring fatty acid ethanolamides, such as palmitoylethanolamide in treating systemic inflammation or blockade of signalling pathways from the periphery to the brain in such settings.

Glial cells not only serve supportive and nutritive roles for neurons, but also respond to protracted stress and insults by up-regulating inflammatory processes. The complexity of studying glial activation in vivo has led to the widespread adoption of in vitro approaches, for example the use of the bacterial toxin lipopolysaccharide (LPS, a ligand for toll-like receptor 4 (TLR4)) as an experimental model of glial activation. Astrocyte cultures frequently contain minor numbers of microglia, which can complicate interpretation of responses. In the present study, enriched (≤5% microglia) astrocytes cultured from neonatal rat cortex and spinal cord were treated with the lysosomotropic agent L-leucyl-L-leucine methyl ester to eliminate residual microglia, as confirmed by loss of microglia-specific marker genes. L-Leucyl-L-leucine methyl ester treatment led to a loss of LPS responsiveness, in terms of nitric oxide and cytokine gene up-regulation and mediator (pro-inflammatory cytokines, nitric oxide) output into the culture medium. Surprisingly, when astrocyte/microglia co-cultures were then reconstituted by adding defined numbers of purified microglia to microglia-depleted astrocytes, the LPS-induced up-regulation of pro-inflammatory gene and mediator output far exceeded that observed from cultures containing the same numbers of microglia only. Similar behaviors were found when examining interleukin-1β release caused by activation of the purinergic P2X7 receptor. Given that astrocytes greatly outnumber microglia in the central nervous system, these data suggest that a similar interaction between microglia and astrocytes in vivo may be an important element in the evolution of an inflammatory pathology.

Alzheimer's disease (AD) is the largest unmet medical need in neurology today. This most common form of irreversible dementia is placing a considerable and increasing burden on patients, caregivers, and society, as more people live long enough to become affected. Current drugs improve symptoms but do not have profound neuroprotective and/or disease-modifying effects. AD is characterized by loss of neurons, dystrophic neurites, senile/amyloid/neuritic plaques, neurofibrillary tangles, and synaptic loss. Beta-amyloid (Aβ) peptide deposition is the major pathological feature of AD. Increasing evidence suggests that overexpression of the amyloid precursor protein and subsequent generation of the 39-43 amino acid residue, Aβ, are central to neuronal degeneration observed in AD patients possessing familial AD mutations, while transgenic mice overexpressing amyloid precursor protein develop AD-like pathology. Despite the genetic and cell biological evidence that supports the amyloid hypothesis, it is becoming increasing clear that AD etiology is complex and that Aβ alone is unable to account for all aspects of AD. The fact that vast overproduction of Aβ peptides in the brain of transgenic mouse models fails to cause overt neurodegeneration raises the question as to whether accumulation of Aβ peptides is indeed the culprit for neurodegeneration in AD. There is increasing evidence to suggest that Aβ/amyloid-independent factors, including the actions of AD-related genes (microtubule-associated protein tau, polymorphisms of apolipoprotein E4), inflammation, and oxidative stress, also contribute to AD pathogenesis. This chapter reviews the current state of knowledge on these factors and their possible interactions, as well as their potential for neuroprotection targets.

Purine nucleotides are well established as extracellular signaling molecules. P2X7 receptors (P2X7Rs) are members of the family of ionotropic ATP-gated receptors. Their activity can be found in a limited number of cell types, but is readily detectable in cells of hemopoietic lineage including macrophages, microglia, and certain lymphocytes, and mediates the influx of Ca2+ and Na+ as well as the release of pro-inflammatory cytokines. Amongst P2X receptors, P2X7Rs behave as a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules with a mass of up to 900 Da. In humans at least, the P2RX7 gene is highly polymorphic, and genetic differences within P2X7R affect receptor pore formation and channel function. ATP can act as a neurotransmitter, while the presence of P2X7Rs on immune cells suggests that they also regulate immune function and inflammatory responses. In addition, activation of the P2X7R has dramatic cytotoxic properties. The role of extracellular ATP and purinoceptors in cytokine regulation and neurological disorders is, in fact, the focus of a rapidly expanding area of research. P2X7Rs may affect neuronal cell death by regulating the processing and release of interleukin-1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7Rs provides an inflammatory stimulus, and P2X7R-deficient mice display a marked attenuation of inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X7R activity, by regulating the release of pro-inflammatory cytokines, may be involved in the pathophysiology of neuropsychiatric disorders. The P2X7R may thus represent a critical communication link between the nervous and immune systems, while providing a target for therapeutic exploitation. In this review we discuss current biology and pharmacology of the P2X7R, as well as insights into the role for this receptor in neurological/psychiatric diseases.

In primary culture of the early postnatal cerebellum, glutamatergic granule cells are highly enriched and recapitulate many properties characteristic of developing granule neurons in vivo. For example, withdrawal of K(+) from differentiated rat primary cerebellar granule neurons results in the apoptotic death of the majority of cells after 48 h. Removal of cerebellar granule neurons from depolarizing culture conditions with high K(+) is thought to reflect the regulation of trophic action of neuronal activity and has found widespread application as a model for studying the mechanisms of survival factor withdrawal-induced neuronal cell apoptosis and the neuroprotective action of trophic agents. This chapter presents a protocol for the culture of postnatal rat cerebellar granule neurons and results in a preparation containing 95% glutamatergic granule cells and its application to the evaluation of corticotropin receptor agonists as neuroprotective agents.

The concentration of nerve growth factor (NGF) is elevated in a number of inflammatory and autoimmune states in conjunction with increased accumulation of mast cells. Mast cells, which are of hematopoietic lineage, and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells themselves are capable of producing and responding to NGF. Here we describe a protocol for the isolation and culture of peritoneal-derived rat mast cells, together with a [(3)H]serotonin release assay which is useful in assessing the effects of antigens and neurotrophic factors on mast-cell activation.

Neurons cultured from rodent central nervous system tissues represent an important tool in the study of neurodegenerative disease mechanisms and neuroregenerative processes, including the survival- and axon growth-promoting properties of neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat and mouse cortical and hippocampal neuron cell cultures using either embryonic or postnatal tissue with enzymatic digestion.

Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate. There are two major types of immunofluorescence staining methods: (1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye and (2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. This chapter describes procedures for the application of indirect immunofluorescence staining to neural cells in culture.

Glial cell activation plays an important role in the pathogenesis of various neurodegenerative disorders. This article presents a protocol for the preparation of cultures consisting of rat embryonic cortical neurons grown in the presence of cortical microglia, in which the glia are present in physical contact with the neurons or separated by a semi-permeable membrane barrier.

Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson's disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the neuroprotective action of brain-derived neurotrophic factor.

Retinal pigment epithelium cells play a key role in maintaining the normal function of retina and can express several neurotrophic factors, which support the neurosensory retina and may also provide trophic signals to the host dopaminergic neurons. The following chapter describes a protocol for the purification and culture of retinal pigmented epithelial cells from postnatal rat.

The neurotrophins are a family of closely related proteins that were first identified as survival factors for sympathetic and sensory neurons and have since been shown to control a number of aspects of survival, development, and function of neurons in both the central and peripheral nervous systems. Limiting quantities of neurotrophins during development control the numbers of surviving neurons to ensure a match between neurons and the requirement for a suitable density of target innervation. Biological effects of each of the four mammalian neurotrophins are mediated through activation of one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB, and TrkC). In addition, all neurotrophins activate the p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily. Neurotrophin engagement of Trk receptors leads to activation of Ras, phosphatidylinositol 3-kinase, phospholipase C-γ1, and signaling pathways controlled through these proteins, including the mitogen-activated protein kinases. Neurotrophin availability is required into adulthood, where they control synaptic function and plasticity and sustain neuronal cell survival, morphology, and differentiation. This chapter will provide an overview of neurotrophin biology, their receptors, and signaling pathways.

Central neurons lose the ability for axonal regrowth during development and typically do not regenerate their axons following axotomy once they become mature unless given a growth-permissive environment, for example, a peripheral nerve graft. Retinal ganglion cells (RGCs) of the optic nerve represent a highly useful cell model for the study of neurotrophic factor responsiveness, although the presence of nonneuronal cells in the retina makes it difficult to interpret the direct effects of tested factors on RGCs. Cultures of purified RGCs thus represent an excellent tool for the study of optic nerve cell trophic responsiveness, in terms of both survival and axonal regeneration.

Glial cells play a key role in nervous system function, providing neurotrophic factor support to neurons as well as taking part in two-way neuron-glia signaling (e.g., neurotransmitter release). White matter-derived glia are important in certain neurodegenerative diseases involving axonal loss, for example in multiple sclerosis. Here we describe procedures for the preparation and culture of mixed nerve cells from postnatal rat optic nerve, followed by protocols which can serve for the purification of individual populations of glia from this tissue, namely O2A progenitors and oligodendrocytes, and astrocytes and astrocyte precursors.

Neurons cultured from rodent central nervous system tissues represent an important tool in the study of neurodegenerative disease mechanisms and neuroregenerative processes, including the survival- and axon growth-promoting properties of neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat and mouse cortical and hippocampal neuron cell cultures, using either embryonic or postnatal tissue.

In retinal diseases characterized by photoreceptor degeneration, the main cause of clinically significant vision loss is cone, rather than rod, loss. Photoreceptor apoptosis can be affected by the availability and/or delivery of neurotrophic proteins, and cultures of photoreceptors are valuable for studying these processes. In the present study, a technique was designed to purify cones to make it possible to screen for neuroprotective molecules. The present chapter describes a technique for preparing cultures of purified rat retina cone photoreceptors based upon panning with peanut agglutinin lectin, which selectively binds to cones. In addition, we describe a protocol for the purification and culture of retinal pigmented epithelial cells from postnatal rat.

Neurotrophic factors released by target tissues maintain the survival and differentiation of innervating neurons. The manner by which these target-derived neurotrophic proteins communicate with innervating neurons has been actively pursued for over three decades. The present chapter describes a technique for preparing and maintaining compartmented chambers for culturing neurons derived from either superior cervical ganglia (sympathetic neurons) or dorsal root ganglia (sensory neurons). This system recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins.

Neurons cultured from rodent central nervous system tissues represent an important tool in the study of neurodegenerative disease mechanisms and neuroregenerative processes, including the survival- and axon growth-promoting properties of neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat and mouse cortical and hippocampal neuron cell cultures using either embryonic or postnatal tissue with enzymatic digestion.