Pietro Giusti

The P2X7 purinergic receptor: from physiology to neurological disorders

Purine nucleotides are well established as extracellular signaling molecules. P2X receptors are ATP‐gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X7, have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in progressive dilation of the channel pore and the development of permeability to molecules as large as 900 Da. The P2X7 receptor was originally described in cells of hematopoietic origin, including macrophages, microglia, and certain lymphocytes, and mediates the influx of Ca2+ and Na+ ions, as well as the release of proinflammatory cytokines. P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin‐1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7 receptors provides an inflammatory stimulus, and P2X7 receptor‐deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X7 receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. The P2X7 receptor may thus represent a critical communication link between the nervous and immune systems, while providing a target for therapeutic exploitation. This review discusses the current biology and cellular signaling pathways of P2X7 receptor function, as well as insights into the role for this receptor in neurological/psychiatric diseases, outstanding questions, and the therapeutic potential of P2X7 receptor antagonism.—Skaper, S. D., Debetto, P., Giusti, P. The P2X7 purinergic receptor: from physiology to neurological disorders. FASEB J. 24, 337–345 (2010). www.fasebj.org

{alpha}-Synuclein and Parkinson's disease

Alpha‐synuclein (α‐syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in α‐syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinsons disease (PD). The physiological functions of α‐syn remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of α‐syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, α‐syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. α‐Syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which α‐syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin‐1, chaperone 14‐3‐3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that α‐syn‐based models represent a paradigm, which is closest to the human pathology.— Recchia, A., Debetto, P., Negro, A., Guidolin, D., Skaper, S. D., Giusti, P. α‐Synuclein and Parkinsons disease.

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.

Melatonin protects against 6-OHDA-induced neurotoxicity in rats: a role for mitochondrial complex I activity

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‐OHDAresults 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 Parkinsons 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 melatonintreated 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.—Dabbeni‐Sala, F., Di Santo, S., Franceschini, D., Skaper, S. D., Giusti, P. Melatonin protects against 6‐OHDA‐induced neurotoxicity in rats: a role for mitochondrial complex I activity. FASEB J. 15, 164–170 (2001)

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

Neuroprotection by melatonin from kainate-induced excitotoxicity in rats.

In this study, we injected 10 mg/kg kainate i.p. into rats. This resulted in a brain injury, which we quantified in the hippocampus, the amygdala, and the pyriform cortex. Neuronal damage was preceded by a set of typical behavioral signs and by biochemical changes (noradrenaline decrease and 5‐hydroxyindoleacetic acid increase) in the af‐fected brain areas. Melatopin (2.5 mg/kg) was injected i.p. four times: 20 min before kainate, immediately after, and 1 and 2 h after the kainate. The cumulative dose of 10 mg/kg melatonin prevented kainate‐induced neuronal death as well as behavioral and biochemical disturbances. A possible mechanism of melatonin‐provided neuroprotection lies in its antioxidant action. Our results suggest that melatonin holds potential for the treatment of pathologies such as epilepsy‐associated brain damage, stroke, and brain trauma.—Giusti, P., Lipartiti, M., Franceschini, D., Schiavo, N., Floreani, M., Manev, H. Neuroprotection by melatonin from kainate‐induced excitotoxicity in rats. FASEB J. 10, 891‐896 (1996)

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.

Melatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl-D-aspartate excitotoxicity.

the antiexcitotoxic efficacy of melatonin, a putative endogenous hydroxyl radical scavenger, was studied in primary cultures of rat cerebellar granule neurons. Excitotoxicity was induced in 7- to 9-day-old cultures by an exposure to glutamate (15 min in the absence of magnesium) or to glutamate receptor agonists, kainate (30 min), and N-methyl-D-aspartate (60 min in the absence of magnesium). Thereafter, cultures were returned to the culture-conditioned medium for 18 h at the end of which time viability was assessed by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Cotreatment with melatonin (500 microM) protected the neurons completely from the toxicity of kainate (up to 1 mM) and shifted the ED50 for glutamate from 55 +/- 2.6 to 97 +/- 3.6 microM. Melatonin cotreatment was ineffective in protecting the neurons from N-methyl-D-aspartate toxicity. When melatonin was added to the cultures only before or after kainate treatment, there was no resultant protection from kainate toxicity. The neuroprotective effect of melatonin does not appear to be related to the direct action of melatonin on ionotropic glutamate receptors. That is, the kainate-stimulated inward currents measured by a patch-clamp technique in voltage clamped neurons and the kainate-stimulated increase in free cytosolic calcium measured at the single-cell level using digital imaging fluorescent microscopy with fura-2 were not affected by melatonin. Moreover, the binding of [3H]glutamate to rat cerebellar membranes was not competed off by melatonin. Further studies are needed to evaluate the pharmacologic relevance of the neuroprotective action of melatonin.

Protective effect of melatonin against hippocampal dna damage induced by intraperitoneal administration of kainate to rats

The pineal hormone melatonin protects neurons in vitro from excitotoxicity mediated by kainate-sensitive glutamate receptors and from oxidative stress-induced DNA damage and apoptosis. Intraperitoneal injection on kainate into experimental animals triggers DNA damage in several brain areas, including the hippocampus. It is not clear whether melatonin is neuroprotective in vivo. In this study, we tested the in vivo efficacy of melatonin in preventing kainate-induced DNA damage in the hippocampus of adult male Wistar rats. Melatonin and kainate were injected i.p. Rats were killed six to 72 h later and their hippocampi were examined for evidence of DNA damage (in situ dUTP-end-labeling, i.e. TUNEL staining) and for cell viability (Nissl staining). Quantitative assay was performed using computerized image analysis. At 48 and 72 h after kainate we found TUNEL-positive cells in the CA1 region of the hippocampus; in the adjacent sections that were Nissl-stained, we found evidence of cell loss. Both the number of TUNEL-positive cells and the loss of Nissl staining were reduced by i.p. administration of melatonin (4 x 2.5 mg/kg; i.e. 20 min before kainate, immediately after, and 1 and 2 h after the kainate). Our results suggest that melatonin might reduce the extent of cell damage associated with pathologies such as epilepsy that involve the activation of kainate-sensitive glutamate receptors.

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.