Sergio Visentin

Metabotropic P2 receptor activation regulates oligodendrocyte progenitor migration and development

To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X1,2,3,4,7) and P2Y (P2Y1,2,4) receptors. Intracellular Ca2+ recording by Fura‐2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPβS = ADP = Benzoyl ATP > ATP > ATPγS > UTP, α,β‐meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of α,βmeATP‐induced inward current in whole‐cell recording, P2X7 and P2Y1 were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y1‐specific agonist ADPβS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet‐derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y1 antagonist MRS2179. By confocal laser scanning microscopy, P2Y1 receptors were localized in NG2‐labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y1 receptors.

Two different ionotropic receptors are activated by ATP in rat microglia

1 Our aim was to assess whether ATP‐induced inward currents in microglia are due to a single or more than one purinergic receptor. The ATP dose‐response curve showed two components, whose presence might be due to the activation of high and low affinity receptors. 2 The P2Z/P2X7 specific receptor agonist benzoylbenzoyl‐ATP (Bz‐ATP) and some P2 receptor agonists were tested. The rank order of potency was Bz‐ATP >> ATP = 2‐methylthio‐ATP (2‐MeSATP) > α,β‐methylene ATP (α,β‐meATP) ≥ ADP. β,γ‐MethyleneATP (β,γ‐meATP), UTP and adenosine were ineffective. 3 The non‐specific P2 receptor antagonist suramin antagonized by 92 ± 2 % the inward current induced by 100 μm ATP, and by 51 ± 8 and 68 ± 6 % those induced by 3 mM ATP and 100 μm Bz‐ATP, respectively. The P2Z/P2X7 antagonist oxidized ATP (oATP) almost abolished the inward current induced by 3 mM ATP or Bz‐ATP, but was ineffective against 100 μm ATP. 4 Inward currents induced by low ATP concentrations (≤ 100 μm) were generally followed by an almost complete and irreversible desensitization, while those elicited by ATP ≥ 1 mM showed only a partial decline. Interestingly, the inward current induced by 100 μm 2‐MeSATP showed a large desensitization, while that induced by Bz‐ATP did not. 5 In voltage‐ramp experiments, the 100 μm ATP‐induced current exhibited a slight inward rectification more visible at negative potentials, while the 3 mM ATP‐induced current did not. 6 ATP induced a fast and large increase in [Ca2+] that promptly recovered in the continuous presence of low ATP doses, but did not recover in high ATP doses. As with desensitization, the response to Bz‐ATP mimicked that of high doses of ATP. 7 When Ca2+ mobilization due to P2Y receptors was blocked by thapsigargin‐induced Ca2+ depletion or by pertussis toxin treatment, 10 μm ATP was still able to induce a Ca2+ transient, which represented the contribution of the Ca2+ influx induced by P2X receptors 8 In conclusion, the inward currents and a fraction of the Ca2+ transients induced by ATP in microglia are due to at least two ATP‐sensitive receptor channel types, whose different properties and sensitivity to ATP may be associated with different functional roles.

TGF‐β and LPS modulate ADP‐induced migration of microglial cells through P2Y1 and P2Y12 receptor expression

J. Neurochem. (2010) 115, 450–459.

Triazine compounds as antagonists at Bv8-prokineticin receptors.

On the basis of a Janssens patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential non peptidic prokineticin receptor antagonists, containing the following substitutions: (N(1) and N(5) link a 4-methoxybenzyl and a 4-ethylbenzyl, respectively; C(2) can link an amino-ethyl-guanidine (reference compound 1) or an ethylendiamine (2) or an amino-ethyl-amino-2-imidazoline (3). New compounds were assessed for PKR1 and PKR2 affinity. Antagonist properties were evaluated as inhibition of 1 nM Bv8-induced intracellular Ca2+ mobilization.

Branched-chain amino acids influence the immune properties of microglial cells and their responsiveness to pro-inflammatory signals.

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids involved in several important brain functions. Although commonly used as nutritional supplements, excessive intake of BCAAs might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of BCAA catabolism such as maple syrup urine disease (MSUD). Recent evidence indicates that BCAAs induce excitotoxicity through mechanisms that require the presence of astrocytes. In the present study, we evaluated the effects of BCAAs on microglia, the main immune cells of the brain. As an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high BCAA medium (H-BCAA). We show that H-BCAA microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the M2 state, with enhanced IL-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. We suggest that such an intermediate M1/M2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. Although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. In addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which BCAAs might turn into toxicants and facilitate neurodegeneration.

Curcumin Protects against NMDA-Induced Toxicity: A Possible Role for NR2A Subunit

PURPOSE Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). RESULTS Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

Multiple Actions of the Human Immunodeficiency Virus Type-1 Tat Protein on Microglial Cell Functions

The human immunodeficiency virus type-1 (HIV-1) regulatory protein Tat is produced in the early phase of infection and is essential for virus replication. Together with other viral products, Tat has been implicated in the pathogenesis of HIV-1–associated dementia (HAD). As HIV-1 infection in the brain is very limited and macrophage/microglial cells are the only cellular type productively infected by the virus, it has been proposed that many of the viral neurotoxic effects are mediated by microglial products. We and others have shown that Tat affects the functional state of microglial cells, supporting the hypothesis that activated microglia play a role in the neuropathology associated with HIV-1 infection. This review describes the experimental evidence indicating that Tat stimulates microglia to synthesize potentially neurotoxic molecules, including proinflammatory cytokines and free radicals, and interferes with molecular mechanisms controlling cAMP levels, intracellular [Ca2+], and ion channel expression.

Arachidonic acid-induced inhibition of microglial outward-rectifying K+ current

In cultured microglial cells pro‐inflammatory substances such as lipopolysaccharide and interferon‐γ induce outward‐rectifying K+ channels (OR) exhibiting features of the Kv1.3 type. Here we studied the modulation of this channel by arachidonic acid (AA). Micromolar doses of AA (0.3–30 μM; ED50 1.55 μM) depressed OR currents at all the potentials tested. Such effect appeared in less than 30 s, reached the maximum in approximately 2 min, and partially reverted upon removal of AA. We then tested whether AA acted by mechanisms involving enzyme activation. The AA effect on OR remained unchanged in the presence of staurosporine (protein kinase C inhibitor), indomethacin (cyclooxygenase inhibitor), nordihydroguaiaretic acid (lipoxygenase inhibitor), and diphenylene iodonium (NADPH‐oxidase inhibitor). The same effect was present in cell‐free membrane patches in the outside‐out configuration. In whole‐cell recording AA induced the following changes in OR current: (a) decrease of OR current peak at all voltages tested; (b) increase of the activation rate and mild shift of the voltage‐dependence of the activation; (c) acceleration of the inactivation rate with a concomitant appearance of a second and faster inactivation component; and (d) shift of the voltage‐dependence of the inactivation curve. We also observed that upon AA application, the acceleration of activation and inactivation developed earlier than the second component of inactivation. We conclude that AA, by acting on both the channel protein and its lipid environment, causes a quick negative modulation of microglial OR current. We propose that a raised free AA may determine a loss of efficiency of OR in controlling the membrane potential and thus influence the functional reactivity of microglia to inflammatory stimuli. GLIA 22:1–10, 1998.

Megalencephalic leukoencephalopathy with subcortical cysts protein 1 functionally cooperates with the TRPV4 cation channel to activate the response of astrocytes to osmotic stress: dysregulation by pathological mutations

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation, has been linked to mutations in the MLC1 gene. This gene encodes a membrane protein that is highly expressed in astrocytes. Based on MLC pathological features, it was proposed that astrocyte-mediated defects in ion and fluid homeostasis could account for the alterations observed in MLC-affected brains. However, the role of MLC1 and the effects of pathological mutations on astrocyte osmoregulatory functions have still to be demonstrated. Using human astrocytoma cells stably overexpressing wild-type MLC1 or three known MLC-associated pathological mutations, we investigated MLC1 involvement in astrocyte reaction to osmotic changes using biochemical, dynamic video imaging and immunofluorescence techniques. We have found that MLC1 overexpressed in astrocytoma cells is mainly localized in the plasma membrane, is part of the Na,K-ATPase-associated molecular complex that includes the potassium channel Kir4.1, syntrophin and aquaporin-4 and functionally interacts with the calcium permeable channel TRPV4 (transient receptor potential vanilloid-4 cation channel) which mediates swelling-induced cytosolic calcium increase and volume recovery in response to hyposmosis. Pathological MLC mutations cause changes in MLC1 expression and intracellular localization as well as in the astrocyte response to osmotic changes by altering MLC1 molecular interactions with the Na,K-ATPase molecular complex and abolishing the increase in calcium influx induced by hyposmosis and treatment with the TRPV4 agonist 4αPDD. These data demonstrate, for the first time, that MLC1 plays a role in astrocyte osmo-homeostasis and that defects in intracellular calcium dynamics may contribute to MLC pathogenesis.

Nonenzymatic oxygenated metabolites of α-linolenic acid B1- and L1-phytoprostanes protect immature neurons from oxidant injury and promote differentiation of oligodendrocyte progenitors through PPAR-γ activation.

Phytoprostanes (PhytoPs) are formed in higher plants from α-linolenic acid via a nonenzymatic free radical-catalyzed pathway and act as endogenous mediators capable of protecting cells from damage under various conditions related to oxidative stress. Humans are exposed to PhytoPs, as they are present in relevant quantities in vegetable food and pollen. The uptake of PhytoPs through the olfactory epithelium of the nasal mucosa, upon pollen grain inhalation, is of interest as the intranasal pathway is regarded as a direct route of communication between the environment and the brain. On this basis, we sought to investigate the potential activities of PhytoPs on immature cells of the central nervous system, which are particularly susceptible to oxidative stress. In neuroblastoma SH-SY5Y cells, used as a model for undifferentiated neurons, B1-PhytoPs, but not F1-PhytoPs, increased cell metabolic activity and protected them from oxidant damage caused by H2O2. Moreover, B1-PhytoPs induced a moderate depolarization of the mitochondrial inner membrane potential. These effects were prevented by the PPAR-γ antagonist GW9662. When SH-SY5Y cells were induced to differentiate toward a more mature phenotype, they became resistant to B1-PhytoP activities. B1-PhytoPs also influenced immature cells of an oligodendroglial line, as they increased the metabolic activity of oligodendrocyte progenitors and strongly accelerated their differentiation to immature oligodendrocytes, through mechanisms at least partially dependent on PPAR-γ activity. However, B1-PhytoPs did not protect oligodendrocyte progenitors against oxidant injury. Taken together, these data suggest that B1-PhytoPs, through novel mechanisms involving PPAR-γ, can specifically affect immature brain cells, such as neuroblasts and oligodendrocyte progenitors, thereby conferring neuroprotection against oxidant injury and promoting myelination.