Claudio Frank

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.

Cholesterol depletion inhibits synaptic transmission and synaptic plasticity in rat hippocampus

Several neurodegenerative disorders are associated with impaired cholesterol homeostasis in the nervous system where cholesterol is known to play a role in modulating synaptic activity and stabilizing membrane microdomains. In the present report, we investigated the effects of methyl-beta-cyclodextrin-induced cholesterol depletion on synaptic transmission and on the expression of 1) paired-pulse facilitation (PPF); 2) paired-pulse inhibition (PPI) and 3) long-term potentiation (LTP) in the CA1 hippocampal region. Results demonstrated that cyclodextrin strongly reduced synaptic transmission and blocked the expression of LTP, but did not affect PPF and PPI. The role of glutamatergic and GABAergic receptors in these cholesterol depletion-mediated effects was evaluated pharmacologically. Data indicate that, in cholesterol depleted neurons, modulation of synaptic transmission and synaptic plasticity phenomena are sustained by AMPA-, kainate-and NMDA-receptors but not by GABA-receptors. The involvement of AMPA-and kainate-receptors was confirmed by fluorimetric analysis of intracellular calcium concentrations in hippocampal cell cultures. These data suggest that modulation of receptor activity by manipulation of membrane lipids is a possible therapeutic strategy in neurodegenerative disease.

Clostridium difficile Toxin B Causes Apoptosis in Epithelial Cells by Thrilling Mitochondria INVOLVEMENT OF ATP-SENSITIVE MITOCHONDRIAL POTASSIUM CHANNELS

Targeting to mitochondria is emerging as a common strategy that bacteria utilize to interact with these central executioners of apoptosis. Several lines of evidence have in fact indicated mitochondria as specific targets for bacterial protein toxins, regarded as the principal virulence factors of pathogenic bacteria. This work shows, for the first time, the ability of the Clostridium difficile toxin B (TcdB), a glucosyltransferase that inhibits the Rho GTPases, to impact mitochondria. In living cells, TcdB provokes an early hyperpolarization of mitochondria that follows a calcium-associated signaling pathway and precedes the final execution step of apoptosis (i.e. mitochondria depolarization). Importantly, in isolated mitochondria, the toxin can induce a calcium-dependent mitochondrial swelling, accompanied by the release of the proapoptogenic factor cytochrome c. This is consistent with a mitochondrial targeting that does not require the Rho-inhibiting activity of the toxin. Of interest, the mitochondrial ATP-sensitive potassium channels are also involved in the apoptotic response to TcdB and appear to be crucial for the cell death execution phase, as demonstrated by using specific modulators of these channels. To our knowledge, the involvement of these mitochondrial channels in the ability of a bacterial toxin to control cell fate is a hitherto unreported finding.

Cholesterol perturbing agents inhibit NMDA‐dependent calcium influx in rat hippocampal primary culture

The present study was carried out to investigate the potential involvement of cholesterol‐rich membrane microdomains in the mobilization of calcium induced by NMDA‐receptors (NMDA‐R). We herein provide evidence that agents interfering with plasma membrane cholesterol (namely, filipin and methyl‐β‐cyclodextrin (Cdex)) inhibit the NMDA‐stimulated influx of calcium in hippocampal cells in culture. Filipin‐treated cells maintained their morphology and were able to respond with a calcium influx to high K+ challenge, whereas Cdex altered both cellular parameters. These results suggest that the NMDA‐R can be located in cholesterol‐rich membrane microdomains or alternatively that the mechanisms coupling their dynamics in the post‐synaptic membrane are dependent on the integrity of the microdomains.

Adenosine A2A receptor blockade differentially influences excitotoxic mechanisms at pre‐ and postsynaptic sites in the rat striatum

Adenosine A2A receptor antagonists are being regarded as potential neuroprotective drugs, although the mechanisms underlying their effects need to be better studied. The aim of this work was to investigate further the mechanism of the neuroprotective action of A2A receptor antagonists in models of pre‐ and postsynaptic excitotoxicity. In microdialysis studies, the intrastriatal perfusion of the A2A receptor antagonist ZM 241385 (5 and 50 nM) significantly reduced, in an inversely dose‐dependent way, the raise in glutamate outflow induced by 5 mM quinolinic acid (QA). In rat corticostriatal slices, ZM 241385 (30–100 nM) significantly reduced 4‐aminopyridine (4‐AP)‐induced paired‐pulse inhibition (PPI; an index of neurotransmitter release), whereas it worsened the depression of field potential amplitude elicited by N‐methyl‐D‐aspartate (NMDA; 12.5 and 50 μM). The A2A antagonist SCH 58261 (30 nM) mimicked the effects of ZM 241385, whereas the A2A agonist CGS 21680 (100 nM) showed a protective influence toward 50 μM NMDA. In rat striatal neurons, 50 nM ZM 241385 did not affect the increase in [Ca2+]i or the release of lactate dehydrogenase (LDH) induced by 100 and 300 μM NMDA, respectively. The ability of ZM 241385 to prevent QA‐induced glutamate outflow and 4‐AP‐induced effects confirms that A2A receptor antagonists have inhibitory effects on neurotransmitter release, whereas the results obtained toward NMDA‐induced effects suggest that A2A receptor blockade does not reduce, or even amplifies, excitotoxic mechanisms due to direct NMDA receptor stimulation. This indicates that the neuroprotective potential of A2A antagonists may be evident mainly in models of neurodegeneration in which presynaptic mechanisms play a major role.

Neuroprotective effects of allopregnenolone on hippocampal irreversible neurotoxicity in vitro

1. The effects of allopregnenolone, a neurosteroid, endowed with GABAmimetic properties, were tested towards two models of irreversible hippocampal neurotoxicity: i) the irreversible depression produced by hypoxia on the CA1 evoked field potentials in rat hippocampal slices, and ii) glutamate-induced irreversible changes in intracellular calcium concentration in primary hippocampal cell coltures. 2. In control conditions during the reoxygenation period after the application of 15 min of hypoxia, the CA1 evoked field potentials were irreversibly suppressed in almost the 50% of the experiments. In the remaining experiments there were a significative (p<0.01) irreversible reduction of the magnitude of the CA1 population spike with respect with the pre-hypoxia values. Allopregnenolone (50-75 microM) perfused 30 min before, during and 30 min after hypoxia produced a significative (p<0.05) decrease both in the hypoxia-induced irreversible suppression of the CA1 PS and both in the irreversible decrease of the CA1 PS at the end of reoxygenation. 3. The exposition of the primary hippocampal cultured cells to glutamate 0.5 mM for 10 min was followed by a sustained elevation of [Ca2+]i, that persisted at 70-80% of maximal increase for the rest of the experiment (60 min). When a pretreatment with 10-50 microM allopregnanolone preceded Glu 0.5 mM application, [Ca2+]i increased to a maximal value during the glutamate application, after which a fast decrease to 50% was observed, followed by a slow recovery within about 30 min. 4. The results showed that the neurosteroid allopregnenolone, endowed with GABAmimetic properties, ameliorated the functional correlates of irreversible hippocampal neurotoxicity.

Selective reduction of hippocampal dentate frequency potentiation in aged rats with impaired place learning

Induction of posttetanic potentiation (PTP) and long-term potentiation (LTP) was analyzed in hippocampal slices obtained from a) young 6-month-old Sprague-Dawley (SD) rats, all of them performing well in the Morris Maze, and b) aged SD 20-month-old and Fischer 344 24-month-old rats showing different degrees of ability in the same test. After the application of an electrical tetanus 1 s, 100 Hz, 50 microA in the stratum radiatum, no significant differences were found in the percent of induction of both PTP and LTP in the CA1 area of hippocampal slices obtained from rats of different strains and ages. After the application of an electrical tetanus 1 s, 100 Hz, 50 microA in the stratum moleculare, a significant difference was found in the percent of dentate PTP induction in hippocampal slices obtained from rats of different ages. Specifically, dentate PTP induction was significantly (p < 0.01) higher in slices obtained from young SD rats, and from old SD rats with a better performance in the Morris maze, escape latency less than 10 s and 150 cm, than in slices obtained from old SD or Fischer 344 rats that had shown poor performance in the Morris Maze. On the contrary, no significant differences were found in the percent of dentate LTP in hippocampal slices obtained from rats of different strains and ages. The data demonstrate that the induction of hippocampal dentate high-frequency PTP is selectively reduced in old rats with impaired Morris Maze performance.

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.

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A2A, cannabinoid CB1 and metabotropic glutamate 5 (mGlu5) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB1R) agonist WIN55,212‐2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A2ARs. Such a permissive effect of A2ARs towards CB1Rs does not seem to occur pre‐synaptically as the ability of WIN55,212‐2 to increase the R2/R1 ratio under a protocol of paired‐pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212‐2 were reduced in slices from mice lacking post‐synaptic striatal A2ARs. The selective mGlu5R agonist (RS)‐2‐chloro‐5‐hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212‐2, and such a potentiation was abolished by A2AR blockade. Unlike the synaptic effects, the ability of WIN55,212‐2 to prevent NMDA‐induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A2ARs regulates the synaptic effects of CB1Rs and that A2ARs may control CB1 effects also indirectly, namely through mGlu5Rs.

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity.

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.