Maurizio Grimaldi

Neuroprotective Antioxidants from Marijuanaa

Abstract: Cannabidiol and other cannabinoids were examined as neuroprotectants in rat cortical neuron cultures exposed to toxic levels of the neurotransmitter, glutamate. The psychotropic cannabinoid receptor agonist Δ9‐tetrahydrocannabinol (THC) and cannabidiol, (a non‐psychoactive constituent of marijuana), both reduced NMDA, AMPA and kainate receptor mediated neurotoxicities. Neuroprotection was not affected by cannabinoid receptor antagonist, indicating a (cannabinoid) receptor‐independent mechanism of action. Glutamate toxicity can be reduced by antioxidants. Using cyclic voltametry and a fenton reaction based system, it was demonstrated that Cannabidiol, THC and other cannabinoids are potent antioxidants. As evidence that cannabinoids can act as an antioxidants in neuronal cultures, cannabidiol was demonstrated to reduce hydroperoxide toxicity in neurons. In a head to head trial of the abilities of various antioxidants to prevent glutamate toxicity, cannabidiol was superior to both a‐tocopherol and ascorbate in protective capacity. Recent preliminary studies in a rat model of focal cerebral ischemia suggest that cannabidiol may be at least as effective in vivo as seen in these in vitro studies.

Functional and molecular diversity of PACAP/VIP receptors in cortical neurons and type I astrocytes

In the present study we determined the mRNA‐expression of pituitary adenylate cyclase activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptors in primary cultures of rat cortical neurons and type I astrocytes, and investigated the effects of PACAP38 on adenylyl cyclase, inositol phospholipid hydrolysis and intracellular calcium homeostasis. PACAP38 elicited a concentration‐dependent (1 n m–100 nm) increase in inositol phosphate levels and [Ca2+]i in neurons but not in type I astrocytes. The PACAP‐induced increase of intracellular calcium concentration, [Ca2+]i, was characterized by a spike, compatible with inositol trisphosphate (IP3) ‐induced calcium mobilization from intracellular stores, and a plateau phase, sustained by activation of capacitative calcium entry triggered by depletion of IP3‐sensitive calcium stores. In the absence of extracellular calcium, only the spike phase was present while the plateau phase was clearly reduced. In addition, thapsigargin pretreatment abolished the PACAP38‐induced [Ca2+]i rise. Treatment with 1 μm VIP did not affect [Ca2+]i in either neurons or type I astrocytes, clearly indicating the coupling of PAC1–HOP subtype to phospholipase‐C in neurons. In addition, as previously reported, PACAP38 stimulated cAMP formation in both neurons and type I astrocytes. Using the reverse transcription polymerase chain reaction, we found mRNA‐expression of PAC1 (PACAP – HOP variant) and VPAC2 in neurons, PAC1 (PACAP – R variant), VPAC1 and VPAC2 in astrocytes. These data indicate both a functional and molecular diversity of PACAP and VIP receptors in these cell types and support the view that the PAC1‐HOP variant may be responsible for phospholipase‐C activation and [Ca2+]i elevation in cortical neurons.

Prevention of beta-amyloid neurotoxicity by blockade of the ubiquitin-proteasome proteolytic pathway.

Abstract: In many neurodegenerative disorders, such as Alzheimers disease, inclusions containing ubiquitinated proteins have been found in the brain, suggesting a pathophysiological role for ubiquitin‐mediated proteasomal degradation of neuronal proteins. Here we show for the first time that the β‐amyloid fragment 1‐40, which in micromolar levels causes the death of cortical neurons, also induces the ubiquitination of several neuronal proteins. Prevention of ubiquitination and inhibition of proteasome activity block the neurotoxic effect of β‐amyloid. These data suggest that β‐amyloid neurotoxicity may cause toxicity through the activation of protein degradation via the ubiquitin—proteasome pathway. These findings suggest possible new pharmacological targets for the prophylaxis and/or treatment of Alzheimers disease and possibly for other related neurodegenerative disorders.

cAMP-induced Cytoskeleton Rearrangement Increases Calcium Transients through the Enhancement of Capacitative Calcium Entry

In this report we investigated the correlation between cell morphology and regulation of cytosolic calcium homeostasis. Type I astrocytes were differentiated to stellate process-bearing cells by a 100-min exposure to cAMP. Differentiation of cortical astrocytes increased the magnitude and duration of calcium transients elicited by phospholipase C-activating agents as measured by single cell Fura-2-based imaging. Calcium imaging showed differences in the spatial pattern of the response. In both differentiated and the control cells, the response originated in the periphery and gradually extended into the center of the cell. However, the elevation of cytosolic calcium concentration ([Ca2+] i ) was particularly evident within the processes and adjacent to the inner cell membrane of the differentiated astrocytes. In addition, differentiation significantly prolonged the duration of the [Ca2+] i elevation. Potentiation of the calcium transients was mimicked by forskolin-induced differentiation and abolished by a specific protein kinase-A blocker. Conversely, the enhancement of the calcium transients was not mimicked by brief exposure to cAMP not causing morphological differentiation, and in PC12 cells that did not undergo morphological changes after 100 min of cAMP treatment. Impairing cAMP-induced cytoskeleton re-organization, by means of cytochalasin D and nocodazole, prevented the potentiation of the calcium transients in cAMP-treated astrocytes. Phospholipase C activity and sensitivity to inositol (1,4,5)-trisphosphate were not involved in the enhancement of the calcium responses. Also, potentiation of the calcium transients was dependent on extracellular calcium. Calcium storage and thapsigargin-depletable intracellular calcium reservoirs were analogously not increased in differentiated astrocytes. Rearrangement of the cell shape also caused a condensation of the endoplasmic reticulum and altered the spatial relationship between the endoplasmic reticulum and the cell membrane. In conclusion, morphological rearrangements of type I astrocytes increase the magnitude and the duration of agonist-induced calcium transients via enhancement of capacitative calcium entry and is associated with a spatial reorganization of the relationship between cell membrane and the endoplasmic reticulum structures.

Somatostatin and SMS 201-995 reverse the impairment of cognitive functions induced by cysteamine depletion of brain somatostatin

The involvement of somatostatin in the organization of cognitive functions was studied. We assessed changes in learning and memory processes by studying the effects of cysteamine, a compound that decreases somatostatin-like immunoreactivity in the brain, somatostatin and the potent somatostatin analogue, SMS 201-995, on active avoidance behaviour, assessed with a shuttle box apparatus, or on passive avoidance behaviour. Cysteamine induced a loss of the conditioned active avoidance response acquired after 3 weeks of daily trials. The effect was observed 2 h (-29%) and 4 h (-51%) after cysteamine treatment (300 mg/kg s.c.) and disappeared after 24 h. Intracerebroventricular administration of somatostatin or SMS 201-995 to cysteamine-treated rats significantly reversed the cysteamine effects on the conditioned avoidance responses. Similar results were obtained on passive avoidance behaviour. We also investigated the effect of cysteamine treatment on brain somatostatin-sensitive adenylate cyclase. We observed that adenylate cyclase activity in the frontal cortex of cysteamine-pretreated animals was more sensitive to inhibition by the SRIF analogue, SMS 201-995, than it was in control animals. This effect was observed at concentrations of SMS 201-995 that were ineffective in control tissue. These results show that disruption of somatostatinergic transmission affects cognitive functions of rats.

Mobilization of calcium from intracellular stores, potentiation of neurotransmitter-induced calcium transients, and capacitative calcium entry by 4-aminopyridine.

In this study we analyzed the effect of 4-aminopyridine (4-AP) on free cytosolic calcium concentration ([Ca2+]i) in basal conditions, after stimulation with neurotransmitters, and during capacitative calcium entry. Using fura-2 ratiometric calcium imaging, we found that 4-AP increased [Ca2+]i in type I astrocytes, neurons, and in skeletal muscle cells. The [Ca2+]i elevation induced by 4-AP was concentration-dependent and consisted of two phases: the first was dependent on intracellular calcium mobilization, and the second was dependent on extracellular calcium influx. 4-AP also increased the second messenger inositol trisphosphate in both neurons and astrocytes. In astrocytes, 4-AP treatment potentiated the sustained phase of the [Ca2+]i elevation induced by ATP and bradykinin. In addition, capacitative calcium entry was potentiated severalfold by 4-AP, in astrocytes and muscle cells but not in neurons. These effects of 4-AP were completely and promptly reversible. 4-AP blocked voltage-sensitive K+ currents in astrocytes. However, voltage-sensitive K+ channel blockers inhibiting these currents did not affect agonist-induced calcium transients or capacitative calcium entry, indicating that 4-AP effects on [Ca2+]i were not caused by the blockade of voltage-gated K+ channels. We conclude that 4-AP is able to affect calcium homeostasis at multiple levels, from increasing basal [Ca2+]ito potentiating capacitative calcium entry. The potentiation of capacitative calcium entry in astrocytes or muscle cells may explain some of the therapeutic activities of 4-AP as a neurotransmission enhancer.

12-Hydroxyeicosatetrenoate (12-HETE) Attenuates AMPA Receptor-Mediated Neurotoxicity: Evidence for a G-Protein-Coupled HETE Receptor

12-Hydroxyeicosatetraenoic acid (12-HETE) is a neuromodulator that is synthesized during ischemia. Its neuronal effects include attenuation of calcium influx and glutamate release as well as inhibition of AMPA receptor (AMPA-R) activation. Because 12-HETE reduces ischemic injury in the heart, we examined whether it can also reduce neuronal excitotoxicity. When treated with 12-(S)HETE, cortical neuron cultures subjected to AMPA-R-mediated glutamate toxicity suffered up to 40% less damage than untreated cultures. The protective effect of 12-(S)HETE was concentration-dependent (EC50 = 88 nm) and stereostructurally selective. Maximal protection was conferred by 300 nm12-(S)HETE; 300 nm15-(S)HETE was similarly protective, but 300 nm 5-(S)HETE was less effective. The chiral isomer 12-(R)HETE offered no protection; neither did arachidonic acid or 12-(S)hydroperoxyeicosatetraenoic acid. Excitotoxicity was calcium-dependent, and 12-(S)HETE was demonstrated to protect by inactivating N and L (but not P) calcium channels via a pertussis toxin-sensitive mechanism. Calcium imaging demonstrated that 12-(S)HETE also attenuates glutamate-induced calcium influx into neurons via a pertussis toxin-sensitive mechanism, suggesting that it acts via a G-protein-coupled receptor. In addition, 12-(S)HETE stimulates GTPγS binding (indicating G-protein activation) and inhibits adenylate cyclase in forskolin-stimulated cultures over the same concentration range as it exerts its anti-excitotoxic and calcium-influx attenuating effects. These studies demonstrate that 12-(S)HETE can protect neurons from excitotoxicity by activating a Gi/o-protein-coupled receptor, which limits calcium influx through voltage-gated channels.

The hop cassette of the PAC1 receptor confers coupling to Ca2+ elevation required for pituitary adenylate cyclase-activating polypeptide-evoked neurosecretion.

We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to [Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid “hop” insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations ([Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108-15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding [Ca2+]i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1null, at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent [Ca2+]i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release [3H]norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.

Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP‐Dependent, Prostaglandin‐Independent Mechanism

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)‐protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose‐dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT‐5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration‐dependent increase in interleukin 6 release that was also inhibited by KT‐5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E2 or F2α. Conversely, forskolin concentration‐dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide‐ or forskolin‐stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E2 and F2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F2α slightly increased interleukin 6 production only at the highest concentrations. 8‐Bromo‐cAMP and dibutyryl‐cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP‐protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide‐ and forskolin‐mediated induction of interleukin 6 production in cultured astrocytes.

Mitochondria regulate Ca2+ wave initiation and inositol trisphosphate signal transduction in oligodendrocyte progenitors

Mitochondria in oligodendrocyte progenitor cells (OPs) take up and release cytosolic Ca2+ during agonist‐evoked Ca2+ waves, but it is not clear whether or how they regulate Ca2+ signaling in OPs. We asked whether mitochondria play an active role during agonist‐evoked Ca2+ release from intracellular stores. Ca2+ puffs, wave initiation, and wave propagation were measured in fluo‐4 loaded OP processes using linescan confocal microscopy. Mitochondrial depolarization, measured by tetramethyl rhodamine ethyl ester (TMRE) fluorescence, accompanied Ca2+ puffs and waves. In addition, waves initiated only where mitochondria were localized. To determine whether energized mitochondria were necessary for wave generation, we blocked mitochondrial function with the electron transport chain inhibitor antimycin A (AA) in combination with oligomycin. AA decreased wave speed and puff probability. These effects were not due to global changes in ATP. We found that AA increased cytosolic Ca2+, markedly reduced agonist‐evoked inositol trisphosphate (IP3) production, and also enhanced phosphatidylinositol 4,5‐bisphosphate (PIP2) binding to the Ca2+ dependent protein gelsolin. Thus, the reduction in puff probability and wave speed after AA treatment may be explained by competition for PIP2 between phospholipase C and gelsolin. Energized mitochondria and low cytosolic Ca2+ concentration may be required to maintain PIP2, a substrate for IP3 signal transduction.