The Neuroprotective Adenosine-Activated Signal Transduction Pathway Involves Activation of Phospholipase C

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid (IAA) followed by “reperfusion” (IAA-R insult), results in severe cytotoxicity, which could be markedly attenuated by prior activation of the adenosine A₁ receptors. We also have demonstrated that adenosine activates a signal transduction pathway (STP), which involves activation of PKCε and opening of K_ATP channels. Here, we provide proof for the involvement also of phospholipase C (PLC) in the neuronal protective adenosine-activated STP. R-PIA, a specific A₁ adenosine receptor agonist, was found to enhance neuronal PLC activity and protect against the IAA-R insult. The PLC inhibitor U73122, abrogated both R-PIA-induced effects. These results demonstrate that activation of PLC is a vital step in the neuronal protective adenosine-induced STP.     

Protein kinase C-epsilon is involved in the adenosine-activated signal transduction pathway conferring protection against ischemia-reperfusion injury in primary rat neuronal cultures

Adenosine activates a signal transduction pathway (STP) in the heart and the brain, conferring protection against ischemia-reperfusion insult. Activation of protein kinase C (PKC), probably mainly PKC-epsilon, has been demonstrated to be part of the heart STP, but its role in the neuronal pathway is less clear. Here, we provide proof for the participation of PKC-epsilon in the neuronal adenosine-activated STP. Primary rat neuronal cultures were exposed to chemical ischemia by iodoacetate, followed by reperfusion. The cultured neurons were protected against this insult by activation of the adenosine mechanism, by N6-(R)-phenylisopropyladenosine [R(-)-PIA], a specific A1 adenosine receptor agonist. Exposure of the cultures to bisindolylmaleimide I, a highly selective PKC inhibitor, abrogated the protection. The exposure of the cultures to R(-)-PIA was found to result in phosphorylation (activation) of PKC-epsilon. Furthermore, insertion into the cells of a specific peptide inhibitor of PKC-epsilon translocation (epsilonV1-2), also abrogated the protection conferred by R(-)-PIA. These results demonstrate that activation of PKC-epsilon is a vital step in the neuronal adenosine-activated STP.     

Different roles of adenosine A1, A2A and A3 receptors in controlling kainate-induced toxicity in cortical cultured neurons

Adenosine is a neuromodulator that can control brain damage through activation of A(1), A(2A) and A(3) receptors, which are located in both neurons and other brain cells. We took advantage of cultured neurons to investigate the role of neuronal adenosine receptors in the control of neurotoxicity caused by kainate and cyclothiazide. Both A(1), A(2A) and A(3) receptors were immunocytochemically identified in cortical neurons. Activation of A(1) receptors with 100 nM CPA did not modify the extent of neuronal death whereas the A(1) receptor antagonist, DPCPX (50 nM), attenuated neurotoxicity by 28 +/- 5%, and effect similar to that resulting from the removal of endogenous adenosine with 2U/ml of adenosine deaminase (27 +/- 3% attenuation of neurotoxicity). In the presence of adenosine deaminase, DPCPX had no further effect and CPA now exacerbated neurotoxicity by 42 +/- 4%. Activation of A(2A) receptor with 30 nM CGS21680 attenuated neurotoxicity by 40 +/- 8%, an effect prevented by the A(2A) receptor antagonists, SCH58261 (50 nM) or ZM241385 (50 nM), which by themselves were devoid of effect. Finally, neither A(3) receptor activation with Cl-IB-MECA (100-500 nM) nor blockade with MRS1191 (5 microM) modified neurotoxicity. These results show that A(1) receptor activation enhances and A(2A) receptor activation attenuates neurotoxicity in cultured cortical neurons, indicating that these two neuronal adenosine receptors directly control neurodegeneration. Interestingly, the control by adenosine of neurotoxicity in cultured neurons is similar to that observed in vivo in newborn animals and is the opposite of what is observed in adult brain preparations where A(1) receptor activation and A(2A) receptor blockade are neuroprotective.     

Molecular mechanisms involved in the adenosine A and A receptor-induced neuronal differentiation in neuroblastoma cells and striatal primary cultures

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.     

IL-5-induced IgA synthesis by LPS-stimulated mouse B cells is prevented by protein kinase C inhibitors.

We have previously demonstrated that activation of protein kinase C (PKC) by phorbol esters induces selectively IgA synthesis by mouse B cells. In this study, we investigated the effects of a number of protein kinase inhibitors on IgA secretion induced by a recombinant murine IL-5 in LPS-stimulated mouse B cells. The results show that PKC inhibitors, such as sphingosine (SPH), staurosporine (STP) and H-7, blocked IL-5-induced IgA synthesis; the protein kinase A inhibitor HA-1004 and the inhibitor of calcium/calmodulin dependent protein kinase W-7 had no effect on IgA secretion induced by IL-5. The proliferation of the IL-5 sensitive B13 cell line in response to IL-5 was also inhibited by addition of SPH or STP or H-7. The data suggest an involvement of the PKC pathway in IL-5-induced B cell differentiation into IgA secreting cells.     

Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current (Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.     

Adenosine in the central nervous system: release mechanisms and extracellular concentrations

Adenosine has several functions within the CNS that involve an inhibitory tone of neurotransmission and neuroprotective actions in pathological conditions. The understanding of adenosine production and release in the brain is therefore of fundamental importance and has been extensively studied. Conflicting results are often obtained regarding the cellular source of adenosine, the stimulus that induces release and the mechanism for release, in relation to different experimental approaches used to study adenosine production and release. A neuronal origin of adenosine has been demonstrated through electrophysiological approaches showing that neurones can release significant quantities of adenosine, sufficient to activate adenosine receptors and to modulate synaptic functions. Specific actions of adenosine are mediated by different receptor subtypes (A(1), A(2A), A(2B) and A(3)), which are activated by various ranges of adenosine concentrations. Another important issue is the measurement of adenosine concentrations in the extracellular fluid under different conditions in order to know the degree of receptor stimulation and understand adenosine central actions. For this purpose, several experimental approaches have been used both in vivo and in vitro, which provide an estimation of basal adenosine levels in the range of 50-200 nM. The purpose of this review is to describe pathways of adenosine production and metabolism, and to summarize characteristics of adenosine release in the brain in response to different stimuli. Finally, studies performed to evaluate adenosine concentrations under physiological and hypoxic/ischemic conditions will be described to evaluate the degree of adenosine receptor activation.     

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.     

Adenosine A2A receptors control the extracellular levels of adenosine through modulation of nucleoside transporters activity in the rat hippocampus

Adenosine, a neuromodulator of the CNS, activates inhibitory-A1 receptors and facilitatory-A2A receptors; its synaptic levels are controlled by the activity of bi-directional equilibrative nucleoside transporters. To study the relationship between the extracellular formation/inactivation of adenosine and the activation of adenosine receptors, we investigated how A1 and A2A receptor activation modifies adenosine transport in hippocampal synaptosomes. The A2A receptor agonist, CGS 21680 (30 nm), facilitated adenosine uptake through a PKC-dependent mechanism, but A1 receptor activation had no effect. CGS 21680 (30 nm) also increased depolarization-induced release of adenosine. Both effects were prevented by A2A receptor blockade. A2A receptor-mediated enhancement of adenosine transport system is important for formatting adenosine neuromodulation according to the stimulation frequency, as: (1) A1 receptor antagonist, DPCPX (250 nm), facilitated the evoked release of [(3)H]acetylcholine under low-frequency stimulation (2 Hz) from CA3 hippocampal slices, but had no effect under high-frequency stimulation (50 Hz); (2) either nucleoside transporter or A2A receptor blockade revealed the facilitatory effect of DPCPX (250 nm) on [3H]acetylcholine evoked-release triggered by high-frequency stimulation. These results indicate that A2A receptor activation facilitates the activity of nucleoside transporters, which have a preponderant role in modulating the extracellular adenosine levels available to activate A1 receptors.     

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.     

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.     

Decrease of PKC precedes other cellular signs of calpain activation in area CA1 of the hippocampus after transient cerebral ischemia

One of the specific features of severe brain injury is an activation of calcium-dependent proteolysis by calpains. We have observed a significant increase of activity as early as 3 h after the insult in a well defined model of delayed ischemic neuronal death in gerbil hippocampus. At 24 h, the enzymatic activity transiently normalized, then increased again, following the place and time of selective cellular death in the CA1 region of hippocampus. The enhanced postischemic proteolysis resulted in concomitant cleavage of calpain-specific endogenous substrates like protein kinase C (PKC), fodrin and microtubule-associated protein-2 (MAP2). These effects were also time-dependent and restricted to the vulnerable, CA1 pyramidal neurons-containing the dorsal part (DP) of the hippocampus. We have also characterized the postischemic changes of six different isoforms of PKC. The vulnerable dorsal part of the hippocampus, but not its relative resistant abdominal part (AbP), exhibited a loss of PKCalpha, beta, gamma, and delta isoforms as early as 3 h after ischemic insult. However, at this time, solely in the soluble fraction of homogenate. Later (72 h), a further loss of the enzyme proteins, comprised the particulate fraction as well and resulted in an about 50% decrease of total PKCs in the vulnerable DP region. In the case of PKCalpha, the immunostaining pattern showed, in addition to the disappearance of the enzyme from the injured area, an extensive translocation into nuclei of the survived, ischemia-resistant neurones. The early decreases of PKC isoforms in the cytosol paralleled the transient calpain activation at 3h postischemia but substantially preceded the proteolysis of any other classical calpain substrates, such as fodrin and MAP2, being evidenced not earlier than 48-72 h after the insult and restricted also to the vulnerable dorsal part. In conclusion, our results of the time-dependent effects of transient global cerebral ischemia on the calpain activity, levels and localization of its several substrates suggest, that calpain-mediated proteolysis is specifically involved in the early (induction) as well as in the late (execution) phases of delayed ischemic neuronal death in the CA1 hippocampus.     

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.     

Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study

In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na⁺ influxes associated with neuronal signalling activate the Na⁺-K⁺ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na⁺. We have simultaneously imaged intracellular Na⁺ and measured adenosine release. The accumulation of intracellular Na⁺ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca²⁺, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na⁺-K⁺ ATPase in the release of adenosine. Under conditions which caused a Na⁺ influx, brief applications of ouabain increased the accumulation of intracellular Na⁺ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na⁺-K⁺ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na⁺-K⁺ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep.     

Why are hippocampal CA1 neurons vulnerable but motor cortex neurons resistant to transient ischemia?

It is well-known that heat-shock protein 70.1 (Hsp70.1), a major protein of the human Hsp70 family, plays cytoprotective roles by both its chaperone function and stabilization of lysosomal membranes. Recently, we found that calpain-mediated cleavage of carbonylated Hsp70.1 in the hippocampal cornu Ammonis1 (CA1) contributes to neuronal death after transient global ischemia. This study aims to elucidate the differential neuronal vulnerability between the motor cortex and CA1 sector against ischemia/reperfusion. Fluoro-Jade B staining and terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labeling analysis of the monkey brain undergoing 20min whole brain ischemia followed by reperfusion, showed that the motor cortex is significantly resistant to the ischemic insult compared with CA1. Up-regulation of Hsp70.1 but absence of its cleavage by calpain facilitated its binding with NF-κB p65/IκBα complex to minimize NF-κB p65 activation, which contributed to a neuroprotection in the motor cortex. In contrast, because activated μ-calpain cleaved carbonylated Hsp70.1 in CA1, the resultant Hsp70.1 dysfunction not only destabilized lysosomal membrane but also induced a sustained activation of NF-κB p65, both of which resulted in delayed neuronal death. We propose that the cascades underlying lysosomal stabilization and regulating NF-κB activation by Hsp70.1 may influence neuronal survival/death after the ischemia/reperfusion.     

Neuroprotection afforded by adenosine A2A receptor blockade is modulated by corticotrophin‐releasing factor (CRF) in glutamate injured cortical neurons

In situations of hypoxia, glutamate excitotoxicity induces neuronal death. The release of extracellular adenosine is also triggered and is accompanied by an increase of the stress mediator, corticotrophin‐releasing factor (CRF). Adenosine A_2A receptors contribute to glutamate excitoxicity and their blockade is effective in stress‐induced neuronal deficits, but the involvement of CRF on this effect was never explored. We now evaluated the interaction between A_2A and CRF receptors (CRFR) function, upon glutamate insult. Primary rat cortical neuronal cultures (9 days in vitro) expressing both CRF₁R and CRF₂R were challenged with glutamate (20–1000 μM, 24 h). CRF₁R was found to co‐localize with neuronal markers and CRF₂R to be present in both neuronal and glial cells. The effects of the CRF and A_2A receptors ligands on cell viability were measured using propidium iodide and Syto‐13 fluorescence staining. Glutamate decreased cell viability in a concentration‐dependent manner. Urocortin (10 pM), an agonist of CRF receptors, increased cell survival in the presence of glutamate. This neuroprotective effect was abolished by blocking either CRF₁R or CRF₂R with antalarmin (10 nM) or anti‐Sauvagine‐30 (10 nM), respectively. The blockade of A_2A receptors with a selective antagonist SCH 58261 (50 nM) improved cell viability against the glutamate insult. This effect was dependent on CRF₂R, but not on CRF₁R activation. Overall, these data show a protective role of CRF in cortical neurons, against glutamate‐induced death. The neuroprotection achieved by A_2A receptors blockade requires CRF₂R activation. This interaction between the adenosine and CRF receptors can explain the beneficial effects of using A_2A receptor antagonists against stress‐induced noxious effects.     

Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury: a crucial role of P2X7 receptor-mediated IL-1beta release

We investigated the consequences of transient application of specific stimuli mimicking inflammation to hippocampal tissue on microglia activation and neuronal cell vulnerability to a subsequent excitotoxic insult. Two-week-old organotypic hippocampal slice cultures, from 7-day-old C57BL/6 donor mice, were exposed for 3 h to lipopolysaccharide (LPS; 10 ng/mL) followed by 3 h co-incubation with 1 mM ATP, or 100 microM 2'3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate triethylammonium, a selective P2X(7) receptor agonist. These treatments in combination, but not individually, induced a pronounced activation and apoptotic-like death of macrophage antigen-1 (MAC-1)-positive microglia associated with a massive release of interleukin (IL)-1beta exceeding that induced by LPS alone. Antagonists of P2X(7) receptors prevented these effects. Transient pre-exposure of slice cultures to a combination of LPS and P2X(7) receptor agonists, but not either one or the other alone, significantly exacerbated CA3 pyramidal cell loss induced by subsequent 12 h exposure to 8 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propinate (AMPA). Potentiation of AMPA toxicity was prevented when IL-1beta production or its receptor signaling were blocked by an inhibitor of interleukin-converting-enzyme or IL-1 receptor antagonist during application of LPS + ATP. The same treatments did not prevent microglia apoptosis-like death. These findings show that transient exposure to specific pro-inflammatory stimuli in brain tissue can prime neuronal susceptibility to a subsequent excitotoxic insult. P2X(7) receptor stimulation, and the consequent IL-1beta release, is mandatory for exacerbation of neuronal loss. These mechanisms may contribute to determine cell death/survival in acute and chronic neurodegenerative conditions associated with inflammatory events.     

Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states

Background

Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.

Results

Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H₂O₂ or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1^S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.

Conclusion

Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.