Depletion of 14-3-3 zeta elicits endoplasmic reticulum stress and cell death, and increases vulnerability to kainate-induced injury in mouse hippocampal cultures

14-3-3 proteins are ubiquitous signalling molecules that regulate development and survival pathways in brain. Altered expression and cellular localization of 14-3-3 proteins has been implicated in neurodegenerative diseases and in neuronal death after acute neurological insults, including seizures. Presently, we examined expression and function of 14-3-3 isoforms in vitro using mouse organotypic hippocampal cultures. Treatment of cultures with the endoplasmic reticulum (ER) stressor tunicamycin caused an increase in levels of 14-3-3 zeta within the ER-containing microsomal fraction, along with up-regulation of Lys-Asp-Glu-Leu-containing proteins and calnexin, and the selective death of dentate granule cells. Depletion of 14-3-3 zeta levels using small interfering RNA induced both ER stress proteins and death of granule cells. Treatment of hippocampal cultures with the excitotoxin kainic acid increased levels of Lys-Asp-Glu-Leu-containing proteins and microsomal 14-3-3 zeta levels and caused cell death within the CA1, CA3 and dentate gyrus of the hippocampus. Kainic acid-induced damage was significantly increased in each hippocampal subfield of cultures treated with small interfering RNA targeting 14-3-3 zeta. The present data indicate a role for 14-3-3 zeta in survival responses following ER stress and possibly protection against seizure injury to the hippocampus.     

Ablation of NMDA Receptors Enhances the Excitability of Hippocampal CA3 Neurons

Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons.     

Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures

The cysteine protease caspase-3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure-induced changes in caspase-3 protein expression and processing, enzyme activity, and the in vivo effect of caspase-3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.1 microg) and were terminated after 45 min by diazepam (30 mg/kg) administration. Animals were killed 0-72 h following diazepam administration. Levels of the 32-kDa proenzyme form of caspase-3 were unaffected by seizures. Levels of the 17-kDa cleaved (active) fragment of caspase-3 were almost undetectable in control brain, but were increased significantly at 4 and 24 h within ipsilateral hippocampus and cortex in seizure animals. Caspase-3-like protease activity was increased within the ipsilateral hippocampus at 8 and 24 h following seizures. Caspase-3 immunoreactivity was increased within the vulnerable ipsilateral CA3/CA4 subfield at 24 and 72 h following seizures and was associated predominantly, but not exclusively, with neurons exhibiting DNA fragmentation. The putatively selective caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly improved neuronal survival bilaterally within the hippocampal CA3/CA4 subfields following seizures. Collectively, these data suggest that caspase-3 may play a significant role in the mechanism by which neurons die following seizures.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Chemotherapy-induced cell death in primary cerebellar granule neurons but not in astrocytes: in vitro paradigm of differential neurotoxicity

The nervous system is frequently the site of symptomatic toxicity of antineoplastic agents. However, there is limited information about the differential vulnerability of neurons, astrocytes and glioma cells. We have analyzed the effects of four chemotherapeutic drugs (lomustine, cisplatin, topotecan and vincristine) on primary cerebellar granule neurons and astrocytes derived from rats. All drugs led to cell death in cerebellar granule neurons in a concentration-dependent manner. Comparison of the EC50 values for cerebellar neurons and astrocytes with the median EC50 values of 12 malignant glioma cell lines demonstrated a large therapeutic range for lomustin and cisplatin. Further, this comparison revealed a 100-fold higher sensitivity of cerebellar neurons towards vincristine and 10-fold higher sensitivity towards topotecan compared with glioma cells. Astrocytes were generally resistant to vincristine. In cerebellar granule neurons, vincristine and to a lesser extent topotecan induced caspase 3 and caspase 9 cleavage, and enhanced caspase activity and Akt-dependent expression of phosphorylated BAD. zVAD-fmk, a caspase inhibitor and brain-derived neurotrophic factor (BDNF), but not MK-801, a non-competitive NMDA receptor antagonist, significantly reduced vincristine- or topotecan-induced cell death.     

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

A role for the protein phosphatase 2B in altered hippocampal synaptic plasticity in the aged rat.

Synaptic plasticity following NMDA application on hippocampal slices from young (3-5 months) and aged (24-27 months) rats was compared. In young rats, NMDA (20 microM) induced opposite effects depending on the duration of the application. A short (1 min) or long (5 min) application induced a long-term depression of synaptic activity while a 3 min application induced a potentiation. In aged rats, however, NMDA application always induced depression, regardless of the duration. To identify mechanisms which could explain the difference observed between young and aged rats, we explored changes in NMDA receptor activation and changes in kinase/phosphatase balance. We first demonstrate that the potentiation present in slices from young rats was not restored in aged rats by exogenous application of the co-agonist of NMDA receptor d-serine (which compensates for the changes in NMDAR activation seen in aged rats). This suggested that alterations in synaptic plasticity activation mainly involve intracellular mechanisms. We next showed that the participation of the kinases PKA and CaMKII in the NMDA-induced potentiation in young rats is negligible. Finally, we determined the consequences of phosphatase inhibition in aged rats. Incubation of slices in okadaic acid (a PP1/PP2B antagonist) did not affect the depression induced by a 3min NMDA application in aged rats. The PP2B antagonist FK506 restored potentiation in aged rats (3 min NMDA application). In hippocampal neurons from aged rats, a depression is always observed, suggesting a preferential activation of PP2B by NMDA in these neurons.     

Effects of MK-801 and CNQX on various neurotoxic responses induced by kainic acid in mice

Effects of MK-801 (a NMDA receptor blocker) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; a non-NMDA receptor blocker) on several neurotoxic responses induced by kainic acid (KA) were examined in ICR mice. In a lethality test, intracerebroventricular (i.c.v.) pretreatment of MK-801 (1 microg), but not CNQX (0.5 microg), attenuated the time to lethality induced by KA (0.5 microg) administered i.c.v. In the memory test (a passive avoidance test), MK-801, but not CNQX, prevented the memory loss induced by KA (0.1 microg). The damage induced by KA (0.1 microg) administered i.c.v. in the hippocampus was markedly concentrated in the CA3 pyramidal neurons. Both MK-801 and CNQX blocked the pyramidal cell death in CA3 hippocampal region induced by KA. In the immunocytochemical study, KA dramatically increased the phosphorylated ERK (p-ERK) and decreased the phosphorylated CREB (p-CREB) in the hippocmapus. Both MK-801 and CNQX attenuated, in part, the increased p-ERK and the decreased p-CREB induced by KA. In addition, both MK-801 and CNQX partially reduced the increased c-Fos and c-Jun protein expression in hippocampus induced by KA. Our results suggest that both NMDA and non-NMDA receptors are involved in supraspinally administered KA-induced pyramidal cell death in CA3 region of hippocampus in the mouse and the p-ERK and the dephosphorylation of CREB protein may play an important role in CA3 region cell death of the hippocampus induced by KA administered supraspinally. Furthermore, c-Fos and c-Jun proteins may serve as third messengers responsible for CA3 pyramidal cell death induced by supraspinally administered KA.     

Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery

A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.     

Differential involvement of cell cycle reactivation between striatal and cortical neurons in cell death induced by 3-nitropropionic acid

Recent evidence suggests that unscheduled cell cycle activity leads to neuronal cell death. 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase and induces cell death in both striatum and cerebral cortex. Here we analyzed the involvement of aberrant cell cycle progression in 3-NP-induced cell death in these brain regions. 3-NP reduced the level of cyclin-dependent kinase inhibitor p27 in striatum but not in cerebral cortex. 3-NP also induced phosphorylation of retinoblastoma protein, a marker of cell cycle progression at late G(1) phase, only in striatum. Pharmacological experiments revealed that cyclin-dependent kinase activity and N-methyl-d-aspartate (NMDA) receptor were cooperatively involved in cell death by 3-NP in striatal neurons, whereas only NMDA receptor was involved in 3-NP-induced neurotoxicity in cortical neurons. Death of striatal neurons was preceded by elevation of somatic Ca(2+) and activation of calpain, a Ca(2+)-dependent protease. Both striatal p27 down-regulation and cell death provoked by 3-NP were dependent on calpain activity. Moreover, transfection of p27 small interfering RNA reduced striatal cell viability. In cortical neurons, however, there was no change in somatic Ca(2+) and calpain activity by 3-NP, and calpain inhibitors were not protective. These results suggest that 3-NP induces aberrant cell cycle progression and neuronal cell death via p27 down-regulation by calpain in striatum but not in the cerebral cortex. This is the first report for differential involvement of cell cycle reactivation in different brain regions and lightens the mechanism for region-selective vulnerability in human disease, including Huntington disease.     

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.     

Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons

We previously reported that ginseng, a well-known herbal medicine, inhibited NMDA receptors in cultured hippocampal neurons. Here, we further examined the detailed mechanism of ginseng-mediated inhibition using its main active ingredient, ginsenoside Rg3. Co-application of ginsenoside Rg3 with increasing concentrations of NMDA did not change the EC50 of NMDA to the receptor, suggesting that ginsenoside Rg3 inhibits NMDA receptors without competing with the NMDA-binding site. Ginsenoside Rg3-mediated inhibition also occurred in a distinctive manner from the well-characterized NMDA receptor open channel blocker, MK-801. However, ginsenoside Rg3 produced its effect in a glycine concentration-dependent manner and shifted the glycine concentration-response curve to the right without changing the maximal response, suggesting the role of ginsenoside Rg3 as a competitive NMDA receptor antagonist. We also demonstrated that ginsenoside Rg3 significantly protected neurons against NMDA insults. Therefore, these results suggest that ginsenoside Rg3 protects NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons.     

Activation of NMDA receptors induces protein kinase A-mediated phosphorylation and degradation of matrin 3. Blocking these effects prevents NMDA-induced neuronal death

Activation of NMDA receptors leads to activation of cAMP-dependent protein kinase (PKA). The main substrates phosphorylated by PKA following NMDA receptor activation remain unidentified. The aim of this work was to identify a major substrate phosphorylated by PKA following NMDA receptor activation in cerebellar neurones in culture, and to assess whether this phosphorylation may be involved in neuronal death induced by excessive NMDA receptor activation. The main PKA substrate following NMDA receptor activation was identified by MALDI-TOFF fingerprinting as the nuclear protein, matrin 3. PKA-mediated phosphorylation of matrin 3 is followed by its degradation. NMDA receptor activation in rat brain in vivo by ammonia injection also induced PKA-mediated matrin 3 phosphorylation and degradation in brain cell nuclei. Blocking NMDA receptors in brain in vivo with MK-801 reduced basal phosphorylation of matrin 3, suggesting that it is modulated by NMDA receptors. Inhibition of PKA with H-89 prevents NMDA-induced phosphorylation and degradation of matrin 3 as well as neuronal death. These results suggest that PKA-mediated phosphorylation of matrin 3 may serve as a rapid way of transferring information from synapses containing NMDA receptors to neuronal nuclei under physiological conditions, and may contribute to neuronal death under pathological conditions.     

Neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport

Recent studies suggest that excitotoxicity may contribute to neuronal damage in neurodegenerative diseases including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. Activated microglia have been observed around degenerative neurons in these diseases, and they are thought to act as effector cells in the degeneration of neural cells in the central nervous system. Neuritic beading, focal bead-like swellings in the dendrites and axons, is a neuropathological sign in epilepsy, trauma, ischemia, aging, and neurodegenerative diseases. Previous reports showed that neuritic beading is induced by various stimuli including glutamate or nitric oxide and is a neuronal response to harmful stimuli. However, the precise physiologic significance of neuritic beading is unclear. We provide evidence that neuritic beading induced by activated microglia is a feature of neuronal cell dysfunction toward neuronal death, and the neurotoxicity of activated microglia is mediated through N-methyl-d-aspartate (NMDA) receptor signaling. Neuritic beading occurred concordant with a rapid drop in intracellular ATP levels and preceded neuronal death. The actual neurite beads consisted of collapsed cytoskeletal proteins and motor proteins arising from impaired neuronal transport secondary to cellular energy loss. The drop in intracellular ATP levels was because of the inhibition of mitochondrial respiratory chain complex IV activity downstream of NMDA receptor signaling. Blockage of NMDA receptors nearly completely abrogated mitochondrial dysfunction and neurotoxicity. Thus, neuritic beading induced by activated microglia occurs through NMDA receptor signaling and represents neuronal cell dysfunction preceding neuronal death. Blockage of NMDA receptors may be an effective therapeutic approach for neurodegenerative diseases.     

Homeostatic NMDA receptor down-regulation via brain derived neurotrophic factor and nitric oxide-dependent signalling in cortical but not in hippocampal neurons

Nitric oxide (NO) has been proposed to down-regulate NMDA receptors (NMDA-Rs) in a homeostatic manner. However, NMDA-R-dependent NO synthesis also can cause excitotoxic cell death. Using bicuculline-stimulated hippocampal and cortical cell cultures, we have addressed the role of the brain-derived neurotrophic factor-NO pathway in NMDA-R down-regulation. This pathway protected cortical cells from NMDA-induced death and led to NMDA-R inhibition. In contrast, no evidence was gained for the presence of this protective pathway in hippocampal neurons, in which NMDA-induced NO synthesis was confirmed to be toxic. Therefore, opposing effects of NO depended on the activation of different signalling pathways. The pathophysiological relevance of this observation was investigated in synaptosomes and post-synaptic densities isolated from rat hippocampi and cerebral cortices following kainic acid-induced status epilepticus. In cortical, but not in hippocampal synaptosomes, brain-derived neurotrophic factor induced NO synthesis and inhibited NMDA-R currents present in isolated post-synaptic densities. In conclusion, we identified a NO-dependent homeostatic response in the rat cerebral cortex induced by elevated activity. A low performance of this pathway in brain areas including the hippocampus may be related to their selective vulnerability in pathologies such as temporal lobe epilepsy.     

BAD is a pro-survival factor prior to activation of its pro-apoptotic function

The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD(L) can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD(S). In addition, caspases convert BAD(L) into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, gamma-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.     

GDNF Selectively Induces Microglial Activation and Neuronal Survival in CA1/CA3 Hippocampal Regions Exposed to NMDA Insult through Ret/ERK Signalling

The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity.     

Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1

We recently identified a novel mechanism for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.