Pituitary adenylate cyclase-activating polypeptide stimulates both c-fos gene expression and cell survival in rat cerebellar granule neurons through activation of the protein kinase A pathway

A high density of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to both adenylyl cyclase and phospholipase C is found in the external granule cell layer of the rat cerebellum during postnatal development. It has recently been reported that synthetic PACAP promotes cell survival and neurite outgrowth in immature granule cells. In the present study, we have investigated the transduction pathways that mediate the neurotrophic activity of PACAP in cultured granule cells from eight-day-old rat cerebellum. The effect of PACAP on cell survival was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate suggesting that only the adenylyl cyclase pathway is involved in the neurotrophic activity of PACAP. PACAP also induced a transient increase in c-fos messenger RNA level. The ability of PACAP to stimulate c-fos gene expression was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate. Similar effects of PACAP on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The protein kinase A inhibitor H89 significantly reduced the effect of PACAP on c-fos messenger RNA level whereas the specific protein kinase C inhibitor chelerythrine did not modify c-fos gene expression. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/protein kinase A pathway. The observation that a short-term stimulation by PACAP can be converted into a long-lasting response indicates that the effect of the peptide on cell survival must involve immediate-early gene activation. The fact that a brief exposure to PACAP causes both c-fos gene expression and promotes cell survival strongly suggests that c-fos is involved in the trophic effect of PACAP on immature cerebellar granule cells.     

Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.     

Activation by thyroid stimulating hormone of nerve growth factor-induced gene-B expression in thyrocytes in culture: relation with proliferation and specific gene expression

Nerve growth factor-induced gene-B (NGFI-B) is an immediate early gene first found as a part of the PC12 cell response to NGF (Milbrandt, J., Science 238 (1987) 797-799). We have previously reported that NGFI-B mRNA is strongly upregulated by thyroid-stimulating hormone (TSH) in dog thyrocytes in culture (Pichon et al., Endocrinology 137 (1996) 4691-4698). In this study, we have analyzed the regulation of NGFI-B mRNA expression by a variety of agents acting on thyrocytes proliferation and/or differentiation. We show that: (1) the induction of NGFI-B mRNA is stronger after stimulation of the cAMP cascade, but it is not restricted to this signaling pathway; (2) the powerful mitogens for thyroid cells EGF and HGF have little or no effect on NGFI-B mRNA induction; (3) NGFI-B mRNA is induced by anisomycin at a subinhibitory concentration for protein synthesis, and is superinduced by the combination of TSH and anisomycin; this treatment decreases the TSH-induced proliferation levels, but does not inhibit the induction of some differentiation markers; and (4) both in dog and in pig thyrocytes, NGFI-B mRNA induction is observed after a variety of treatments stimulating differentiation, but without proliferative effects. Our results therefore suggest that NGFI-B mRNA induction might not be related to TSH-induced thyrocyte proliferation, but could participate in the differentiation program triggered by TSH.     

Changes in immune parameters seen in Gulf War veterans but not in civilians with chronic fatigue syndrome

Primary cultures of rat cerebral cortical cells and cerebellar granule cells die by an apoptotic mechanism after more than 2 weeks in cultures in the absence of medium change and glucose supplement, a process termed age-induced apoptosis of cultured neurons. Our preliminary study has shown that age-induced apoptosis of cerebellar granule cells is protected by pretreatment with tetrahydroaminoacridine (THA), an antidementia drug. In this study, we systematically compared the neuroprotective effects of THA with those of (S)-1-[N-(4-chlorobenzyl)succinamoyl]pyrrolidine-2-carbaldehyde (ONO-1603), a novel prolyl endopeptidase inhibitor and potential antidementia drug. Both ONO-1603 and THA effectively delay age-induced apoptosis of cerebral and cerebellar neurons, as demonstrated morphologically with toluidine blue and fluorescein diacetate/propidium iodide staining or biochemically by DNA laddering analysis on agarose gels. ONO-1603 is about 300 times more potent than THA, with a maximal protective effect at 0.03 and 10 microM, respectively. ONO-1603 shows a wide protective range of 0.03 to 1 microM in contrast to a narrow effective range of 3 to 10 microM for THA. Moreover, ONO-1603 is nontoxic to neurons, even at the high concentration of 100 microM, whereas THA elicits severe neurotoxicity at a dose of >/=30 microM. Both ONO-1603 and THA robustly suppress overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) mRNA and accumulation of GAPDH protein in a particulate fraction of cultured neurons undergoing age-induced apoptosis. Because we documented that GAPDH overexpression participates in neuronal apoptosis induced by various insults, we conclude that the neuroprotective actions of ONO-1603 and THA appear to be mediated by suppression of this protein overexpression.     

Conditional ablation of cerebellar astrocytes in postnatal transgenic mice

Astrocytes have been proposed to have multiple roles in the development and maintenance of the vertebrate CNS. To facilitate documentation of these roles, we designed a transgene to enable their ablation at selectable times. The transgene consists of the coding region for the herpes simplex virus-thymidine kinase (HSV-TK) under the control of the human glial fibrillary acidic protein gene promoter. The HSV-TK is innocuous but converts the antiherpetic agent ganciclovir (GCV) to a toxic product that interferes with DNA replication in proliferating cells. In a developmental study, transgenic mice were treated with GCV during the first postnatal week, with evaluation at P19. Treated mice displayed severe ataxia. Histological examination revealed disrupted astrocyte development, particularly in the cerebellum, with marked secondary effects on other cell types. Cerebellar defects included a loss in the numbers of astrocytes and an overall reduction in cerebellar size and disruption of the normally well defined cellular layers. Radial glia were disordered, Purkinje cells were ectopically distributed and displayed abnormal dendritic trees, and granule cells were markedly depleted. These effects were more severe in animals treated on postnatal day 1 versus treatment at day 5. A major factor causing granule cell death was excitotoxicity attributable to activation of NMDA receptors. These results suggest a critical role for astrocytes in cerebellar development.     

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells

1. We have examined the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) responses in bovine aortic endothelial (BAE) cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. Exchange of medium on BAE cells in the absence of agonist was found to be a stimulus for Ins(1,4,5)P3 generation. BAE cells stimulated with 100 microM ATP, 30 microM 2MeSATP (an agonist at P2Y-purinoceptors but not nucleotide receptors) or 100 microM UTP (an agonist at nucleotide receptors but not P2Y-purinoceptors) gave Ins(1,4,5)P3 responses above that caused by exchange of medium. The time course was rapid, with peak response within the first 5 s and levels returning close to basal after 30 s of stimulation. 3. Significant differences in Ins(1,4,5)P3 responses to 100 microM UTP and 30 microM 2MeSATP stimulation were observed. The response to UTP was reproducibly more sustained than that to 2MeSATP. 4. Stimulation of BAE cells with 100 microM UTP plus 30 microM 2MeSATP produced a response statistically indistinguishable from that predicted by addition of the responses to the two agonists in isolation. 5. The Ins(1,4,5)P3 response to UTP was attenuated to 25% of control by pretreatment of BAE cells with pertussis toxin. Responses to 2MeSATP and ADP were essentially unaffected. ATP stimulation was reduced to 65% of control. 6. Activation of protein kinase C with tetradecanoyl phorbol acetate (TPA) profoundly inhibited Ins(1,4,5)P3 responses to 2MeSATP and ADP but had no effect on UTP stimulation. The protein kinase C inhibitor, Ro 31-8220, enhanced responses to 2MeSATP, ADP and ATP but no effect was observed on UTP stimulation. 7. These observations show that nucleotide and P2Y-receptors mobilise the second messenger Ins(1,4,5)P3 by separate routes resulting in different patterns of generation and suggest that while ATP activates both receptors, ADP principally influences these cells by interacting with the P2Y-purinoceptors.     

Depolarization of cerebellar granule cells increases phosphorylation of rabphilin-3A

Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using 32P-labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 +/- 13%) (mean +/- SEM) on depolarization of the cells with K+ (56 mM) in the presence of external Ca2+ (1 mM). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 +/- 21%). Inhibitors of Ca2+/calmodulin-stimulated protein kinases or protein kinase C reduced the depolarization-enhanced phosphorylation of rabphilin-3A, indicating that rabphilin-3A is one of the targets for Ca2+-activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13-dibutyrate and K+ depolarization produced an increased level of phosphorylation of rabphilin-3A compared with either stimulus alone (287 +/- 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin-3A is phosphorylated in vivo and undergoes synaptic activity-dependent phosphorylation during Ca2+-activated K+ depolarization.     

Change of ganglioside accessibility at the plasma membrane surface of cultured neurons, following protein kinase C activation

While the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degreesC), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 microM glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 microM glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state.