Binge-like ethanol consumption increases corticosterone levels and neurodegneration whereas occupancy of type II glucocorticoid receptors with mifepristone is neuroprotective.

Excessive ethanol (EtOH) use leads to impaired memory and cognition. Using a rat model of binge-like intoxication, we tested whether elevated corticosterone (Cort) levels contribute to the neurotoxic consequences of EtOH exposure. Rats were adrenalectomized (Adx) and implanted with cholesterol pellets, or cholesterol pellets containing Cort in order to achieve basal, medium, or high blood concentrations of Cort. Intragastric EtOH or an isocaloric control solution was given three times daily for 4 days to achieve blood alcohol levels ranging between 200 and 350 mg/dl. Mean 24-hour plasma levels of Cort were ∼110 and ∼40 ng/ml in intact EtOH-treated and intact control animals, respectively. Basal Cort replacement concentrations in EtOH-treated Adx animals did not exacerbate alcohol-induced neurodegeneration in the hippocampal dentate gyrus (DG) or the entorhinal cortex (EC) as observed by amino-cupric silver staining. In contrast, Cort replacement pellets resulting in plasma Cort levels twofold higher (medium) than normal, or greater than twofold higher (high) in Adx-Cort-EtOH animals increased neurodegeneration. In separate experiments, pharmacological blockade of the Type II glucocorticoid (GC) receptor was initiated with mifepristone (RU38486; 0, 5, 15 mg/kg/day, i.p.). At the higher dose, mifepristone decreased the number of degenerating hippocampal DG cells in binge-EtOH-treated intact animals, whereas, only a trend for reduction was observed in 15 mg/kg/day mifepristone-treated animals in the EC, as determined by fluoro-jade B staining. These results suggest that elevated circulating Cort in part mediates EtOH-induced neurotoxicity in the brain through activation of Type II GC receptors.

Meta-analysis of microarray-derived data from PACAP-deficient adrenal gland in vivo and PACAP-treated chromaffin cells identifies distinct classes of PACAP-regulated genes.

Initial PACAP-regulated transcriptomes of PACAP-treated cultured chromaffin cells, and the adrenal gland of wild-type versus PACAP-deficient mice, have been assembled using microarray analysis. These were compared to previously acquired PACAP-regulated transcriptome sets from PC12 cells and mouse central nervous system, using the same microarray platform. The Ingenuity Pathways Knowledge Base was then employed to group regulated transcripts into common first and second messenger regulatory clusters. The purpose of our meta-analysis was to identify sets of genes regulated distinctly or in common by the neurotransmitter/neurotrophin PACAP in specific physiological contexts. Results suggest that PACAP participates in both the basal differentiated expression, and the induction upon physiological stimulation, of distinct sets of transcripts in neuronal and endocrine cells. PACAP in both developmental and acute regulatory paradigms acts on target genes also regulated by either TNFalpha or TGFbeta, two first messengers acting on transcription mainly through NFkappaB and Smads, respectively.

Neuroprotection by endogenous and exogenous PACAP following stroke.

We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c.v. 1 h after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 h later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice. A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at 1 h post-MCAO (acute response transcripts), 142 were up-regulated only at 24 h post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than half of these are transcripts not previously reported to be altered in ischemia. A larger percentage of genes up-regulated at 24 hr than at 1 hr required endogenous PACAP, suggesting a more prominent role for PACAP in later response to injury than in the initial response. This is consistent with a neuroprotective role for PACAP in late response to injury, i.e., even when administered 1 hr or more after MCAO. Putative injury effector transcripts regulated by PACAP include beta-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin.

Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult.

The rodent cerebellum is richly supplied with PACAPergic innervation. Exogenous pituitary adenylate cyclase-activating polypeptide (PACAP) increases cerebellar granule cell survival and differentiation in culture, and enhances the number of neuroblasts in the molecular and internal granule cell layers (IGL) when injected postnatally into the cerebellum in vivo. Here, we have investigated the role of endogenous PACAP during cerebellar development by comparing the morphology of normal and PACAP-deficient mouse cerebellum, and the response of cerebellar granule cells from normal and PACAP-deficient mice subjected to neurotoxic insult in culture. There was no difference in cerebellar volume or granule cell number, in 11-day-old wild type versus PACAP-deficient mice. Cultured cerebellar neurons from PACAP-deficient and wild type mice also showed no apparent differences in survival and differentiation either under depolarizing conditions, or non-depolarizing conditions in the presence or absence of either dibutyryl cAMP or 100 nM PACAP. However, cultured cerebellar neurons from PACAP-deficient mice were significantly more sensitive than wild type neurons to ethanol- or hydrogen peroxide-induced toxicity. Differential ethanol toxicity was reversed by addition of 100 nM exogenous PACAP, suggesting that endogenous PACAP has neuroprotective activity in the context of cellular insult or stress. The neuroprotective action of PACAP was mimicked by dibutryl cAMP, indicating that it occurred via activation of adenylate cyclase. These results indicate that PACAP might act to protect the brain from paraphysiological insult, including exposure to toxins or hypoxia.

Comparison of cannabidiol, antioxidants, and diuretics in reversing binge ethanol-induced neurotoxicity.

Binge alcohol consumption in the rat induces substantial neurodegeneration in the hippocampus and entorhinal cortex. Oxidative stress and cytotoxic edema have both been shown to be involved in such neurotoxicity, whereas N-methyl-d-aspartate (NMDA) receptor activity has been implicated in alcohol withdrawal and excitoxic injury. Because the nonpsychoactive cannabinoid cannabidiol (CBD) was previously shown in vitro to prevent glutamate toxicity through its ability to reduce oxidative stress, we evaluated CBD as a neuroprotectant in a rat binge ethanol model. When administered concurrently with binge ethanol exposure, CBD protected against hippocampal and entorhinal cortical neurodegeneration in a dose-dependent manner. Similarly, the common antioxidants butylated hydroxytoluene and alpha-tocopherol also afforded significant protection. In contrast, the NMDA receptor antagonists dizocilpine (MK-801) and memantine did not prevent cell death. Of the diuretics tested, furosemide was protective, whereas the other two anion exchanger inhibitors, L-644,711 [(R)-(+)-(5,6-dichloro2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy acetic acid] and bumetanide, were ineffective. In vitro comparison of these diuretics indicated that furosemide is also a potent antioxidant, whereas the nonprotective diuretics are not. The lack of efficacy of L-644,711 and bumetanide suggests that the antioxidant rather than the diuretic properties of furosemide contribute most critically to its efficacy in reversing ethanol-induced neurotoxicity in vitro, in our model. This study provides the first demonstration of CBD as an in vivo neuroprotectant and shows the efficacy of lipophilic antioxidants in preventing binge ethanol-induced brain injury.

A restrictive element 1 (RE-1) in the VIP gene modulates transcription in neuronal and non-neuronal cells in collaboration with an upstream tissue specifier element.

The vasoactive intestinal peptide (VIP) gene has been studied extensively as a prototype neuronal gene containing multiple cis-active elements that confer responsiveness to cell lineage, neurotrophic, and activity-dependent intrinsic and extrinsic cues. However, reporter genes containing the presumptive complete regulatory region 5' to the start of transcription do not confer tissue-specific gene expression in vivo. We therefore sought cis-regulatory elements downstream of the transcriptional start that might confer additional tissue-specific and tissue-restrictive properties to the VIP transcriptional unit. We report here a repressor element, similar to the canonical restrictive element-1 (RE-1), located within the first non-coding exon of the human VIP gene. The ability of this element to regulate VIP reporter gene expression in neuroblastoma and fibroblastic cells was examined. Endogenous VIP expression is high in SH-EP neuroblastoma cells, low but inducible in SH-SY5Y cells, and absent in HeLa cells. Endogenous RE-1 silencer factor (REST) expression was highest in SH-EP and HeLa cells, and significantly lower in SH-SY5Y cells. Transient transfection of a VIP reporter gene containing a mutated RE-1 sequence revealed an RE-1-dependent regulation of VIP gene expression in all three cell types, with regulation greatest in cells (SH-EP, HeLa) with highest levels of REST expression. Serial truncation of the VIP reporter gene further revealed a specific interaction between the RE-1 and a tissue-specifier element located 5 kb upstream in the VIP gene. Thus, REST can regulate VIP gene expression in both neuroblastic and non-neuronal cells, but requires coupling to the upstream tissue specifier element.

Analysis of the PC12 cell transcriptome after differentiation with pituitary adenylate cyclase-activating polypeptide (PACAP).

Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neurite outgrowth and inhibits proliferation of rat pheochromocytoma (PC12) cells. Characterizing the PACAP-differentiated PC12 cell transcriptome should provide genetic insight into how these processes occur in these cells, and in neuronal precursors in vivo. For this purpose, RNA samples were collected from PC12 cells before or after a 6-h treatment with PACAP, from which a labeled cDNA was hybridized to a high-density cDNA array containing 15 365 genes. The genomic response to PACAP involves at least 73 genes. Among the genes differentially expressed in the presence of PACAP, 71% were up regulated, and 29% down regulated, 2-fold or more. Sixty-six percent of the messages affected by PACAP code for functionally categorized proteins, most not previously known to be regulated during PC12 cell differentiation. PACAP has been shown to induce PC12 cell neurite outgrowth through the mitogen-activated protein kinase kinase (MEK) pathway independently of protein kinase A (PKA). Therefore treatments were conducted in the absence or presence of the PKA inhibitor H89, or the MEK inhibitor U0126 in order to identify subsets of genes involved in specific aspects of PC12 cell differentiation. Co-treatment of PC12 cells with PACAP plus H89 revealed a cluster of five genes specifically regulated through the PKA pathway and co-treatment of the cells with PACAP and U0126 revealed a cluster of 13 messages specifically activated through the MEK pathway. Many of the known genes regulated by PACAP have been associated with neuritogenesis (i.e. villin 2 or annexin A2) or cell growth (i.e. growth arrest specific 1 or cyclin B2). Thus, some of the expressed sequence tags (ESTs) that exhibit the same regulation pattern (i.e. AU016391 or AW552690) may also be involved in the neuritogenic and anti-mitogenic effects of PACAP in PC12 cells. Among the 73 PACAP regulated genes, 10 are disqualified on pharmacological grounds as actors in PACAP-mediated neurite outgrowth or growth arrest, leaving 63 new PACAP-regulated genes implicated in neuronal differentiation. Thirteen of these are candidates for mediating ERK-dependent neurite outgrowth, and 47 are possibly involved in the ERK-independent growth arrest induced by PACAP.

Role of protein kinases in neuropeptide gene regulation by PACAP in chromaffin cells: a pharmacological and bioinformatic analysis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an adrenomedullary cotransmitter that along with acetylcholine is responsible for driving catecholamine and neuropeptide biosynthesis and secretion from chromaffin cells in response to stimulation of the splanchnic nerve. Two neuropeptides whose biosynthesis is regulated by PACAP include enkephalin and vasoactive intestinal polypeptide (VIP). Occupancy of PAC1 PACAP receptors on chromaffin cells can result in elevation of cyclic AMP, inositol phosphates, and intracellular calcium. The proenkephalin A and VIP genes are transcriptionally responsive to signals generated within all three pathways, and potentially by combinatorial activation of these pathways as well. The characteristics of PACAP regulation of enkephalin and VIP biosynthesis were examined pharmacologically for evidence of involvement of several serine/threonine protein kinases activated by cAMP, IP3, and/or calcium, including calmodulin kinase II, protein kinase A, and protein kinase C. Evidence is presented for the differential involvement of these protein kinases in regulation of enkephalin and VIP biosynthesis in chromaffin cells, and for a prominent role of the mixed-function (tyrosine and serine/threonine) MAP kinase family in mediating transcriptional activation of neuropeptide genes by PACAP.

Coincident elevation of cAMP and calcium influx by PACAP-27 synergistically regulates vasoactive intestinal polypeptide gene transcription through a novel PKA-independent signaling pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes calcium influx, intracellular calcium release, and elevation of cAMP in chromaffin cells. Calcium influx is required for PACAP-stimulated secretion of catecholamines and neuropeptides. The role of cAMP elevation in the action of PACAP at either sympathetic or adrenomedullary synapses, however, is unknown. Here, we show that PACAP-27-induced calcium influx through voltage-sensitive calcium channels (VSCCs), together with elevation of intracellular cAMP, was sufficient to stimulate vasoactive intestinal polypeptide (VIP) biosynthesis at least 40-fold. Combined treatment of chromaffin cells with 40 mm KCl, which elevates intracellular calcium, and 25 micrometer forskolin, which elevates intracellular cAMP, caused an increase in VIP peptide and mRNA much greater than that elicited by either agent alone, and comparable to the increase caused by 10-100 nm PACAP-27. Elevation of VIP mRNA by either KCl plus forskolin, or PACAP, (1) was independent of new protein synthesis, (2) was blocked by inhibition of calcium influx through voltage-sensitive calcium channels, (3) was calcineurin dependent, and (4) was dependent on MAP kinase activation but not activation of protein kinase A. The degree of activation of two different second-messenger pathways, calcium influx and cAMP elevation, appears to determine the magnitude of transcriptional activation of the VIP gene in chromaffin cells. Maximal stimulation of VIP biosynthesis by PACAP appears to require the coincident activation of both of these pathways.

Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an "emergency response" cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.