PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice.

BACKGROUND: Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE-/-) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE-/- mouse model of atherosclerosis and under the influence of a high-fat diet. METHODS: For this purpose, PACAP-/-/ApoE-/-, PAC1-/-/ApoE-/-, and ApoE-/- mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified. RESULTS: ApoE-/- mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1-/-/ApoE-/- mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE-/- mice. In contrast, PACAP deficiency in ApoE-/- mice did not affect the chondrogenic signature under either diet. CONCLUSIONS: Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models.

AIMS/HYPOTHESIS: Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. METHODS: We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. RESULTS: The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. CONCLUSIONS/INTERPRETATION: Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a 'null' model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas.

PACAP controls adrenomedullary catecholamine secretion and expression of catecholamine biosynthetic enzymes at high splanchnic nerve firing rates characteristic of stress transduction in male mice.

The neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) is a cotransmitter of acetylcholine at the adrenomedullary synapse, where autonomic regulation of hormone secretion occurs. We have previously reported that survival of prolonged metabolic stress in mice requires PACAP-dependent biosynthesis and secretion of adrenomedullary catecholamines (CAs). In the present experiments, we show that CA secretion evoked by direct high-frequency stimulation of the splanchnic nerve is abolished in native adrenal slices from male PACAP-deficient mice. Further, we demonstrate that PACAP is both necessary and sufficient for CA secretion ex vivo during stimulation protocols designed to mimic stress. In vivo, up-regulation of transcripts encoding adrenomedullary CA-synthesizing enzymes (tyrosine hydroxylase, phenylethanolamine N-methyltransferase) in response to both psychogenic and metabolic stressors (restraint and hypoglycemia) is PACAP-dependent. Stressor-induced alteration of the adrenomedullary secretory cocktail also appears to require PACAP, because up-regulation of galanin mRNA is abrogated in male PACAP-deficient mice. We further show that hypoglycemia-induced corticosterone secretion is not PACAP-dependent, ruling out the possibility that glucocorticoids are the main mediators of the aforementioned effects. Instead, experiments with bovine chromaffin cells suggest that PACAP acts directly at the level of the adrenal medulla. By integrating prolonged CA secretion, expression of biosynthetic enzymes and production of modulatory neuropeptides such as galanin, PACAP is crucial for adrenomedullary function. Importantly, our results show that PACAP is the dominant adrenomedullary neurotransmitter during conditions of enhanced secretory demand.

Pituitary adenylate cyclase-activating polypeptide controls stimulus-transcription coupling in the hypothalamic-pituitary-adrenal axis to mediate sustained hormone secretion during stress.

External and internal stimuli that threaten homeostasis trigger coordinated stress responses through activation of specialised neuroendocrine circuits. In mammals, the hypothalamic-pituitary-adrenal (HPA) axis mediates responses to stressors such as restraint, ultimately enhancing adrenocortical hormone secretion. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in central control of the HPA axis, and we have recently shown PACAP-dependent expression of corticotropin-releasing hormone (CRH) and secretion of corticosterone in response to restraint. We now provide a more detailed analysis of PACAP-dependent HPA axis stimulation in the mouse, indicating that the hypothalamic paraventricular nucleus (PVN) is the primary site of action. We demonstrate by quantitative polymerase chain reaction and in situ hybridisation that up-regulation of mRNAs encoding CRH and inducible transcription factors, from the Nr4a family (Nur77, Nor1) in the PVN is PACAP-dependent. Furthermore, CRH hnRNA is rapidly up-regulated in cultured hypothalamic neurones after treatment with PACAP. Induction of Nr4a factors (Nur77, Nurr1) in response to restraint is attenuated in the pituitary gland of PACAP-deficient mice. In the adrenal glands, restraint elicits a marked PACAP-dependent increase in adrenocortical mRNA levels of all three Nr4a transcription factors, steroidogenic factor 1 (Nr5a1), steroidogenic acute regulatory protein and steroid 21-hydroxylase. Taken together, our results show that PACAP controls HPA responses to restraint primarily at the level of the hypothalamus by up-regulating CRH, possibly involving transcription factors such as Nur77 and Nor1. Subsequent adrenocortical steroidogenesis also appears to involve PACAP-dependent stimulus-transcription coupling, suggesting a mechanism by which PACAP exerts control over HPA axis function during stress.

Stress hormone synthesis in mouse hypothalamus and adrenal gland triggered by restraint is dependent on pituitary adenylate cyclase-activating polypeptide signaling.

Stress responses are elicited by a variety of stimuli and are aimed at counteracting direct or perceived threats to the well-being of an organism. In the mammalian central and peripheral nervous systems, specific cell groups constitute signaling circuits that indicate the presence of a stressor and elaborate an adequate response. Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed in central and peripheral parts of these circuits and has recently been identified as a candidate for regulation of the stress axis. In the present experiments, we tested the involvement of PACAP in the response to a psychological stressor in vivo. We used a restraint paradigm and compared PACAP-deficient mice (PACAP-/-) to wild-type controls (PACAP+/+). Acute secretion of corticosterone elicited by 1 h of restraint was found to be identical between genotypes, whereas sustained secretion provoked by 6 h of unrelieved restraint was 48% lower in PACAP-/-mice. Within the latter time frame, expression of messenger RNA (mRNA) encoding corticotropin-releasing hormone (CRH) was increased in the hypothalamus of wild type, but not PACAP-deficient mice. Expression of the activity-regulated transcription factors Egr1 (early growth response 1) and Fos (FBJ osteosarcoma oncogene) in the hypothalamus was rapidly and transiently induced by restraint in a PACAP-dependent fashion, a pattern that was also found in the adrenal glands. Here, abundance of transcripts encoding enzymes required for adrenomedullary catecholamine biosynthesis, namely TH (tyrosine hydroxylase) and PNMT (phenylethanolamine N-methyltransferase), was higher in PACAP+/+ mice after 6 h of unrelieved restraint. Our results suggest that sustained corticosterone secretion, synthesis of the hypophysiotropic hormone CRH in the hypothalamus, and synthesis of the enzymes producing the hormone adrenaline in the adrenal medulla, are controlled by PACAP signaling in the mouse. These findings identify PACAP as a major contributor to the stimulus-secretion-synthesis coupling that supports stress responses in vivo.

Cellular distribution of chromogranin A in excitatory, inhibitory, aminergic and peptidergic neurons of the rodent central nervous system.

Immunoreactivity for both processed and unprocessed forms of chromogranin A (CGA) was examined, using an antibody recognizing the WE14 epitope, among terminal fields and cell bodies of anatomically defined GABAergic, glutamatergic, cholinergic, catecholaminergic, and peptidergic cell groups in the rodent central nervous system. CGA is ubiquitous within neuronal cell bodies, with no obvious anatomical or chemically-coded subdivision of the nervous system in which CGA is not expressed in most neurons. CGA expression is essentially absent from catecholaminergic terminal fields in the CNS, suggesting a relative paucity of large dense-core vesicles in CNS compared to peripheral catecholaminergic neurons. Extensive synaptic co-localization with classical transmitter markers is not observed even in areas such as amygdala, where CGA fibers are numerous, suggesting preferential segregation of CGA to peptidergic terminals in CNS. Localization of CGA in dendrites in some areas of CNS may indicate its involvement in regulation of dendritic release mechanisms. Finally, the ubiquitous presence of CGA in neuronal cell somata, especially pronounced in GABAergic neurons, suggests a second non-secretory vesicle-associated function for CGA in CNS. We propose that CGA may function in the CNS as a prohormone and granulogenic factor in some terminal fields, but also possesses as-yet unknown unique cellular functions within neuronal somata and dendrites.

Sweat gland innervation is pioneered by sympathetic neurons expressing a cholinergic/noradrenergic co-phenotype in the mouse.

Classic neurotransmitter phenotypes are generally predetermined and develop as a consequence of target-independent lineage decisions. A unique mode of target-dependent phenotype instruction is the acquisition of the cholinergic phenotype in the peripheral sympathetic nervous system. A body of work suggests that the sweat gland plays an important role to determine the cholinergic phenotype at this target site. A key issue is whether neurons destined to innervate the sweat glands express cholinergic markers before or only after their terminals make target contact. We employed cholinergic-specific over-expression of the vesicular acetylcholine transporter (VAChT) in transgenic mice to overcome sensitivity limits in the detection of initial cholinergic sweat gland innervation. We found that VAChT immunoreactive nerve terminals were present around the sweat gland anlage already from the earliest postnatal stages on, coincident selectively at this sympathetic target with tyrosine hydroxylase-positive fibers. Our results provide a new mechanistic model for sympathetic neuron-target interaction during development, with initial selection by the target of pioneering nerve terminals expressing a cholinergic phenotype, and subsequent stabilization of this phenotype during development.

Signaling pathways for PC12 cell differentiation: making the right connections.

A key issue in signal transduction is how signaling pathways common to many systems-so-called canonical signaling cassettes-integrate signals from molecules having a wide spectrum of activities, such as hormones and neurotrophins, to deliver distinct biological outcomes. The neuroendocrine cell line PC12, derived from rat pheochromocytoma, provides an example of how one canonical signaling cassette-the Raf --> mitogen-activated protein kinase kinase (MEK) --> extracellular signal-regulated kinase (ERK) pathway-can promote distinct outcomes, which in this case include neuritogenesis, gene induction, and proliferation. Two growth hormones, epidermal growth factor (EGF) and nerve growth factor (NGF), use the same pathway to cause PC12 proliferation and differentiation, respectively. In addition, pituitary adenylate cyclase-activating polypeptide (PACAP), a neurotransmitter that also causes differentiation, uses the same canonical cassette as NGF but in a different way. The Connections Map for PC12 Cell Differentiation brings into focus the complex array of specific cellular responses that rely on canonical signal transduction systems.

Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis.

We present evidence that regulation of dense-core secretory granule biogenesis and hormone secretion in endocrine cells is dependent on chromogranin A (CGA). Downregulation of CGA expression in a neuroendocrine cell line, PC12, by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected prohormone, and reduction of secretory granule proteins. Transfection of bovine CGA into a CGA-deficient PC12 clone rescued the regulated secretory phenotype. Stable transfection of CGA into a CGA-deficient pituitary cell line, 6T3, lacking a regulated secretory pathway, restored regulated secretion. Overexpression of CGA induced dense-core granules, immunoreactive for CGA, in nonendocrine fibroblast CV-1 cells. We conclude that CGA is an "on/off" switch that alone is sufficient to drive dense-core secretory granule biogenesis and hormone sequestration in endocrine cells.

Independent patterns of transcription for the products of the rat cholinergic gene locus.

The cholinergic phenotype requires the expression of the vesicular acetylcholine transporter and choline acetyltransferase proteins. Both genes are encoded at one chromosomal location called the cholinergic gene locus. We have identified by in situ hybridization histochemistry distinct patterns of transcription from the cholinergic gene locus in the subdivisions of the rat cholinergic nervous system. The vesicular acetylcholine transporter and choline acetyltransferase are co-expressed in cholinergic neurons at all developmental stages in all major types of cholinergic neurons. The relative levels of vesicular acetylcholine transporter and choline acetyltransferase transcripts, however, change substantially during development in the CNS. They also differ dramatically in distinct subdivisions of the mature cholinergic nervous system, with vesicular acetylcholine transporter mRNA expressed at high levels relative to choline acetyltransferase mRNA in the peripheral nervous system, but at equivalent levels in the CNS. Expression of the R-exon, the presumptive first non-coding exon common to both the vesicular acetylcholine transporter and choline acetyltransferase, was not detectable at any developmental stage in any of the cholinergic neuronal subtypes in the rat nervous system. Thus, in contrast to less complex metazoan organisms, production of the vesicular acetylcholine transporter and choline acetyltransferase via a common differentially spliced transcript does not seem to occur to a significant extent in the rat. We suggest that separate transcriptional start sites within the cholinergic gene locus control vesicular acetylcholine transporter and choline acetyltransferase transcription, while additional elements are responsible for the specific transcriptional control of the entire locus in cholinergic versus non-cholinergic neurons. Independent transcription of the vesicular acetylcholine transporter and choline acetyltransferase genes provides a mechanism for regulating the relative expression of these two proteins to fine-tune acetylcholine quantal size in different types of cholinergic neurons, both centrally and peripherally.

Chemical neuroanatomy of the vesicular amine transporters.

Acetylcholine, catecholamines, serotonin, and histamine are classical neurotransmitters. These small molecules also play important roles in the endocrine and immune/inflammatory systems. Serotonin secreted from enterochromaffin cells of the gut epithelium regulates gut motility; histamine secreted from basophils and mast cells is a major regulator of vascular permeability and skin inflammatory responses; epinephrine is a classical hormone released from the adrenal medulla. Each of these molecules is released from neural, endocrine, or immune/inflammatory cells only in response to specific physiological stimuli. Regulated secretion is possible because amines are stored in secretory vesicles and released via a stimulus-dependent exocytotic event. Amine storage-at concentrations orders of magnitude higher than in the cytoplasm-is accomplished in turn by specific secretory vesicle transporters that recognize the amines and move them from the cytosol into the vesicle. Immunohistochemical visualization of specific vesicular amine transporters (VATs) in neuronal, endocrine, and inflammatory cells provides important new information about how amine-handling cell phenotypes arise during development and how vesicular transport is regulated during homeostatic response events. Comparison of the chemical neuroanatomy of VATs and amine biosynthetic enzymes has also revealed cell groups that express vesicular transporters but not enzymes for monoamine synthesis, and vice versa: their function and regulation is a new topic of investigation in mammalian neurobiology. The chemical neuroanatomy of the vesicular amine transporters is reviewed here. These and similar data emerging from the study of the localization of the recently characterized vesicular inhibitory and excitatory amino acid transporters will contribute to understanding chemically coded synaptic circuitry in the brain, and amine-handling neuroendocrine and immune/inflammatory cell regulation.

Somatomotor neuron-specific expression of the human cholinergic gene locus in transgenic mice.

We examined the expression pattern of the vesicular acetylcholine transporter in the mouse nervous system, using rodent-specific riboprobes and antibodies, prior to comparing it with the distribution of vesicular acetylcholine transporter expressed from a human transgene in the mouse, using riboprobes and antibodies specific for human. Endogenous vesicular acetylcholine transporter expression was high in spinal and brainstem somatomotor neurons, vagal visceromotor neurons, and postganglionic parasympathetic neurons, moderate in basal forebrain and brainstem projection neurons and striatal interneurons, and low in intestinal intrinsic neurons. Vesicular acetylcholine transporter expression in intrinsic cortical neurons was restricted to the entorhinal cortex. The sequence of the mouse cholinergic gene locus to 5.1kb upstream of the start of transcription of the vesicular acetylcholine transporter gene was determined and compared with the corresponding region of the human gene. Cis-regulatory domains implicated previously in human or rat cholinergic gene regulation are highly conserved in mouse, indicating their probable relevance to the regulation of the mammalian cholinergic gene locus in vivo. Mouse lines were established containing a human transgene that included the vesicular acetylcholine transporter gene and sequences spanning 5kb upstream and 1.8kb downstream of the vesicular acetylcholine transporter open reading frame. In this transgene, the intact human vesicular acetylcholine transporter was able to act as its own reporter. This allowed elements within the vesicular acetylcholine transporter open reading frame itself, shown previously to affect transcription in vitro, to be assessed in vivo with antibodies and riboprobes that reliably distinguished between human and mouse vesicular acetylcholine transporters and their messenger RNAs. Expression of the human vesicular acetylcholine transporter was restricted to mouse cholinergic somatomotor neurons in the spinal cord and brainstem, but absent from other central and peripheral cholinergic neurons. The mouse appears to be an appropriate model for the study of the genetic regulation of the cholinergic gene locus, and the physiology and neurochemistry of the mammalian cholinergic nervous system, although differences exist in the distribution of cortical cholinergic neurons between the mouse and other mammals. The somatomotor neuron-specific expression pattern of the transgenic human vesicular acetylcholine transporter suggests a mosaic model for cholinergic gene locus regulation in separate subdivisions of the mammalian cholinergic nervous system.

Expression of vesicular monoamine transporters in endocrine hyperplasia and endocrine tumors of the oxyntic stomach.

BACKGROUND: Gastric enterochromaffin-like (ECL) cells selectively express the vesicular monoamine transporter (VMAT) VMAT2, and enterochromaffin (EC) cells the VMAT1 isoform. AIMS: We investigated whether VMAT isoform selection indicates the origin of endocrine hyperplasia and neoplasia from oxyntic ECL or EC cells and may be of prognostic significance in different types of gastric carcinoids. METHODS: Tissue from patients with chronic atrophic gastritis (CAG), Zollinger-Ellison-syndrome (ZES), gastric carcinoids and neuroendocrine carcinoma (NEC) was investigated by immunohistology and in situ hybridization. RESULTS: Endocrine cells forming diffuse, linear, and micronodular hyperplasia in CAG and ZES, as well as oxyntic microcarcinoids expressed both VMAT2 and chromogranin A (CgA) but neither VMAT1 nor serotonin. In five of six sporadic carcinoids VMAT2 and CgA but not VMAT1 were detected. One carcinoid was copositive for VMAT1 and serotonin but negative for VMAT2. Electron microscopy confirmed the VMAT2-positive tumors as ECLoma and the VMAT1-immunoreactive carcinoid as EComa. CONCLUSIONS: VMAT2 and VMAT1 are reliable markers for differentiation of gastric endocrine hyperplasia and neoplasia from ECL and EC cells, respectively. The significance of VMAT2 and VMAT1 as prognostic markers lies in the relatively poor prognosis for EComa compared to ECLoma, characterized by VMAT2 positivity. The absence of both VMAT2 and VMAT1 in NEC may indicate poor prognosis.

Pituitary adenylate cyclase-activating polypeptide regulation of vasoactive intestinal polypeptide transcription requires Ca2+ influx and activation of the serine/threonine phosphatase calcineurin.

A >15-fold increase in vasoactive intestinal polypeptide (VIP) mRNA and VIP peptide levels occurred in primary chromaffin cells following exposure to the neurotrophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-27 with an EC50 of approximately 2 nM. PACAP induction of VIP expression was blocked by methoxyverapamil or by a combination of nimodipine and omega-conotoxin MVIIC, indicating a requirement for PACAP-initiated calcium entry through voltage-dependent calcium channels for regulation of VIP biosynthesis. Ascomycin, which inhibits calcineurin through formation of an ascomycin/FKBP12/calcineurin ternary complex, abolished the PACAP-evoked increase in VIP expression, whereas rapamycin, which also binds to FKBP12 but does not cause inhibition of calcineurin, did not. Cyclosporin A, which inhibits calcineurin through formation of a cyclosporin A/cyclophilin/calcineurin complex, also abolished PACAP-evoked VIP biosynthesis. These data indicate that PACAP regulates the expression of VIP via a signaling pathway that requires calcium influx and activation of calcineurin.

Two separate cis-active elements of the vasoactive intestinal peptide gene mediate constitutive and inducible transcription by binding different sets of AP-1 proteins.

Vasoactive intestinal peptide (VIP) gene expression is highly restricted throughout the neuroaxis and regulated by extracellular factors that activate tyrosine- or serine/threonine-directed protein kinase pathways. Cytokine, cyclic AMP, and tissue-specific response elements on the VIP gene have been characterized. Those mediating responsiveness to protein kinase C have not. The endogenous VIP gene and a 5.2-kilobase pair (kb) VIP-luciferase reporter gene, are up-regulated by phorbol 12-myristate 13-acetate (PMA) in SK-N-SH neuroblastoma cells. PMA stimulation was abolished by deletion of sequences at -1.37 to -1.28 or -1.28 to -0.904 kb, but not by removal of the single phorbol ester response element (TRE; TGACTCA) located at -2.25 kb. Mutation of sites at -1.32 or -1.20 that mediate neurotrophin responsiveness of the VIP gene (Symes, A., Lewis, S., Corpus, L., Rajan, P., Hyman, S. E., and Fink, J. S. (1994) Mol. Endocrinol. 8, 1750-1763) each reduced PMA induction in SK-N-SH cells by >50%, and double mutation abolished it. The two mutations also reduced basal VIP reporter gene transcription in SH-EP neuroblastoma cells expressing VIP constitutively. Both cis-active elements bound pre-existing AP-1 proteins in SH-EP- or PMA-stimulated SK-N-SH cell nuclear extracts. The AP-1 complex at both sites contained a Fos-related protein with c-Jun in SH-EP cells and c-Fos with a Jun-related protein in SK-N-SH cells. Recruitment of combinatorially distinct AP-1 complexes to these elements may underlie cell type-specific regulation of the VIP gene.

Increased expression of nitric oxide synthase and dendritic injury in simian immunodeficiency virus encephalitis.

OBJECTIVES: Widespread dendritic injury may be one mechanism involved in the neurologic impairment that occurs in HIV-1 infection. The objectives of this study were to quantitate the extent of dendritic injury in a primate model of central nervous system (CNS) infection, investigate the role of nitric oxide (NO) as a mediator of neuropathologic changes, and evaluate the relation of these changes to cognitive and motor function. STUDY DESIGN/METHODS: Cognitive and motor function was assessed in rhesus macaque monkeys infected with simian immunodeficiency virus (SIV). In situ hybridization, immunohistochemistry, and quantitative image analysis were employed to assess the relations among productive infection, NO synthase (iNOS), and dendritic injury. RESULTS: Productive infection of cells of the macrophage lineage in CNS is associated with inflammation, increased expression of iNOS, and dendritic injury. The tests of cognitive and motor function employed were abnormal in both animals that had evidence of productive infection and those that did not. CONCLUSIONS: Increased NO accompanying productive infection and encephalitis may be one cause of neuronal injury in lentivirus infections of the CNS. Extension of tests of cognitive and motor function to late-stage AIDS in rhesus monkeys is needed to assess the potential role of NO-induced dendritic damage in lentiviral encephalopathy/AIDS dementia complex.

Both inducible and constitutive activator protein-1-like transcription factors are used for transcriptional activation of the galanin gene by different first and second messenger pathways.

We investigated trans-acting factors mediating galanin (GAL) gene activation by protein kinase-dependent signal transduction pathways in chromaffin cells. GAL mRNA up-regulation via the protein kinase A (PKA) pathway (25 microM forskolin) required new protein synthesis. Stimulation via protein kinase C (0.1 microM phorbol myristate acetate) did not. The involvement of activator protein-1(AP-1) and cAMP response element-binding protein (CREB) in serine/threonine protein kinase activation of GAL gene transcription was assessed. Cotransfection of a GAL reporter gene along with expression plasmids encoding c-Jun plus c-Fos, or the catalytic subunit of PKA (PKAbeta), resulted in a 4- to 8-fold enhancement of GAL reporter gene transcription. Transcriptional activation required the galanin 12-O-tetradecanoylphorbol-13-acetate (phorbol-12-myristate-13-acetate) response element (GTRE) octamer sequence (TGACGCGG) in the proximal enhancer of the GAL gene, previously shown to confer phorbol ester responsiveness in chromaffin cells. CREB coexpression did not stimulate GAL gene transcription or increase transcriptional activation by PKAbeta. The GTRE preferentially bound in vitro synthesized Jun and Fos-Jun, compared with CREB, in electrophoretic mobility shift assays. The GTRE preference for binding AP-1-immunoreactive protein compared with CREB was even more pronounced in chromaffin cell nuclear extracts, in which the majority of GTRE-bound protein in electrophoretic mobility shift assays was supershifted with anti-Fos and anti-Jun antibodies. Thus, GAL gene regulation mediated by protein kinase activation appears to involve both constitutively expressed and inducible AP-1-related proteins. Elevated potassium stimulation of GAL mRNA was completely blocked, but pituitary adenylyl cyclase-activating polypeptide and histamine stimulations were only partially blocked, by cycloheximide. Both inducible and constitutive pathways are therefore used by physiologically relevant first messengers that stimulate GAL biosynthesis in vivo.

The SIV-infected rhesus monkey model for HIV-associated dementia and implications for neurological diseases.

The neuropathogenesis of human immunodeficiency virus (HIV)-associated dementia has remained elusive, despite identification of HIV as the causal agent. Although a number of contributing factors have been identified, the series of events that culminate in motor and cognitive impairments after HIV infection of the central nervous system (CNS) are still not known. Rhesus monkeys infected with simian immunodeficiency virus (SIV) manifest immunosuppression and CNS disease that is pathologically [L. R. Sharer et al. (1991) J. Med. Primatol. 20, 211-217] and behaviorally [E. A. Murray et al. (1992) Science 255, 1246-1249] similar to humans. The SIV model of HIV-associated dementia (HAD) is widely recognized as a highly relevant model in which to investigate neuropathogenesis. With better understanding of neuropathogenesis comes the opportunity to interrupt progression and to design better treatments for HAD. This becomes increasingly important as patients live longer yet still harbor HIV-infected cells in the CNS. The use of the SIV model has allowed the identification of neurochemical markers of neuropathogenesis important not only for HAD, but also for other inflammatory neurological diseases.