James A. Waschek

Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons

The mammalian suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It is not known which SCN neurons are autonomous pacemakers or how they synchronize their daily firing rhythms to coordinate circadian behavior. Vasoactive intestinal polypeptide (VIP) and the VIP receptor VPAC2 (encoded by the gene Vipr2) may mediate rhythms in individual SCN neurons, synchrony between neurons, or both. We found that Vip−/− and Vipr2−/− mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC2 also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. Critically, daily application of a VPAC2 agonist restored rhythmicity and synchrony to VIP−/− SCN neurons, but not to Vipr2−/− neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons.

Pharmacology and functions of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide: IUPHAR Review 1

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) are members of a superfamily of structurally related peptide hormones that includes glucagon, glucagon‐like peptides, secretin, gastric inhibitory peptide (GIP) and growth hormone‐releasing hormone (GHRH). VIP and PACAP exert their actions through three GPCRs – PAC1, VPAC1 and VPAC2– belonging to class B (also referred to as class II, or secretin receptor‐like GPCRs). This family comprises receptors for all peptides structurally related to VIP and PACAP, and also receptors for parathyroid hormone, corticotropin‐releasing factor, calcitonin and related peptides. PAC1 receptors are selective for PACAP, whereas VPAC1 and VPAC2 respond to both VIP and PACAP with high affinity. VIP and PACAP play diverse and important roles in the CNS, with functions in the control of circadian rhythms, learning and memory, anxiety and responses to stress and brain injury. Recent genetic studies also implicate the VPAC2 receptor in susceptibility to schizophrenia and the PAC1 receptor in post‐traumatic stress disorder. In the periphery, VIP and PACAP play important roles in the control of immunity and inflammation, the control of pancreatic insulin secretion, the release of catecholamines from the adrenal medulla and as co‐transmitters in autonomic and sensory neurons. This article, written by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC‐IUPHAR) subcommittee on receptors for VIP and PACAP, confirms the existing nomenclature for these receptors and reviews our current understanding of their structure, pharmacology and functions and their likely physiological roles in health and disease. More detailed information has been incorporated into newly revised pages in the IUPHAR database (http://www.iuphar‐db.org/DATABASE/FamilyMenuForward?familyId=67).

Multiple Actions of Pituitary Adenylyl Cyclase Activating Peptide in Nervous System Development and Regeneration

Pituitary adenylyl cyclase activating peptide (PACAP) is widely expressed in the embryonic brain at the onset of neurogenesis, and is strongly upregulated in several models of nerve injury. Moreover, high-affinity PACAP receptors are expressed in proliferative zones in the embryonic and postnatal nervous system suggesting that PACAP regulates the development of both neuronal and glial precursors. Tissue culture studies indicate that PACAP exerts a variety of growth factor-like actions that depend on the origin of the cell population and developmental stage. These effects include regulation of proliferation, survival, maturation, neurite outgrowth, and expression of trophic factors, cytokines and trk receptors. The presence of other growth factors can also markedly affect these actions of PACAP, for example, reversing PACAP’s effect from proliferative to antiproliferative. In vivo models now provide additional evidence that PACAP acts in neural development and regeneration.

Moderate Pulmonary Arterial Hypertension in Male Mice Lacking the Vasoactive Intestinal Peptide Gene

Background— Vasoactive intestinal peptide (VIP), a pulmonary vasodilator and inhibitor of vascular smooth muscle proliferation, has been reported absent in pulmonary arteries from patients with idiopathic pulmonary arterial hypertension (PAH). We have tested the hypothesis that targeted deletion of the VIP gene may lead to PAH with pulmonary vascular remodeling. Methods and Results— We examined VIP knockout (VIP−/−) mice for evidence of PAH, right ventricular (RV) hypertrophy, and pulmonary vascular remodeling. Relative to wild-type control mice, VIP−/− mice showed moderate RV hypertension, RV hypertrophy confirmed by increased ratio of RV to left ventricle plus septum weight, and enlarged, thickened pulmonary artery and smaller branches with increased muscularization and narrowed lumen. Lung sections also showed perivascular inflammatory cell infiltrates. No systemic hypertension and no arterial hypoxemia existed to explain the PAH. The condition was associated with increased mortality. Both the vascular remodeling and RV remodeling were attenuated after a 4-week treatment with VIP. Conclusions— Deletion of the VIP gene leads to spontaneous expression of moderately severe PAH in mice during air breathing. Although not an exact model of idiopathic PAH, the VIP−/− mouse should be useful for studying molecular mechanisms of PAH and evaluating potential therapeutic agents. VIP replacement therapy holds promise for the treatment of PAH, and mutations of the VIP gene may be a factor in the pathogenesis of idiopathic PAH.

Maternal Embryonic Leucine Zipper Kinase Is a Key Regulator of the Proliferation of Malignant Brain Tumors, Including Brain Tumor Stem Cells

Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up‐regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high‐grade brain tumors.

IMPAIRED NERVE REGENERATION AND ENHANCED NEUROINFLAMMATORY RESPONSE IN MICE LACKING PITUITARY ADENYLYL CYCLASE ACTIVATING PEPTIDE

Peripheral nerve injury models are used to investigate processes that can potentially be exploited in CNS injury. A consistent change that occurs in injured peripheral neurons is an induction in expression of pituitary adenylyl cyclase activating peptide (PACAP), a neuropeptide with putative neuroprotective and neuritogenic actions. PACAP-deficient mice were used here to investigate actions of endogenous PACAP after facial nerve injury. Although motor neuron survival after axotomy was not significantly different in PACAP deficient vs. wild type mice, recovery of axon regeneration after crush injury was significantly delayed. The impaired regeneration was associated with 8- to 12-fold increases in gene expression of proinflammatory cytokines tumor necrosis factor-alpha, interferon-gamma, interleukin (IL) -6, and a 90% decrease in the anti-inflammatory cytokine IL-4 at the injury site. Similar cytokine changes and an increased microglial response were observed in the brainstem facial motor nucleus. Because immunocompromised animals such as SCID mice are known to exhibit peripheral nerve regeneration defects, the observations raise the novel hypothesis that PACAP is critically involved in a carefully controlled immune response that is necessary for proper nerve regeneration after injury.

Pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance and protects against experimental autoimmune encephalomyelitis.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a widely expressed neuropeptide originally discovered in the hypothalamus. It closely resembles vasoactive intestinal peptide (VIP), a neuropeptide well known to inhibit macrophage activity, promote Th2-type responses, and enhance regulatory T cell (Treg) production. Recent studies have shown that administration of PACAP, like VIP, can attenuate dramatically the clinical and pathological features of murine models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However, specific roles (if any) of endogenous VIP and PACAP in the protection against autoimmune diseases have not been explored. Here, we subjected PACAP-deficient mice to myelin oligodendrocyte glycoprotein (MOG35–55)-induced EAE. MOG immunization of PACAP-deficient mice triggered heightened clinical and pathological manifestations of EAE compared to wild-type mice. The increased sensitivity was accompanied by enhanced mRNA expression of proinflammatory cytokines (TNFα, IL-6, IFN-γ, IL-12p35, IL-23p19, and IL-17), chemokines (MCP-1/CCL2, MIP-1α/CCL3, and RANTES/CCL5), and chemotactic factor receptors (CCR1, CCR2, and CCR5), but downregulation of the anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) in the spinal cord. Moreover, the abundance of CD4+CD25+FoxP3+ Tregs in lymph nodes and levels of FoxP3 mRNA in the spinal cord were also diminished. The reduction in Tregs was associated with increased proliferation and decreased TGF-β secretion in lymph node cultures stimulated with MOG. These results demonstrate that endogenous PACAP provides protection in EAE and identify PACAP as an intrinsic regulator of Treg abundance after inflammation.

Vasoactive Intestinal Peptide: An Important Trophic Factor and Developmental Regulator?

It has been proposed that vasoactive intestinal peptide (VIP) or a very closely related peptide has important actions very early in embryonic development. Recent data supporting this hypothesis are that subnanomolar concentrations of VIP significantly increased the growth rate of cultured embryonic day-9.5 (E9.5) mouse embryos, and that embryos at this and later stages exhibit a high degree of VIP binding in the brain stem and spinal cord. It is not known whether VIP is derived from the fetus, placenta, or mother at these early stages, or whether VIP acts in this culture system in place of a related peptide. The earliest reported expression of VIP in rat embryos is at E13.5, when the peptide and mRNA are expressed transiently in a high percentage of cells in the rat stellate ganglia. The time course of events mapped in other sympathetic ganglia at this stage suggest that transient expression of VIP in the ganglia might function to regulate neuroblast and/or glial cell proliferation, maturation or survival. Tissue culture studies indicate that VIP can support many of these trophic functions at concentrations that are the same or lower than that necessary to increase cAMP levels by way of classical VIP receptors. For example, VIP at 10(-10) M stimulates the release of neurotrophic factors from glial cells and maximally stimulates the proliferation of astrocytes. Two VIP receptors encoded on different genes have now been cloned. Both are members of the seven transmembrane G-protein-coupled receptor family and, when expressed in mammalian cells, mediate an increase in cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

VIP and PACAP: neuropeptide modulators of CNS inflammation, injury, and repair

Inflammatory processes play both regenerative and destructive roles in multiple sclerosis, stroke, CNS trauma, amyotrophic lateral sclerosis and aging‐related neurodegenerative diseases such as Alzheimers, Parkinsons and Huntingtons. Endogenous defence mechanisms against these pathologies include those that are directly neuroprotective, and those that modulate the expression of inflammatory mediators in microglia, astrocytes, and invading inflammatory cells. While a number of mechanisms and molecules have been identified that can directly promote neuronal survival, less is known about how the brain protects itself from harmful inflammation, and further, how it co‐opts the healing function of the immune system to promote CNS repair. The two closely related neuroprotective peptides, vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase‐activating peptide (PACAP), which are up‐regulated in neurons and immune cells after injury and/or inflammation, are known to protect neurons, but also exert powerful in vivo immunomodulatory actions, which are primarily anti‐inflammatory. These peptide actions are mediated by high‐affinity receptors expressed not only on neurons, but also astrocytes, microglia and peripheral inflammatory cells. Well‐established immunomodulatory actions of these peptides are to inhibit macrophage and microglia production and release of inflammatory mediators such as TNF‐α and IFN‐γ, and polarization of T‐cell responses away from Th1 and Th17, and towards a Th2 phenotype. More recent studies have revealed that these peptides can also promote the production of both natural and inducible subsets of regulatory T‐cells. The neuroprotective and immunomodulatory actions of VIP and PACAP suggest that receptors for these peptides may be therapeutic targets for neurodegenerative and neuroinflammatory diseases and other forms of CNS injury.

Noncompensation in peptide/receptor gene expression and distinct behavioral phenotypes in VIP- and PACAP-deficient mice

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are closely related neurotrophic peptides of the secretin/glucagon family. The two peptides are derived from a common ancestral gene and share many functional attributes in neuronal development/regeneration which occur not only from overlapping receptor subtype signaling but also through common mechanisms regulating their expression. Although PACAP or VIP null mice have been generated for study, it is unclear whether the expression of the complementary peptide or their receptor systems are altered in a compensatory manner during nervous system development. By radioimmunoassay and quantitative PCR measurements, we first show that PACAP and VIP have very different temporal patterns of expression in developing postnatal mouse brain. In wild‐type animals, PACAP transcript and peptide levels increased rapidly 2‐ and 5‐fold, respectively, within 1 week of age. These levels at 1 week of age were maintained through adulthood. VIP transcript and peptide levels, by contrast, increased 25‐ and 50‐fold, respectively, over a later time course. In parallel studies of development, there were no apparent compensatory increases in brain VIP expression in the PACAP knockout animals, PACAP expression in the VIP‐deficient animals, or receptor mRNA levels in either genotype. To the contrary, there was evidence for developmental delays in the expression of peptide and receptor transcripts in the knockout animals. A series of behavioral and neurological tests demonstrated differences between the knockout genotypes, revealing some functional distinctions between the two genes. These results suggest that the PACAP and VIP have evolved to possess distinct biological activities and intimate that the respective knockout phenotypes represent deficits unmitigated by the actions of the complementary related peptide.