James Arvid Waschek - US grants

DESCRIPTION: Vasoactive intestinal peptide (VIP) is a potent regulator of neuroblast proliferation and survival in vitro. Administration of VIP antagonist to pregnant mice produced a pronounced microcephaly condition in offspring, providing a new model for human brain disease. VIP effects in tissue culture can also be brought about by the structurally-related neuropeptide pituitary adenylate cyclase activating peptide (PACAP). Despite the demonstrated capacity of these peptides to regulate CNS development, investigators have been unable to show that VIP or PACAP is expressed in embryonic CNS of mice at early stages. Thus, the current model contends that VIP is derived transplacentally from the mother, and thereby acts as a global regulator of embryonic CNS development. To challenge this model , sensitive in situ hybridization protocols to detect mRNA for these ligands and their receptors was developed. This combined with data obtained by Northern analyses suggest that the VIP/PACAP ligand/receptor system may be operational within the embryo at the earliest stages of neurogenesis. It is thus proposed that VIP and PACAP are derived from the embryo and act in local domains to regulate CNS development. Two novel potential target populations for VIP and PACAP were identified by this analysis, neuroepithelial cells and microglia. It is proposed that VIP and PACAP regulate the proliferation of these cells. This will be tested using dispersed cell and short-term cultures of intact embryos. In addition, the unexplained ability of these peptides to induce proliferation in some cell populations and cell cycle withdrawal in others will be examined. It is hypothesized that the differences are due to G-protein coupling of receptors to different signal transduction pathways. This will be tested using a combination of pharmacological, molecular, and biochemical approaches, including analysis of the Rs/Raf/MAP kinase pathway. The results are expected to provide important mechanistic information on how these neuropeptides play important roles in CNS development, as well as in nerve injury, neurological tumors, and degenerative diseases of the nervous system.

Vasoactive intestinal peptide (VIP) and the related neuropeptide pituitary adenylate cyclase activating peptide (PACAP) are potent regulators of neuroblast proliferation and survival in vitro. Administration of a VIP antagonist to pregnant mice produced a microcephaly condition in offspring, providing a new model for a human disease that is nearly always associated with mental retardation. In addition, VIP has been found to be highly upregulated in nerve injury models, and PACAP has been found to reduce the degree of cell death in the hippocampus following experimental forebrain ischemia. Thus, the VIP/PACAP ligand/receptor system may be important in CNS morphogenesis and injury. With respect to development, the current model contends that VIP is derived transplacentally from the mother, and thereby acts as a global regulator of embryonic CNS growth. However, key data from this laboratory indicate that the VIP gene is expressed in the mouse hindbrain as early as embryonic day 11, suggesting that the peptides are derived from the embryo and act in local domains (rather than globally) to regulate CNS development. In this proposal, the temporal and spatial expression patterns of VIP and PACAP, and their receptors during normal development will be determined using in situ hybridization and immunohistochemistry. The phenotype of potential target cell types will be identified by colocalizing receptors with markers for various developing neural lineages. It will also be determined if the VIP/PACAP ligand/receptor system is upregulated in an injury model. Based on the patterns of expression of peptide and receptor during development and after injury, in vitro model will be established to study the growth- or injury- related actions of these peptides on relevant cell populations Signal transduction pathways leading to these actions will be examined using pharmacological, biochemical, an molecular approaches. The results are expected to provide important mechanistic information on the role of peptides such as VIP and PACAP in normal and abnormal CNS development and after injury to the developing brain.

DESCRIPTION (provided by applicant): Circadian rhythms have been extensively studied as a behavioral model because of their high degree of predictability and reproducibility within a species, and because several components and regulatory mechanisms are conserved from Drosophila to humans. Molecular dissection of this process has begun in mice using knockout and transgenic strategies. In general, information from these approaches can be maximized if expression or excision of a gene can be restricted to a desired tissue and controlled. These approaches have been successfully used to investigate learning and memory, but have not yet been applied to the regulation of circadian rhythms. A major reason for this is that a reliable strategy has not yet been developed that can specifically target expression of a desired gene to the retinorecipient cells of the suprachiasmatic nucleus (SCN), a primary circadian regulator in mammals. A targeting approach will be developed here to achieve relatively specific expression of a gene product in the retinorecipient neurons of SCN, i.e. the primary cells in which the circadian clock is reset in response to environmental signals such as light. To show the utility of the targeting strategy, CRE recombinase will be expressed in these cells. Tissue-specific CRE-mediated gene excision will be demonstrated using an established reporter system. Once validated, the CRE-expressing mice will be breed with existing foxed NMDA1 receptor mice to test the hypothesis that NMDA receptors in retinorecipient neurons are critically required for light-induced phase shifts. The same targeting system may be adapted in the future to study the putative involvement of other signaling proteins in light-induced resetting of the circadian clock, such as CAM kinase II, NOS, cGMP- and cAMP-dependent protein kinases, MAP kinases and CREB. Moreover, the same targeting system should have utility in investigating individual components of the clock, such as PER, CRY, BMAL and CLOCK proteins.

DESCRIPTION (provided by applicant): Pituitary adenylyl cyclase activating peptide (PACAP) is a neuropeptide in the secretin family with potential significance in a range of brain afflictions, including mental retardation, autism, neurodegenerative diseases, CNS injury, and brain tumors. Because PACAP and PACAP receptor gene expression is widespread at very early stages of neurogenesis, several hypotheses regarding specific developmental actions of this peptide have been proposed. For example, in vitro and in vivo studies indicate that PACAP acts as an autocrine or paracrine factor to control neural cell proliferation, survival and phenotype in several regions of the developing nervous system. It is propose here that PACAP acts to control processes in the developing hindbrain and cerebellum by antagonizing the action of the mitogen/patterning factor sonic hedgehog (Shh) via protein kinase A activation.In this proposal, genetic models will be used to identify and investigate the essential developmental roles of PACAP and the specific mechanisms involved. These models include 1) mice in which the PACAP gene have been disrupted by homologous recombination, 2) transgenic mice that express an overactive Shh signaling system due to a mutation in the Shh receptor/tumor suppressor gene ptc-1, 3) a Xenopus embryo mRNA injection system which overexpresses PACAP, and 4) PACAP-responsive embryonic hindbrain and postnatal cerebellar granule cell precursor cultures. The action of PACAP will be investigated in these models using morphologic and phenotypic markers, gene expression, and indicators of cell proliferation and apoptosis. The results are expected to reveal critical developmental functions of PACAP and provide information on the relevant signaling pathways. The work may lead to new strategies for the diagnosis and treatment of diseases of brain development, degenerative diseases and brain tumors.

Abstract Not Provided.

DESCRIPTION (provided by applicant): Medulloblastoma is the most common malignant brain tumor in children, accounting for 20-25% of pediatric brain tumors. The survival rate for patients with this tumor is about 50%, however the therapy required to eradicate the tumor in children during brain development results in significant additional morbidity. Overactivty of the sonic hedgehog (Shh) signaling cascade appears to occur at high frequency in these tumors. Thus, animal models with medulloblastoma tumors overactive in this signaling pathway are a potentially valuable resource to investigate the initiation and propagation of these tumors, and can also be used to test potential therapies. This proposal investigates a new mouse model in which sonic hedgehog (Shh) signaling is enhanced in the cells that give rise to medulloblastoma. These mice contain two targeted mutations, one in the gene encoding the Shh receptor/tumor suppressor patched-1 (ptc-1), and other in the gene encoding the secreted neuropeptide PACAP (pituitary adenylyl cyclase activating peptide). PACAP receptors are colocalized in the germinal areas the brain that are thought to give rise to medulloblastoma. It is proposed that PACAP normally inhibits Shh signaling and Shh mitogenic action via protein kinase A (PKA). Preliminary data indicate that ptc-1/PACAP double heterozygous mice have a medulloblastoma incidence of 66% (compared to 15% in ptc-1 mice) with an average onset that is significantly more rapid (16 weeks vs. 28 weeks in ptc-1). In this proposal, we will further characterize this model, and plan to use it along with the derived cell lines to better understand the significance of the PACAP/PKA pathway in medulloblastoma and the mechanism by which PKA interacts with the hedgehog pathway.

DESCRIPTION (provided by applicant): Medulloblastoma is the most common malignant brain tumor in children, accounting for 20-25% of pediatric brain tumors. The survival rate for patients with this tumor is about 50%, however the therapy required to eradicate the tumor in children during brain development results in significant additional morbidity. Overactivty of the sonic hedgehog (Shh) signaling cascade appears to occur at high frequency in these tumors. Thus, animal models with medulloblastoma tumors overactive in this signaling pathway are a potentially valuable resource to investigate the initiation and propagation of these tumors, and can also be used to test potential therapies. This proposal investigates a new mouse model in which sonic hedgehog (Shh) signaling is enhanced in the cells that give rise to medulloblastoma. These mice contain two targeted mutations, one in the gene encoding the Shh receptor/tumor suppressor patched-1 (ptc-1), and other in the gene encoding the secreted neuropeptide PACAP (pituitary adenylyl cyclase activating peptide). PACAP receptors are colocalized in the germinal areas the brain that are thought to give rise to medulloblastoma. It is proposed that PACAP normally inhibits Shh signaling and Shh mitogenic action via protein kinase A (PKA). Preliminary data indicate that ptc-1/PACAP double heterozygous mice have a medulloblastoma incidence of 66% (compared to 15% in ptc-1 mice) with an average onset that is significantly more rapid (16 weeks vs. 28 weeks in ptc-1). In this proposal, we will further characterize this model, and plan to use it along with the derived cell lines to better understand the significance of the PACAP/PKA pathway in medulloblastoma and the mechanism by which PKA interacts with the hedgehog pathway.

[unreadable] DESCRIPTION (provided by applicant): Periventricular white matter injury (PWMI), a major cause of cerebral palsy associated with premature birth, results in significant suffering to the afflicted and their families, and is a major financial burden to society. The establishment of animal models is critical to understanding the pathogenesis of PWMI and for examining new treatment modalities. Historically, the most prevalent type of PWMI was focal in nature, and thought to be due to rupture of prematurely developed brain blood vessels, local hypoxia and ischemia. The final result was necrotic death and cystic accumulation of all cell types in the affected white matter. Recent advances in perinatal care have led to a dramatically increased survival of preterm infants and a significant reduction in focal PWMI. However, the incidence of a diffuse form of PWMI, primarily affecting the myelinating cells of the CNS (oligodendrocytes), has not decreased, and is now the most prevalent type of PWMI leading to mental retardation. The investigators propose here to establish a new mouse model of PWMI in which injury is induced at a developmental time point at which OL progenitors are most vulnerable. Because epidemiological data indicate an association of PWMI with infectious diseases in pregnancy, they will determine if an inflammatory insult is sufficient to induce PWMI, and if so, the potential mechanisms involved. Among future applications, they anticipate using the model to study the protective roles of two neuropeptides with potent in vivo anti-inflammatory actions, VIP and PACAP (vasoactive intestinal peptide and pituitary adenylyl cyclase activating peptide, respectively). [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Medulloblastoma is the most common malignant brain tumor in children, accounting for 20-25% of pediatric brain tumors. The survival rate for patients with this tumor is about 50%, however the therapy required to eradicate the tumor in children during brain development results in significant additional morbidity. Overactivty of the sonic hedgehog (Shh) signaling cascade appears to occur at high frequency in these tumors. Thus, animal models with medulloblastoma tumors overactive in this signaling pathway are a potentially valuable resource to investigate the initiation and propagation of these tumors, and can also be used to test potential therapies. This proposal investigates a new mouse model in which sonic hedgehog (Shh) signaling is enhanced in the cells that give rise to medulloblastoma. These mice contain two targeted mutations, one in the gene encoding the Shh receptor/tumor suppressor patched-1 (ptc-1), and other in the gene encoding the secreted neuropeptide PACAP (pituitary adenylyl cyclase activating peptide). PACAP receptors are colocalized in the germinal areas the brain that are thought to give rise to medulloblastoma. It is proposed that PACAP normally inhibits Shh signaling and Shh mitogenic action via protein kinase A (PKA). Preliminary data indicate that ptc-1/PACAP double heterozygous mice have a medulloblastoma incidence of 66% (compared to 15% in ptc-1 mice) with an average onset that is significantly more rapid (16 weeks vs. 28 weeks in ptc-1). In this proposal, we will further characterize this model, and plan to use it along with the derived cell lines to better understand the significance of the PACAP/PKA pathway in medulloblastoma and the mechanism by which PKA interacts with the hedgehog pathway.

DESCRIPTION (provided by applicant): Periventricular white matter injury (PWMI) associated with premature birth results in significant suffering to the afflicted and their families and is a major financial burden to society. Appropriate animal models need to be established and refined to serve important tools to understanding the pathogenesis of PWMI and for testing preclinical treatment modalities. Historically, the most prevalent type of PWMI was focal in nature, and thought to be due to rupture of prematurely developed brain blood vessels, local hypoxia and ischemia. The final result was necrotic death of multiple cell types and cystic accumulation in the affected white matter. Recent advances in perinatal care have led to a dramatically increased survival of preterm infants and a significant reduction in focal PWMI. However, the incidence of a diffuse form of PWMI, primarily affecting the myelinating cells of the CNS (oligodendrocytes), has not decreased, and is now the most prevalent type of PWMI leading to motor and cognitive disabilities, occurring in about 45-65% of pre-term births. We have modeled this increasingly- prevalent form of PWMI in mice by inducing injury at a developmental time point at which oligodendrocyte progenitors are most vulnerable. In light of epidemiological data which indicate an association of PWMI with infectious diseases during pregnancy and preterm infants, we have determined that a single systemic inflammatory insult administered at this time of peak vulnerability mimics many of the anatomical and behavioral features of this newly-emerging form of PWMI. We propose here to use this new model to dissect the possible mechanisms of oligodendrocyte damage and white matter loss in PWMI using a transgenic lineage tracing system which labels oligodendrocyte progenitors and their progeny, as well as a conditional knockout mouse in which the astrogliotic response is blocked. PUBLIC HEALTH RELEVANCE: Brain injury, including cerebral palsy and mental retardation is associated with premature birth and results in significant suffering to the afflicted and their families and is a major financial burden to society. As epidemiological studies indicate that a likely cause for this condition is maternal infection, we have established a mouse model which recapitulates the disease, and propose to use the model to determine how maternal infection leads to these defects. The proposed studies are expected to reveal new strategies that will allow the development of both predictors that help identify infants at risk and therapeutic agents reduce the consequences in afflicted children.

DESCRIPTION (Provided by Applicant): The Hedgehog (Hh) pathway is involved in many aspects of mammalian development, and it also has been implicated in the growth and metastasis of multiple types of cancer. It has been shown that Hh signaling is critically dependent on the primary cilium, an antenna-like organelle that extends from the cell surface into the surrounding environment. However, little is known about how, on the molecular level, different components of the pathway interact at the cilium. The overall goal of this application is to elucidate the role of primary cilium in the negative regulation of the Hh pathway by protein kinase A (PKA) in vertebrates. The primary hypothesis is that the Hh pathway is subject to negative modulation by two separate, differentially regulated pools of PKA, one sequestered in the cilium and responsible for keeping the Hh pathway silenced when the Hh ligand is absent, and the other in the cytoplasm and regulated by heterotrimeric G protein-coupled receptors. This hypothesis will be tested by targeting various proteins to ciliary or extra-ciliary compartments in cultured cells through genetic manipulation, visualizing their subcellular compartmentalization by confocal fluorescence microscopy, and then assaying their effect on PKA activity and on posttranslational modifications of the Hh regulated transcription factor Gli3, a protein enriched in cilia and whose phosphorylation by PKA and proteolytic cleavage is blocked by Hh signaling. These studies have the potential to greatly improve our understanding of how the primary cilium participates in the regulation of cellular signaling. More specifically, the results are expected to provide evidence for a fully functional phosphorylation pathway in the cilium, which is remarkable in view of extreme spatial constraints that exist within this organelle. PROJECT NARRATIVE: The Hedgehog (Hh) pathway is involved in many aspects of organ development and it also has been implicated in the growth and metastasis of multiple types of cancer. The molecular events triggered by the Hh pathway are antagonized by protein kinase A (PKA), but the significance is poorly understood, hampering the development of useful pharmacologic agents that target PKA. This application tests a novel hypothesis with respect to the Hh/PKA interaction that, if shown to be true, is expected to lay the groundwork for developing entirely new sets of therapeutic agents to block the cancer-promoting actions of the Hh pathway.

DESCRIPTION (provided by applicant): Understanding the biological mechanisms and biomarkers of psychiatric disease is critical for understanding, assessing risk, and designing treatments of disorders such as post traumatic stress disorder (PTSD). In this regard, it was recently reported that the neuropeptide PACAP and its plasma membrane receptor PAC1 are linked to PTSD at both genetic and epigenetic levels. These findings complement a considerable set of prior evidence implicating PACAP/PAC1 signaling in stress and fear circuitries. Experiments in the proposal will use gene targeting approaches to dissect at the cellular, molecular, and behavioral levels, the involvement of PACAP-PAC1 signaling in the circuitry regulating fear in mice. The results are hoped to lay a mechanistic foundation for the development of an entirely new set of therapeutic targets for PTSD based on PAC1 signaling and downstream actions, and may also lead to the discovery of novel and robust biomarkers associated with this pathway.