James R. Howe - US grants

granule cell; glutamate receptor; cerebellum; developmental neurobiology; AMPA receptors; neural plasticity; receptor expression; NMDA receptors; kainate; cell migration; green fluorescent proteins; transfection /expression vector; confocal scanning microscopy; laboratory mouse; electrophysiology; Herpesviridae; tissue /cell culture;

glutamate receptor; receptor expression; membrane channels; brain electrical activity; recombinant proteins; protein sequence; gene mutation; granule cell; green fluorescent proteins; transfection; laboratory mouse; voltage /patch clamp; tissue /cell culture;

Glutamate-mediated excitotoxic injury has been implicated in ischemic neuronal death and a variety of neurodegenerative disorders. Sensitivity to excitotoxic injury is known to vary between different types of CNS neurons, to change during development, and to be influenced by various neurotrophic factors. This proposal will use two well-characterized model systems, primary cultures of cerebellar granule cell neurons and spinal motor neurons, to determine whether and to what degree alterations in the expression, subunit composition, or phenotypic properties of glutamate receptors contribute to differences we have found in the sensitivity of these two types of neurons to glutamate-induced cell death. The proposal has three specific aims. 1.) Mature cerebellar granule cells are resistant to excitotoxic injury. Immature granule cells express the NMDA- receptor subunit NR2B, whereas mature granule cells express the NR2C subunit. To test directly whether changes in NR2 expression influence the sensitivity of granule cells to excitotoxic death, we will compare the sensitivity of granule cells cultured under conditions where NR2B or NR2C expression predominates and use replication-deficient viral vectors to alter the expression of these two subunits. 2.) We have found that exposure of purified cultures of spinal motor neurons to brain-derived neurotrophic factor (BDNF) greatly increases the vulnerability of these cells to excitotoxic death. To test whether this effect is the result of changes in glutamate-receptor (GluR) phenotype, we Will use electrophysiological methods to compare motor neurons that have been grown in the presence or absense of BDNF. We will also compare the GluR phenotype of motor neurons from BDNF-treated cultures that have or have not been exposed to glutamate. 3.) The effect of BDNF on the GluR expression and sensitivity of motor neurons to excitotoxic injury will be studied in primary cultures of spinal cord from transgenic animals in which motor neurons can be identified by cell-type specific expression of the Green Fluorescent Protein (GFP). The results will be compared with those from experiments on spinal cord cultures from mutant mice we will generate by crossbreeding the GFP-transgenic animals with BDNF knockout mice.

DESCRIPTION (provided by applicant) The purpose of the program is to give both predoctoral and postdoctoral training in neuropharmacology to future research workers and future faculty members. The program will capitalize on the wide diversity of talent in the training faculty, who include individuals whose approaches would normally be found not just in a department of Pharmacology, but also approaches more commonly found in departments of Anatomy, Biochemistry, Biophysics, Physiology and Psychiatry. The approach thus will be multidisciplinary: Predoctoral trainees will be recruited from recently graduated college students, usually with majors in the biological or physical sciences; and postdoctoral trainees will be drawn from recently graduated Ph.D's in a variety of fields (Biochemistry, Physiology, Biophysics and Molecular Biology, for example) and M.D's. Training in neuropharmacology is offered with general emphasis on drugs as they elucidate, and modify: the mechanism of synthesis, storage and release of neurotransmitters and neuromodulators; the molecular basis of conduction; the relations between metabolism and function in nervous tissue; and the importance of these parameters in health and disease. The specific areas of research training will all be in the areas of expertise of the senior neuropharmacologists in the department. In addition, advanced seminar courses in neuropharmacology as well as seminar programs in general pharmacology are available.

DESCRIPTION (provided by applicant): Juvenile Polyposis (JP) is a hamartomatous gastrointestinal polyposis syndrome in which affected patients are at significant risk for developing colorectal cancer. We have previously shown that JP is caused by germline mutations of SMAD4 and BMPR1A, implicating the bone morphogenetic and TGF-beta superfamily signaling pathways in JP-associated tumorigenesis. Microscopically, juvenile polyps have a markedly expanded lamina propria, with abundant stroma, an inflammatory infiltrate, and normal overlying epithelium. However, Jp-associated cancers are epithelial, and the process by which these polyps develop and how they transform to adenocarcinoma represents a unique and largely unexplored mechanism of carcinogenesis. The long-term goal of these studies is to define how cancers arise from juvenile polyps of the gastrointestinal tract. The objective of this application is to determine the molecular genetic events involved in juvenile polyp formation. The central hypothesis is that germline mutations in TGF-beta superfamily genes predispose patients to JP, and gastrointestinal polyps develop as a consequence of further alterations in these and related signaling pathways. We propose to achieve our objective through two Specific Aims: 1) To discover new JP genes by genetic linkage and positional candidate approaches; and 2) To examine patterns of global gene expression in the transcriptomes of juvenile polyps in order to define the common genetic pathways involved in their formation, and the specific contributions of different cell types within polyps to these expression patterns. Collectively, these studies will define the molecular genetic changes occurring in JP patients, beginning at birth with germline mutations and progressing through somatic alterations in the gastrointestinal tract, leading to juvenile polyps. Identification of a new JP gene will further define the specific TGF-beta superfamily pathways whose altered signaling brings about this phenotype, and gene expression studies will delineate the secondary events leading to juvenile polyps, and those genes whose transcription is affected by the germline mutations. The insights gained from this project will lay the groundwork for future studies to determine the specific genetic events required for the transformation of juvenile polyps into cancers, and to study the contribution of this novel mechanism of carcinogenesis in sporadic colorectal tumors. These studies will also benefit JP patients by improving presymptomatic genetic testing, and identification of new therapeutic targets for polyp regression and cancer prevention in JP.

DESCRIPTION (provided by applicant): The AMPA subtype of ionotropic glutamate receptors (GluRs) mediate the vast majority of excitatory synaptic transmission in the mammalian CNS and participate in a variety of plastic changes in synaptic strength. One hallmark feature of these channels is that they desensitize in a few milliseconds, and at some CNS synapses desensitization contributes to shaping the decay of EPSCs. Because concentrations of glutamate in the low micromolar range can produce nearly complete desensitization, at most synapses desensitization likely occurs during single EPSCs and may even be significant at ambient levels of glutamate. Thus the rate at which channels recover from desensitization is a key determinant of the fidelity with which CNS neurons are able to follow high-frequency presynaptic firing. Here we propose a series of biophysical experiments designed to answer three questions about the kinetic and structural features that control recovery from AMPA-receptor desensitization. 1) What are the relative contributions of re-sensitization and ligand dissociation to the kinetics of recover and do they vary for different agonists? 2) To what extent is the ligand binding domain closed during desensitization and does this vary for individual subunits? 3) What are the structural determinants of the rate of recovery?

DESCRIPTION (provided by applicant): Juvenile Polyposis (JP) is a hamartomatous gastrointestinal polyposis syndrome in which affected patients are at significant risk for developing colorectal cancer. We have previously shown that JP is caused by germline mutations of SMAD4 and BMPR1A, implicating the bone morphogenetic and TGF-beta superfamily signaling pathways in JP-associated tumorigenesis. Microscopically, juvenile polyps have a markedly expanded lamina propria, with abundant stroma, an inflammatory infiltrate, and normal overlying epithelium. However, Jp-associated cancers are epithelial, and the process by which these polyps develop and how they transform to adenocarcinoma represents a unique and largely unexplored mechanism of carcinogenesis. The long-term goal of these studies is to define how cancers arise from juvenile polyps of the gastrointestinal tract. The objective of this application is to determine the molecular genetic events involved in juvenile polyp formation. The central hypothesis is that germline mutations in TGF-beta superfamily genes predispose patients to JP, and gastrointestinal polyps develop as a consequence of further alterations in these and related signaling pathways. We propose to achieve our objective through two Specific Aims: 1) To discover new JP genes by genetic linkage and positional candidate approaches; and 2) To examine patterns of global gene expression in the transcriptomes of juvenile polyps in order to define the common genetic pathways involved in their formation, and the specific contributions of different cell types within polyps to these expression patterns. Collectively, these studies will define the molecular genetic changes occurring in JP patients, beginning at birth with germline mutations and progressing through somatic alterations in the gastrointestinal tract, leading to juvenile polyps. Identification of a new JP gene will further define the specific TGF-beta superfamily pathways whose altered signaling brings about this phenotype, and gene expression studies will delineate the secondary events leading to juvenile polyps, and those genes whose transcription is affected by the germline mutations. The insights gained from this project will lay the groundwork for future studies to determine the specific genetic events required for the transformation of juvenile polyps into cancers, and to study the contribution of this novel mechanism of carcinogenesis in sporadic colorectal tumors. These studies will also benefit JP patients by improving presymptomatic genetic testing, and identification of new therapeutic targets for polyp regression and cancer prevention in JP.

Glutamate-receptor ion channels (iGluRs) participate in brain functions that range from fast synaptic transmission to activity-dependent changes that underlie certain forms of learning and memory. These receptors are also implicated in a variety of excitotoxic pathologies and neurodegenerative diseases. The AMPA subtype of iGluRs (AMPARs) gives rise to the fast component of excitatory postsynaptic currents (EPSCs) at virtually all brain synapses examined, but there are few direct measurements of the properties of individual channel molecules. In addition, there are no direct structural data on the determinants of protein-protein interactions that influence localization of the receptors at synapses. We propose a series of biophysical studies that will characterize the unitary properties of AMPARs by analyzing single-channel currents through native and recombinant channels. The studies of recombinant channels will be combined with crystallographic investigations of the ligand-binding domain of the GluR2 subunit (GluR2-S1S2). Other crystallographic studies will further define the molecular determinants of AMPAR interactions with important trafficking, scaffolding, and cytoskeletal proteins. The proposal has four main goals. 1.) In Aim 1 we will determine the kinetics of native AMPARs in one channel patches from cerebellar neurons in situ. The results will provide information crucial to evaluating the impact of receptor properties on synaptic transmission. 2.) Aim 2 will employ single-channel recording to elucidate the molecular mechanisms underlying the effect of mutations that reduce steady-state desensitization of AMPARs and determine to what extent the mutations also alter activation gating. 3.) In Aim 3, single-channel recording and x-ray crystallography will be used to understand how detailed interactions within the binding cleft influence the stability of binding cleft closure and in turn the affinity and efficacy of receptor agonists. 4.) The number and location of AMPARs at synapses are key determinants of the gain and fidelity of information transfer in the brain. Aim 4 will use x-ray crystallography to determine the structural basis of important protein-protein interactions that regulate receptor trafficking.

Juvenile Polyposis (JP) is an autosomal dominant syndrome predisposing to the development of hamartomatous polyps of the colon, rectum, and stomach. Affected patients have an approximately 50% risk of developing gastrointestinal cancer. Work in our laboratory has identified 2 genes that cause JP, both of which are members of the transforming growth factor beta (TGF-[unreadable]) super family. One of these genes, SMAD4, is the common intracellular mediator of signaling through the TGF-[unreadable], bone morphogenetic protein (BMP), and activin pathways. The other, BMPR1A, is a cell surface receptor which transduces BMP signals from the cell membrane into the cytoplasm. Mutations in the coding sequence of each gene have been found in the germline of approximately 20% of JP cases, respectively. The cause for the other 60% of JP cases is unknown, and could include other undiscovered genes predisposing to JP, larger deletions of the known genes not detectable by sequencing, and changes in non-coding regions of these genes, thereby altering gene expression. The focus of our JP studies over the past several years has been to discover new JP genes through a linkage-based genome screen of a large JP kindred, and direct sequencing of other genes in the TGF-[unreadable] super family for mutations in a large number of JP families. We have also explored the prevalence of exonic deletions, which accounted for another 4% of JP cases. A recent provocative finding came from this genome screen and deletion studies. In a large JP family without coding mutations of SMAD4 or BMPR1A, it was discovered that a third JP gene was not the cause of JP, but rather, a deletion of the putative promoter region and first non-coding exon of BMPR1A was inherited through the germline. This has led us to focus our studies upon investigating how the known JP genes are regulated and lead to altered BMP signaling. Another JP patient has been found with a deletion of the putative promoter and first 2 non-coding exons of SMAD4, lending further support to this approach. Therefore, in this grant we propose to examine factors which affect the expression of JP genes and proteins, a largely unexplored and potentially very important mechanism underlying the genetic basis of autosomal dominant cancer syndromes. Our specific aims are: 1) to characterize the promoter region and non-coding exons of SMAD4 and BMPR1A and evaluate JP patients for germline changes;and 2) to determine the influence of mutations in coding and non-coding regions of JP genes on RNA and protein expression.

The goal of the Career Development Program of the Iowa Neuroendocrine Tumor SPORE is to develop knowledgeable, well-trained scientists experienced in multidisciplinary research for future studies in translational neuroendocrine tumor research. The provision of ample opportunities for training and career enhancement is a top priority of the University of Iowa scientific community. The Career Development Program is a critical component of our long-term commitment to research in neuroendocrine tumors that allows us to recruit bright, energetic new investigators into translational SPORE research. The extensive research and training base available at the University of Iowa provides strong support for the SPORE Career Development Program. The primary objective of the Iowa Neuroendocrine Tumor SPORE Career Development Program is to train young investigators in translational, multidisciplinary basic, clinical or population-based research in neuroendocrine tumors. This is an ideal point in their careers to develop a collaborative, translational focus. One career development awardee will be chosen each year and supported for 1-2 years. The SPORE Career Development award will provide up to $100,000 for one year ($50,000 from SPORE funds and a matching $50,000 from the University of Iowa). An additional year of support is possible based on progress and successful re-competition. The Career Development Program Director will maintain: (1) a stringent candidate selection system; (2) comprehensive trainee guidance by a mentor; (3) support through a scientific mentor group (the Individual Trainee Mentorship Committee) comprised of investigators with expertise in each trainee's area of interest; (4) multi-disciplinary research courses and on-going training activities such as the Multidisciplinary Cancer Seminar Series and Neuroendocrine Tumor Group meetings; (5) attendance at Investigator Group meetings and yearly SPORE retreat; (6) encouragement of collaboration with investigators at both Iowa and outside institutions; and (7) opportunities for research enhancement through the Developmental Research Program.

The incidence of Neuroendocrine Tumors (NETs) has increased five-fold over the last three decades, and many patients do not develop symptoms until the tumors have metastasized. Although patients with these tumors may have prolonged survival despite advanced stage, further understanding of the molecular biologic basis of NETs holds the promise for improved diagnosis, imaging, and therapy. We hypothesize that analyzing the gene expression profiles of these tumors and their exomes will allow us to identify important genes that will facilitate clinical advances for patients with these tumors. This contribution will be significant because it will allow us to determine the tumor site of origin in patients presenting with liver metastases and unknown primaries, which will lead to more directed surgical exploration and resection; and knowledge of cell surface receptors or other genes significantly over-expressed in NETs relative to normal tissues will facilitate the development of new targets for detection, imaging, and medical management. Novel targets for therapy will also be suggested by the identification of frequently mutated or deleted genes in these tumors or the germline of patients with familial NETs.