Martin Grumet - US grants

Interactions among neurons and between neurons and glia are critical in neural development and in the maintenance of the vertebrate brain. Specific cell surface molecules that mediate cell-cell adhesion have been identified and evidence for their specific roles in neural histogenesis is emerging. The long-term objective of this work is to understand the molecular basis for adhesion between neurons and glia in normal and aberrant brain development and function. Ng-CAM is a glycoprotein found on neurons but not on astroglia. Perturbation experiments with specific antibodies indicated that Ng-CAM is involved in neuron- astroglia adhesion and in the migration of neurons along Bergmann glia. The molecule is also involved in neuron-neuron adhesion and the fasciculation of neurites. Our recent results suggest that the ligands for Ng-CAM on neurons and astroglia are different. The specific aims of this proposal are to characterize structural features of the Ng-CAM molecule that are involved in binding, to identity and, characterize astroglial and neuronal ligands for Ng- CAM and to analyze other molecules that influence neuron-astroglial cell adhesion. Protein chemical and immunological techniques will be used to analyze the structure and binding function of Ng-CAM polypeptides. Ligands for Ng-CAM on neurons and astroglia will be identified by their binding to immobilized Ng-CAM. Glial ligands will also be isolated by selecting molecules that neutralize the inhibition by anti-astroglial antibodies of Ng-CAM binding to astroglial cells. Specific antibodies against ligands for Ng-CAM will be prepared and used to determine their localization in tissues and to evaluate their role in neuron-astroglial cell ad- hesion and granule cell migration in cerebellar explants in vitro. Other molecules that may be involved in neuron-astroglia interactions include cytotactin (a matrix, molecule secreted by astroglia), a neuronal proteoglycan that binds to cytotactin, laminin, fibronectin, and N-CAM. To understand the roles of these molecules in neuron-astroglia interactions in tissue, specific antibodies and binding fragments of the adhesion molecules will be used to perturb cerebellar morphogenesis in vitro and the results of functional assays will be correlated with the amount and distribution of each molecule in the tissues. The findings of these and related studies will form a basis for evaluating the role of Ng-CAM and other adhesion molecules in specific neurological disorders by comparison to the normal phenotype using biochemical, histological and cell biological criteria.

The long term goal of our research is to understand the mechanism of signal transduction by neuropeptide receptors. Substance-K (neurokinin-A) receptors play important roles in both normal and pathological processes, including memory retention and rheumatoid arthritis [l, 2, 3]. The goal of the research proposed here is to determine structure-function relationships of neuropeptide receptors using Neurokinin A receptor as a model system for neuropeptide receptors, which have seven hydrophobic membrane spanning regions. Following ligand binding, neuropeptide receptors couple with G-proteins to transduce their signals. A key breakthrough in analyzing structure-function is the cloning of the receptor. The human Neurokinin A receptor has been cloned by us and will be used in this analysis. Specific aims are 1) To determine functional domains of the substance-K receptor involved with G-protein interaction by producing chimeras between the neurokinin A receptor and the beta2- adrenergic receptor which utilize different G-proteins. Precedence from the adrenergic and muscarinic receptor systems will guide the choice of domains to be swapped. 2) To determine the role of post-translational modification of the Neurokinin A receptor. a) There are potential myristylation and palmitoylation sites on the Neurokinin A receptor. The use and importance of these sites will be analyzed by metabolic labelling experiments and site directed mutagenesis. b) The response to neurokinin A is rapidly desensitized. In the adrenergic system, desensitization has been shown to controlled by phosphorylation of the C-terminal tail. The role of phosphorylation of the C-terminal tail of the Neurokinin A receptor will be analyzed by in vivo labelling with 32p and by deletion mutagenesis of the C-terminal tail. A concise understanding of the sites important in the interaction between neuropeptide receptors and their transducing proteins (G-proteins) is necessary for understanding the mechanism of signal transduction by neuropeptide receptors. The site of G-protein interaction and the role of post-translational modifications of the Neurokinin A receptor are keys in understanding the mechanism of stimulation or desensitization of the response to Neurokinin A, important concepts in normal regulation of the proper receptor function. The understanding of mechanism of signal transduction by these receptors may lead to more potent drugs for treating diseases such as rheumatoid arthritis and for analysis of memory retention induced b Neurokinin A.

9310731 Grumet Ng-CAM is a neuronal cell adhesion molecule (CAM) that is a potent promoter of cell adhesion and axon growth. An Ng-CAM molecule on one cell can bind to another Ng-CAM molecule on an adjacent cell (homophilic binding). Ng-CAM can also bind to other cell surface proteins. The goal of this project is to identify regions in the Ng-CAM molecule that mediate homophilic binding. To identify regions in Ng-CAM that mediate homophilic binding, established assays will be used to test the properties of Ng-CAM polypeptides representing different regions of the molecule. Different regions of Ng-CAM will be generated either by genetic engineering or by cleaving the native protein into small subunits. Various regions within the molecule will be tested for their ability to bind to intact Ng-CAM and eventually for binding to fragments of the molecule. Neuronal adhesion and axon growth to surfaces coated with the various forms of Ng-CAM will then be measured. Identification of homophilic binding region(s) in Ng-CAM is an essential foundation for analyzing molecular interactions that mediate or control neuronal adhesion and axon growth. These studies will therefore allow a better understanding of the types of molecular interactions that control key aspects of brain development. *** %T%U%V%W%X%Y%Z% %\% %^%_%`%a% c% f%g%h%i%j%k%l%m%n%o%p%q%r%s%t%u%v%w%x%y%z%{%|%}%~% % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % %9310731 Grumet Ng-CAM is a neuronal cell adhesion molecule (CAM) that is a potent promoter of cell adhesi on and axon growth ! ! ! F % % ( Times New Roman Symbol & Arial # # # " h A grumet William Proctor, IBN William Proctor, IBN

Cell adhesion molecules (CAMs) and extracellular matrix (ECM) molecules play important roles in cell adhesion and migration during neural histogenesis. The long-term objective of this work is to understand the roles of these proteins in adhesion and migration of neural cells during development. Ng-CAM is expressed on neurons and Schwann cells but not on astroglia. Perturbation experiments with specific antibodies indicate that Ng-CAM is involved in neuron.astroglia adhesion and migration of neurons along Bergmann glia. Its function is also important for neuron-neuron adhesion, axonal growth, and axonal fasciculation. Ng-CAM binds to Ng-CAM on neurons and to distinct ligands on astroglia (heterophilic binding). Recent studies indicate that Ng-CAM interacts with ECM molecules that are associated with astroglia including the 1D1 and 3F8 chondroitin sulfate proteoglycans and laminin. The specific aims of thiN proposal are a) to characterize heterophilic ligands for Ng-CAM and other neural CAMs, b) to identify and analyze regions of the Ng-CAM molecule that are involved in heterophilic binding, and c) to analyze the potential roles of these interactions in adhesion and migration of neural cells. The specificity of interactions between neural CAMs and these ECM molecules will be further investigated using other ECM molecules including proteoglycans, laminin, cytotactin, and fibronectin. Specific antibodies against ECM ligands for Ng-CAM will be prepared and used to compare their localization in tissues with Ng-CAM and N-CAM, and to determine their cellular origin using biosynthesis experiments in culture. To map different structural and functional regions within the Ng-CAM molecule, protein chemical, molecular genetic, and immunological techniques will be used to identify domains in Ng-CAM that bind to specific ligands including the 1D1 and 3F8 proteoglycans, and laminin. To evaluate potential roles of specific domains of Ng-CAM and the heterophilic ligands, protein fragments, for example, that represent defined regions of Ng-CAM and bind to certain ligands, as well as specific antibodies against Ng-CAM and ECM proteins, will be used in assays for neuronal adhesion, neurite growth, and migration of granule cells in cerebellar explants. The recent discovery that the 1D1 and 3F8 proteoglycans bind to neural CAMs and inhibit neuronal adhesion raises the possibility that CAMs on the cell surface function as receptors for proteoglycans and may be involved in signal transduction. Therefore, binding of radiolabeled proteoglycans to cells will be measured and the involvement of neural CAMs will be tested using specific antibodies against the CAMs; the results may provide clues for further studies of molecular mechanisms of cell "repulsion." The findings of these and related studies will form a basis for evaluating the role of interactions between neural CAMs and ECM proteins during normal development, and some of the reagents generated may be useful in therapeutic protocols to improve neuronal regeneration.

Cellular tyrosine phosphorylation plays a crucial role in the control of normal development and neoplasia. RPTP beta is a receptor protein tyrosine phosphatase that is expressed in glia in a pattern suggesting a role in morphogenesis and plasticity of the nervous system. Moreover, this protein binds to the extracellular matrix protein tenascin and to the neural cell adhesion molecules N-CAM and Ng. CAM/L1. The goal of this project is to analyze interactions of RPTP beta with various ligands and to study the consequences of these interactions on cell adhesion, and on transmembrane signalling that may modulate glial differentiation and interactions with neurons. The first specific aim is to characterize the expression patterns of RPTP beta in tissues and cells, and its interactions with different ligands during development. Then to analyze the binding properties of the different extracellular domains in RPTP beta cDNA constructs encoding different regions in RPTP beta will be used to express secreted and membrane-anchored forms of RPTP beta by transfection into mammalian cells. Molecular binding assays for secreted forms and cellular adhesion assays for forms expressed on the cell surface will be used to analyze which domains are important for binding of RPTP beta to different ligands such as tenascin and Ng-CAM/L1. To test potential functions of RPTP beta and its different domains in cells, molecular cloning techniques will be used to express or suppress expression of RFTP beta. In each case, effects of the treatment will be analyzed to detect changes in RPTP beta expression which will be correlated with alterations in cellular responses to ligands (i.e. tenascin and Ng-CAM) including cell adhesion, cell shape and cell division. RPTP beta is the first receptor/phosphatase with identified heterophilic ligands, and therefore it is important to investigate whether ligand binding to RPTP beta is involved in signal transduction by altering the specific activity of the phosphatase, by causing a redistribution of the phosphatase to alter its activity locally, or by changing the pattern of RPTP beta expression. This experimental approach will provide new information on the structure of binding regions of RPTP beta that may be involved in adhesion and growth regulation of normal and transformed astroglial cells. The results may also provide important clues for understanding development of radial glial cells and astrocytes, and their interactions with developing neurons, as well as potential roles of RPTP beta in growth of brain tumors.

Radial glial cells appear transiently during neural development and play key roles in cell migration and differentiation. However, they have been difficult to study because they are unstable in culture and cell lines with radial glial properties have not been available. We have derived a stable cell line (C6-R) from the rat C6 glioma cell line by transfection with a mutant phosphatase receptor. C6-R has radial morphology and stimulates neuronal migration in culture and in vivo. Although both C6-R and C6 can proliferate rapidly in culture, C6-R is unable to form tumors in rat brain like C6. Since expression of the transfected mutant receptor in many other C6 clones can not account for the phenotypic differences between C6-R and C6, it is likely that the alteration is due to genetic disruption at a single integration site that has been detected in Southern blots of C6-R genomic DNA. We hypothesize that this integration event disrupted genes that either promote glial tumor formation or suppress gliogenesis. The goals of this proposal are to understand the cellular properties of C6-R and the molecular basis of this unique phenotype. In addition, given the ability of C6-R cells to migrate and align along white matter in mature CNS and to support neuronal migration, they will be implanted in the CNS to explore their ability to promote growth of nerves in various situations including following injury. To understand the reduced ability of C6-R to form tumors in vivo, its cellular properties, including proliferation and death, will be compared with the parental C6 cell line. Molecular analysis will be directed to identify the genetic alterations responsible for transformation C6 into C6-R cells and the resulting differences in expression that are responsible for their phenotypic differences. Plasmid rescue and genomic cloning methods will be used to recover DNA from the locus of plasmid insertion. Genes at this locus, and other identified by subtractive suppression hybridization (SSH) using mRNA from C6-R and C6 will be analyzed to determine their roles in gliogenesis and formation of gliomas. These studies will lead to a better understanding of genes that are involved in glial development and in formation of high grade tumors. Studies of the reversion of C6-R to cells that can form tumors in vivo may have implications for understanding how gliomas progress to high grade malignancies. Finally, characterization of radial glial cell lines may yield new methods for promoting nerve growth following injury.

This IGERT program at Rutgers University, focused on integratively engineered biointerfaces, will be an intimately collaborative effort of 32 selected faculty from graduate programs in Molecular Biosciences, Physical Sciences (Physics, Chemistry & Chemical Biology), and Engineering (Biomedical Engineering, Ceramics and Materials Engineering, Chemical and Biochemical Engineering, Mechanical and Aerospace Engineering).

Intellectual Merit: The program derives strength from the highly cross-disciplinary nature of over fifteen research project areas identified at the cutting edge of the field of biointerfaces, and programmatic partnerships with five strategic centers of excellence to promote cohesive access for the IGERT community to state-of-the-art research infrastructure. A wide range of thesis project themes is planned for the IGERT trainees, developed around three research and educational thrusts, (1) living cell-based interfaces, (2) microengineered and nanoengineered biointerfaces, (3) biosensing and bioresponsive interfaces. The five major partnering Centers for the IGERT program are: Keck Center for Collaborative Neuroscience, Center for Nanomaterials Research, New Jersey Center for Biomaterials, the Laboratory for Surface Modification, and the Rutgers Center for Computational Design. The educational core of the proposed IGERT program will intimately support the research program, and includes graduate courses in the integrative areas of biointerfacial engineering, as well as course modules on responsible conduct of research, technical communications, entrepreneurship and effective teaching/learning methods.

Broader Impact: The IGERT curriculum is designed to foster a community featuring the next generation of biointerfacial and biomaterials engineers by offering IGERT graduate fellows a range of interactive experiences at multiple levels: multi-disciplinary coursework, lab rotations in two cross-cutting research groups, biannual participation in symposia, and participation in a national/international conference resulting in a white paper. To maximize its impact, the IGERT program will offer varied programmatic pathways to promote diverse modes of professional development of IGERT graduate fellows: (1) Summer research internships at selected international sites for academically inclined students; and (2) Translational research and industrial summer internships for students interested in industrial and entrepreneurial careers. Through a partnership with the Robert Davis Learning Institute of the Rutgers Graduate School of Education Institute, the IGERT program will establish a COLTS (Community of Learners and Thought Shapers) program, inspired by communication-driven cognition models, to encourage IGERT fellows to develop as learners by dynamically communicating their research on integratively engineered biointerfaces.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In this sixth year of the program, awards are being made to institutions for programs that collectively span the areas of science and engineering supported by NSF.

The overarching objective of the work proposed here is to support a productive interdisciplinary collaboration that will simultaneously generate meaningful quantitative research results in the neurosciences and foster the acquisition of the physiological background that will permit me to develop an independent research program in quantitative modeling of neuronal morphogenesis. The proposed work will be performed at the Center for Collaborative Neuroscience at Rutgers University, and will focus on developmental and regenerative mechanisms associated with Dorsal Root Ganglion (DRG) neurons and their associated glia. This work will involve two research projects to be performed under the guidance of Prof. Wise Young, MD PhD and Chair of Cell Biology & Neuroscience, and Prof. Martin Grumet, PhD and Director of the Center, in close collaboration with appropriate faculty, postdoctoral fellows, graduate students, and visiting scientists affiliated with the Center. The aims of this work are to quantitative test the hypotheses that morphogenesis of Dorsal Root Ganglion (DRG) neurites in vitro can be well described by a stochastic model, and that the function of such stochastic morphogenesis is to efficiently explore space in search of targets. The approach that will be used to achieve these aims will involve direct numerical simulations of axonal and glial structures that are validated and modified to accord with 3D imaging of neuronal cultures. Throughout this work, the guiding objective will be to use modeling to derive concrete, testable predictions.

This IGMS project is jointly supported by the MPS Office of Multidisciplinary Activities (OMA) and the Division of Mathematical Sciences (DMS).

This Integrative Graduate Education and Research Traineeship program (IGERT) renewal award establishes a training program focused on the science and engineering of stem cells. New cross-cutting thrusts for Ph.D. research, together with a new multidisciplinary graduate curriculum, will integrate stem cell biology with research in biomaterials, process engineering, and computational modeling. Trainees will participate in an IGERT Research Interchange Forum to develop their abilities to communicate across disparate disciplines. Professional development activities encompassing teaching, mentoring, and outreach will enable IGERT trainees to better realize the impact of their technological know-how. Each IGERT trainee will be guided by an advisory constellation of scholars drawn from over 30 faculty members from Engineering, Molecular Biosciences, Physical Sciences, Business, Public Policy, and Management. The IGERT program will leverage Rutgers' active "diversity infrastructure" to help broaden the participation of underrepresented minority students. In addition to providing research opportunities for visiting underrepresented undergraduates, the IGERT will offer two new initiatives: a teacher-student summer institute at Rutgers, and, a bridge-to-IGERT program. New outreach programs at the intersection of stem cell science and engineering with public policy and business include: (1) an initiative with the School of Management and Labor Relations to "bundle" IGERT research and curriculum into portable modules for scientific workforce training; (2) Rutgers Business School-mediated interactions with pharmaceutical management students and industry; (3) public policy workshops with policy makers, facilitated by the Eagleton Institute of Politics. IGERT trainees will acquire global perspectives through internships and workshops with leading stem cell researchers at over 15 sites in Europe and Asia. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.