Diane M. Jaworski - US grants

DESCRIPTION: The elucidation of the mechanisms of cell migration during central nervous system (CNS) development is important to number of issues in neurobiology. Although the phenomenon of neuronal cell migration has been appreciated for more than a century, identification of The molecular components regulating neuronal cell migration is only beginning to emerge. While neurogenesis occurs in a systematic laminar progression, the mechanisms underlying glial cell dispersion within the CNS are not well understood, and the molecules involved in glial cell migration are yet to be elucidated. In the last several years, our appreciation of the extracellular matrix (ECM) of the CNS has grown. The brain ECM consists of a heterogeneous mixture of glycoconjugates, including the glycosaminoglycan hyaluronan (HA). HA regulates the local cellular environment by facilitating permeability and retention of low molecular weight solutes, while excluding other macromolecules. In a number of systems, including the developing CNS, HA has been implicated in cell and tissue specific functions, including the regulation of cell proliferation, differentiation and migration. HA is the only glycosaminoglycan not covalently associated with core protein; therefore, functions ascribed to HA are likely mediated by proteins which bind HA. We recently identified the gene for a new ECM protein, BEHAB (Brain Enriched HyAluronan-Binding). BEHAB represents the only tissue specific HA-binding protein reported to date. BEHAB mRNA expression is restricted to the CNS. BEHAB mRNA is expressed at high levels when astrocytes proliferate and migrate - during rat brain development, in the reactive gliosis induced by CNS injury and during glioma invasion. These observations have led to the hypothesis we propose to test: that the expression of BEHAB plays a role in regulating astrocyte differentiation and motility in the CNS. Thus far, we have characterized the expression of the BEHAB gene at the mRNA level. The BEHAB cDNA encodes putative secretedHA-binding protein. To ascertain the function of BEHAB, it will be necessary to determine the expression pattern of the extracellular protein product of the BEHAB gene. The first specific aim of this project is to characterize th distribution and regulation of the BEHAB protein during CNS development. BEHAB mRNA is expressed in primary rat astrocyte cultures. However, different levels of BEHAB are expressed by astrocytes derived from different brain areas and from different developmental stages. The second specific aim of this project is to determine how BEHAB expression is regulated. BEHAB mRNA is expressed at high levels during periods of astrocyte cell differentiation and migration. The third specific aim of this project is to test whether BEHAB plays a role in astrocyte differentiation and migration.

tissue inhibitor of metalloproteinases; DNA; cell differentiation;

DESCRIPTION (provided by applicant): Information about the developmental regulation of the extracellular matrix in the CNS is incomplete and the function of proteins such as the tissue inhibitor of matrix metalloproteinases (TIMPS) is poorly understood. We recently characterized the expression of the four known TIMPs during CNS development in vivo. The expression of TIMP-2 by post-mitotic neurons correlates with neuronal differentiation. We demonstrate herein that TIMP-2 induces cell cycle arrest and neuronal differentiation in a cell autonomous manner. Strikingly, TIMP-2 's effect was independent of its MMP-inhibitory activity. The punctate labeling of TIMP-2 on the cell surface combined with the interaction of TIMP-2 with alpha3 beta1 integrin suggests that TIMP-2 may exert its MMP-independent activities via integrins. The persistence of nestin-positive progenitors in the neocortical ventricular zone and the reduced neurite length of Timp-2 -null neurons suggest that neuronal differentiation is delayed in the absence of TIMP-2. This is the first report to detail the mechanism of TIMP-2 action in neuronal ceils and demonstrates a novel function for TIMP- 2 in neurons independent of MMP inhibition. Primary cultured Timp-2 -/- and wild-type cerebral cortical neurons will be used to test the hypothesis that TIMP- 2 plays a role in neuronal differentiation independent of MMP-inhibitory activity via interaction with integrins. In aim 1, the number, location and affinity of TIMP-2 receptors will be determined and the Timp-2 null neurite length phenotype will be rescued by exogenous application of TIMP-2 protein with and without MMP-inhibitory activity. In aim 2, the integrin(s) to which TIMP-2 binds will be identified and competitive binding assays will be performed to block the phenotypic rescue by TIMP-2. Aim 3 will determine whether Timp-2 deletion alters cortical progenitor cell cycle parameters or laminar fate in vivo using two BrdU injection paradigms. Unlike somatic cells, neurons irreversibly withdraw from the cell cycle and permanently remain quiescent. The mechanisms responsible for the maintenance of neuronal quiescence are poorly understood. The identification of molecules responsible for neuronal quiescence has significant implications for the ability of the adult brain to generate new neurons in response injury as well as in neurological deficits such as Alzheimer's and Parkinson's disease.