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.