Defense Date

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Anatomy & Neurobiology

First Advisor

Dr. Helen L. Fillmore

Abstract

Membrane-type 5 matrix metalloproteinase (MT5-MMP) is unique among MMP family members as it is predominately expressed in the CNS. Its expression is ubiquitous during brain development and restricted to regions of neurogenesis and neuroplasticity in the adult. MT5-MMP is a mediator of pericellular proteolysis and is thought to have a functional impact on neurite outgrowth. The studies presented in this work were designed to examine MT5-MMP expression in cultured NT2 cells, a model of newogenesis and neuronal differentiation, and in adult neurogenic brain regions. We further sought to overexpress MT5-MMP and test the hypothesis that it plays a role in substrate-specific cell motility.MTS-MMP mRNA was expressed in NT2 cells and was significantly higher in differentiated neuronal hNT cells. MT5-MMP cDNA cloned from NT2 cells unexpectedly revealed a novel sequence (MTS-MMPvar) which was 92% homologous with the published MT5-MMP and was characterized by a 162 bp deletion. Both transcripts could be identified in NT2, hNT and adult human hippocampus. The newly cloned MT5-MMPvar cDNA translated into an approximately 52 kDa protein as seen in in vitro expression studies. Using an MTS-MMPvar specific antibody designed to span the 162 bp deletion, MT5-MMPvar protein could be detected in NT2 cells and these protein levels increased in their neuronal counterparts, hNT cells. MT5-MMPvar protein was also expressed in adult human hippocampal tissue. MT5-MMPvar protein was shown to be expressed in a murine region of neurogenesis and plasticity, suggesting the existence of a murine homolog of this variant.Based on bioinformatic analysis, the MT5-MMPvar transcript was predicted to lack a sufficient signal peptide and to remain a Type-I membrane protein. This computer assisted modeling suggests that the most significant functional implication of MTS-MMPvar sequence variations is to affect its direction into the ER for processing. Functional studies using COS-7 cells genetically modified to overexpress MT5- MMPvar demonstrated no difference in cellular motility compared to parental or vector control cells. Preliminary studies show MT5-MMPvar expression in COS-7 cells associated with perinuclear structures and the cell membrane. Adult human neural progenitor cells stimulated to differentiate into immature neurons demonstrated MT5-MMPvar expression associated with the cell membrane and process outgrowths. This work has identified a novel transcript variant of the human MTS-MMP gene and shown that the protein product of this gene is significantly higher in differentiated NT2 cells. This, combined with preliminary results suggesting MT5- MMPvar cellular redistribution in more mature cell types, indicates a role for MTSWvar in neural differentiation and function.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008

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