| Laminins are important components of the extracellular matrix (ECM), and participate in neuronal development, survival and regeneration. The tissue plasminogen activator (tPA)/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse-Transcription-PCR (RT-PCR) demonstrated the expression of all laminin chains in the hippocampus, and RNA in situ hybridization confirmed the presence of most of these chains in a neuronal pattern. Immunostaining supported the conclusion that most of the laminin chains are present in the hippocampus, although only α5, β1 and γ1 are observed in association with both neurons and blood vessels, with other chains showing immunoreactivity within the vasculature only. Immunoblotting confirmed the hippocampal expression of the chains α1, α5, β1 and γ1.; At early timepoints following intrahippocampal injection of kainate, α5, β1 and γ1 chain immunoreactivities are lost. In addition, tPA-deficient mice, which are resistant to kainate-induced neuronal death, show no change in laminins α5, β1 and γ1 after intrahippocampal kainate injection. This result suggests that laminin-10 (α5-β1-γ1) comprises a neuronal laminin in the mouse hippocampus, and is degraded early during excitotoxic injury, in a manner that precedes neuronal death. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the ECM in the hippocampus and further defines a pathway for mechanisms of neuronal death.; The matrix metalloproteinase (MMP) family of extracellular proteases comprises a large group of enzymes that can degrade virtually any type of ECM protein, including laminins. Studies were performed on mice deficient in the MMPs-7 (matrilysin) and -9 (gelatinase B), and show that these enzymes are not significantly involved in excitotoxin-induced neurodegeneration. Infusions of a naturally found protein inhibitor of MMPs (N-TIMP-1) was also tested and found to not affect kainate-induced neuronal death in the hippocampus. A novel gel zymography substrate (laminin) was also used to show that plasmin can effectively cleave laminin, and this protocol holds promise for use in identifying other laminin-degrading enzymes.; In addition, studies were performed that tested the relationship between laminin degradation and neuronal death. Laminin peptide infusions were performed in an attempt to find laminin peptide sequences important for neuronal survival. The p20 peptide, a potent stimulator of neurite outgrowth in vitro , was tested, but did not produce any increase in vulnerability to kainate-induced neurodegeneration in tPA-deficient mice. Plasminogen-deficient mice were also used to show that the relevant source of plasminogen which contributes to neurodegeneration following kainate injection is intracerebral, as intracerebral plasminogen infusion (by microosmotic pump) was able to make plasminogen-deficient mice susceptible to kainate-induced neuronal death, but bolus intravenous plasminogen was not.; The investigation of the extracellular matrix molecule laminin and the proteases affecting it has revealed important roles for laminins in influencing neuronal survival, and illustrate the importance of such ubiquitous and highly biologically active molecules. Further study of the extracellular matrix in the nervous system should be pursued, and will allow us to better understand the role of laminins in the ECM and how they influence CNS function and dysfunction. |
