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In situ Localization of MT3 and Associated Proteins in Mouse Brain Tissue

In situ Localization of MT3 and Associated Proteins in Mouse Brain Tissue. Andrew Davis, 2004-05. Background. Third isoform of metallothionein (MT3) protein implicated in Alzheimer’s disease growth inhibitory factor

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In situ Localization of MT3 and Associated Proteins in Mouse Brain Tissue

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  1. In situ Localization of MT3 and Associated Proteins in Mouse Brain Tissue Andrew Davis, 2004-05

  2. Background • Third isoform of metallothionein (MT3) protein implicated in Alzheimer’s disease • growth inhibitory factor • Alzheimer’s disease affects 5% of humans between the ages of 65 and 74 and about 50% of those 85 years and older (http://adear.niapublications.org/pubs/adfact.html )

  3. Background • Armitage, Martin et. al. found that MT3 part of multi-protein complex with: -creatine kinase (CK) -heat shock protein 84 (HSP84) -heat shock protein 70 -DHP-like protein 2 • MT3 and neuronal nitrous oxide synthase (nNOS) reduced in Alzheimer’s brains

  4. Goals Probe further into the protein complex by examining: 1. Localization of proteins in intact tissue 2. Specific protein:protein interactions -MT3 on nNOS -MT3 on CK 3. The existence of the complex

  5. Hypotheses 1. MT3, CK, nNOS, and HSP84 co-localize in brain tissue 2. MT3 neither activating nor inhibiting effect on nNOS and CK 3. The protein complex confirmed

  6. Methods • Immunohistochemistry http://www.nichirei.co.jp/nio/english/protocol/ssmp/procedure.html

  7. Figure 1: nNOS, HSP84, and Negative Control Ridge inside Hippocampus

  8. Figure 2: Controls, MT3, CK, nNOS, and HSP84 Ridge above Hippocampus

  9. Methods • Enzyme assays -enzymatic rates of nNOS and CK with varied concentrations of MT3 -nNOS activity using Cytochrome c Reductase procedure by Martasek et. al. -CK activity by monitoring CK as a catalyst in the reaction of ADP with creatine phosphate to yield creatine and ATP

  10. Enzyme Assays

  11. p-values

  12. Methods • Size-exclusion chromatography -separated proteins based on molecular mass -ran homogenized mouse brains through column -ran pure MT3 through column

  13. Chromatography Results

  14. Summary • Significant co-localization of proteins in and around hippocampus • MT3 neither activating nor inhibiting effect on nNOS and CK • Protein complex not confirmed due to diffusion through column and possible oxidative cross-linking of MT3

  15. Future Studies • Co-localization using fluorescently- tagged antibodies • Confirmation of the protein complex and replication of the enzyme assays using monomeric MT3

  16. Significance • Finding proteins localized in the hippocampus • Discovering co-localization of proteins step toward finding how MT3 may play a role in Alzheimer’s disease

  17. Acknowledgements • Dr. Ian Armitage and Dr. Bruce Martin • Abigail Tolkheim • Ms. Lois Fruen • Mr. Brad Peterson • Team Research

  18. In situ Localization of MT3 and Associated Proteins in Mouse Brain Tissue by Andrew Davis, 2005

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