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