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

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 Alzheimers disease
• growth inhibitory factor
• Alzheimers 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.htm
  l )

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 Alzheimers brains

4
Goals

• Probe further into the protein complex by
  examining
• 1. Localization of proteins in intact tissue
• 2. Specific proteinprotein 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/ssm
p/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
  Alzheimers 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