RE|NOUS Meeting November 21, 2013; 6pm Presenters: Joseph Conovaloff, Navneet Ramesh, Julia Tong

1. RE|NOUS Meeting November 21, 2013; 6pm Presenters: Joseph Conovaloff, Navneet Ramesh, Julia Tong Impact factor of Journal of Neuroscience: 7.12 Xie H et al. J. Neurosci. 2013;33:17042-17051

2. Outline • Background information • Mitochondria • Results of Experiments • Conclusion • Plans for the future

3. Mitochondria (mt) • 1957: Coined the “Power House” of Cells • Neurons are high energy cells, need aerobic metabolism • The center of metabolic process to get ATP • Citric Acid Cycle • Electron Transport Chain • Dynamic: divide, fuse, form cable-like structure along neuronal projections • Deficiency neurodegeneration

4. Electron Transport Chain I • System of linked electron carriers • Series of REDOX reactions • Re-oxidation of FADH2 and NADH from TCA • Impermeable inner membrane and Proton Pump complexes • ATP

5. ETC II Inner mitochondrial membrane- impermeable H+ transport, Mitochondrial membrane potential (MMP): electrochemical gradient

6. COX: Cytochrome c Oxidase • COX catalyzes a four electron Redox Rxn • Increases the proton gradient- taking 8 protons out of the matrix: • Full ox. Of O2 • Proton (wire) pump • COX associated with Aβ plaques • Deficiency in COX in dystrophic neurites of senile plaques (Perez-Gracia et al., 2008)

7. Transgenic mice for AD The mice used were APPSWE/humanPS1-ΔE9 APPSWE is a form of APP that has been identified in humans to cause familial AD. It is a two amino acid change: K670N/M671L. This mutation was found in two Swedish families and has been used since the 90’s as a mutation in animal models to cause AD. It causes an overproduction of Aβ but does not affect memory until later in life, at the equivalent time that it does in a human life span. “In the Swedish mutation APP, the N-terminal β-secretase cleavage of APP occurs in Golgi-derived vesicles, most likely within secretory vesicles. Therefore, this cleavage occurs in the same compartment as the α-secretase cleavage, which normally prevents Aβ production, explaining the increased Aβ generation by a competition between α- and β-secretase.” Haass, C. et. al., Nat. Med., 1291-6 (1995). PS1-ΔE9 (PS1 with deletion of exon 9) induces function of PS1. PS1 is tied with the activity of γ-secretase.

8. Aβ plaques cause decrease in local mitochondria density Staining: mtGTP: stains mitochondria Methoxy: stains amyloid plaques Note: mice are 7 months old, when several Aβ plaques are present Decreased density of mitochondria near the amyloid plaque Quantification: Took concentric circles around the amyloid plaque and determined the area dyed green (mitochondria) and divided by total area for percent mtGFP occupance Control: does their staining of mitochondria work?

9. Aβ plaques cause swelling of local mitochondria and cytoplasm in neurites Staining: mtGTP: stains mitochondria GTP: represents cytoplasm Methoxy: stains amyloid plaques Note: mice are 7 months old, when several Aβ plaques are present In the vicinity of 26/109 of the plaques that they studied (in 6 mice) showed mitochondrial swelling They also found cytoplasmic swelling in the areas around the plaques, showing dystrophic morphology in nearly 100% of all nearby cells

10. Mitochondrial membrane potential (MMP) of mitochondria near Aβ plaques affected near plaques Now the researchers looked at MMP as one means of mitochondrial function MMP-sensitive dyes: MTR: sensitive to high MMP TMRE: sensitive to high MMP DiOC6: data not shown, but similar to MTR JC-1: changes with MMP (with high MMP is red, with low MMP is green) MMP-insensitive dyes: MTG: used as internal control MitoFluo Red 589: weak and photobleached, therefore not used in further experiments 20 micrometers away from the plaque seems to be the threshold for a stabilized MMP Y-axis: ratio of J-a/J-m Gives some comparison of the mitochondria with high MMP and low MMP (low MMP: decoupling of ETC and ATP synthase) Staining: J-aggregate: high MMP J-monomer: low MMP Methoxy: stains amyloid plaques Note: mice are 7 months old, when several Aβ plaques are present

11. Mitochondrial membrane potential (MMP) of mitochondria near Aβ plaques 20 micrometers away from the plaque seems to be the threshold for a stabilized MMP. The farther away from the amyloid plaques, the higher the intensity of TMRE (meaning there is MMP maintained more efficiently). This correlates to improved mitochondrial function. Staining: TRME: sensitive to high MMP Methoxy: stains amyloid plaques Note: mice are 7 months old, when several Aβ plaques are present

12. MMP of mitochondria near dystrophic neurites Cortical Neurons (Away from Plaques) Control: Does AAV-GFP Transfection Alter MMP? No  Levels of TMRE are equal in both GFP + and GFP - neurites

13. MMP severely altered near dystrophic neurites Cortical Neurons (Away from Plaques) Dystrophic neurites near Aβ plaques Increased variation of TMRE levels in dystrophic vs. normal neurites TMRE staining decreased

14. Conclusions • Decreased density of mitochondria near Aβ plaques • Dystrophic morphology of most cells surrounding plaques • Mitochondria Membrane Potential (MMP) impaired near plaques • Bottom-line: Focal Mitochondrial dysfunction

15. Future Directions • Sequence of events that leads to mitchondrial dysfunction • Methods to restore mitochondrial function • Protect mitochondria structure/function • Reduce Aβ plaques • Biomarker? • Measure mitochondrial function to assess progression of disease