Upstream Signaling Pathway Leading to Activation of Serine/Threonin Protein Kinase in Beta-Amyloid Peptide Neurotoxicity

1. Upstream signaling pathway leading to the activation of double-stranded RNA-dependent serine/threonin protein kinase in beta-amyloid peptide neurotoxicity Presented by Finda Mendez Written by : Suen, KC., MS. Yu, KF. So, RCC. Chang and J. Hugon. 2003. Journal of Bio. Chem. Sep 15 [http://www.jbc.org/cgi/reprint/M306503200v1]

2. ALZHEIMER’S INFORMATION • What is Alzheimer’s disease? Is a common condition in which the cell of the brain die, as a result the signals are not properly transmitted. • It causes many problems including loss of memory, confusion changes in personality and behavior are among these problems.

3. What is the difference? • normal Alzheimer’s • http://www.lifesciences.napier.ac.uk/courses/projects00/alzheimers/alzheime.htm

4. What is the pathology involved in Alzheimer’s disease? • The pathological “hallmark is accumulation of senile plaques (it block the pathway) • These plaques are primarily beta-amyloid peptides. • These peptides tend to induce apoptosis (cell suicide) of the brain cells and sometimes even necrosis (dead cells follow by injured tissue)

5. UPSTREAM SIGNALING • The inhibition of caspase-3 (proteases that degrade other cellular proteins) decreases the activity of the phosphorilation of protein kinase receptors • Endoplasmic reticulum releases Ca++ activating caspase-8 • After being produced by a virus double-stranded RNA –dependent protein kinase can produce activation of the protein kinase receptor • Protein kinase receptor can be activated even without the double-stranded RNA presence.

6. AMYLOID PRECURSOR PROTEIN • This view shows part of the amyloid precursor protein (APP), with alpha helices in blue and beta sheets in red.  Beta-amyloid usually is comprised entirely of alpha-helices, but in the toxic form, beta sheets are present as well.  Since beta sheets are less soluble than alpha-helices, the toxic form of beta-amyloid falls out of solution, forming plaques. • A:\ALZHEIMER'S DISEASE AMYLOID BETA-PEPTIDE.htm

7. Experimental Procedures • Primary cell cultures of cortical neurons--- Suen and colleagues used cerebral cortices of 17-day-old Sprague-Dawley rats to perform tests. • Neurons were cultured for 7 days • The pretreated with cell-permeable caspase-3 inhibitor, cell-permeable caspase-8 or inositol 1,4,5-trisphosphate receptor antagonist XestosponginC • followed by Beta-amyloid peptide 25-35 or Beta-amyloid peptide 1-42 • Incubated in autoclaved at 37 degrees Celsius for 3 days

8. Western-blot analysis • Neuron were scratch and lysed in ice-cold lysis buffer containing several different chemical. Mixed in and inhibitor cocktail. • Protein was separated by SDS-polyacrylamine gel electrophoresis, and other steps were performed

9. Continue Experimental Procedures • Co-immunoprecipitation Assay was performed. Basically the same procedure as with the Western-blot but with some slight differences. • Caspase activity assay- supernatant from a protein was used for different caspase. • Meausring of intracellular free calcium level- was determined by fluorescence imaging with acetoxymythyl-fura 2

10. Cont. of Experiment • Statistical analysis- data from duplication was compared by one-way analysis of variance (ANOVA) • level significance was p<0.05 • results were expressed as the means+/- standard error from at least 3 independent experiments.

11. Results • Caspase-3 specific activity indicated that its significantly activated neurons 1 hour after the treatment of beta-amyliod 25-35 peptide (p<0.05)Western-blot analysis showed that beta-amyliod peptide induced an increase in phosphorylation of protein kinase peptide at threonine-446 and 451 in neurons in comparison with the control of each one.

12. These toxic beta amyloid fragments can build up outside of the cell. Beta sheets are less soluble than alpha helices, causing them to fall out of the solution, forming toxic plaques in the brain. • These plaques are made of beta-amyloid, other associated proteins, and non-nerve cells that gradually build up outside and around neurons. • The plaques are the first physiological signs of Alzheimer’s Disease. • Beta-amyloid peptide 1-42 and 25-35 can induce neuronal apoptosis. • Fibril formation has been suggested to be required for beta-amyolid peptide neurotoxicity.

13. CONCLUSION • Blockage of early activation of caspase-3 can reduce phosphorilation of protein kinase peptide and eukaryotic initiation factor 2 alpha, suggests that caspase-3 is an important candidate in mediating early cellualr signaling events associated with beta-amyoild peptide neurotoxicity. • Calcium release from the endoplasmic reticulum and caspase-8 activation are upstream of caspase-3 protein kinase receptor activation in beta-amyloid peptide neurotoxicity. • According to Suen and colleagues the results of the experiment demostrated a new mode of protein kinase receptor activation and explored a new upstream signaling pathway for beta-amyloid peptide neurotoxicity. • Understanding the mechanism of neuronal apoptosis may pave the way for future therapeutic interventions against neurona; death in Alzheimer’s (Suen et.al, 2003)

14. REFERENCES • Bush, AI. 2003. The metallobiology of Alzheimer’s disease. Trends in Neuroscience. 26(4):207-214 • Caricasole, A., A. Copani, A. Caruso, F. Caraci, L. Iacovelli, MA. Sortino, GC> Terstappen and F. Nicoletti. 2003. The Wnt pathway, cell-cycle activation and Beta-amyloid: novel theraputic strategies in Alzheimer’s disease?. Trends in Pharmacological Science. 24(5):233-238. • Dobson, C., MA. Wozniak and RF. Itzhaki. 2003. Do infectious agents play a role in dementia?. Trends in Microbiology. 11(7):312-317 • Scherk, D. 2000. A parter for presenilin. Nature. 407:34-35 • St.George-Hyslop, PH. 2000. Piecing together Alzheimer’s. Scientific America. 283(Dec. 2000):76-82 • Suen, KC., MS. Yu, KF. SO, R.Chang and J. Hugon, 2003. Upstrean signaling pathway leading to the activation of double-stranded RNA-dependent serine/threonine proteine kinase in beta-amyloid peptide neurotoxicity. Journal of Biological Chemistry. Sept. 15, 2003.

15. Did I forgot something?

16. Thank you • Any Question????????