Forms and Functions of Beta- Amyloids

1. Forms and Functions of Beta-Amyloids By: Aileen Li, Andrea Zhao, Matthew Dytoc, Rose Hrvatin PHM142 Fall 2012 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson

2. What is Amyloid? • Amyloid is a general term for protein fragments that the body produces normally • 3D structures of these proteins in their native states vary, but abnormal amyloid structures exhibit a characteristic “cross b” structure • Associated with a variety of human diseases and neurodegenerative disorders Figure 1. “Cross-b” structure Retrieved from: http://www.emergentuniverse.org/#/evidence

3. What is Beta-Amyloid? • Beta-amyloid is a small piece of a larger protein called amyloid precursor protein (APP) • The two main forms of beta-amyloid are Aß42 and Aß40 • Composed of 42 and 40 amino acids respectively • Aß42 has a greater propensity to agglutinate and thereby form amyloid plaques Fig 2. Structural differences between Aß42 and Aß40 Retrieved from: http://www.emergentuniverse.org/#/evidence

4. Formation of Beta-Amyloid • APP is a transmembrane protein that is highly present in the CNS that helps neurons grow, survive, and repair themselves • APP and beta-amyloids are normal components of an organism • When APP is “activated” to do its normal job, it is cut by other proteins into smaller sections • There are several different ways APP can be cut and sometimes it will form beta-amyloids

5. Formation of Beta-Amyloid (Cont’d) • For the beta-amyloid peptide to be released, APP must be cleaved at two locations by two special types of enzymes 1. Beta-secretases cut the APP at a certain distance outside the cell membrane 2. Then gamma-secretases make another cut, this time inside the cell membrane, thus releasing the beta-amyloid peptide • The length of this peptide varies from 38 to 42 amino acids Fig 3. Formation of beta-amyloids Retrieved from: http://thebrain.mcgill.ca/flash/d/d_08/d_08_m/d_08_m_alz/d_08_m_alz.html

6. The Amyloid-Cascade Hypothesis • In 1992, Hardy and Higgins assigned a central role to beta-amyloid and its precursor, APP • According to this hypothesis, the production and subsequent aggregation of beta-amyloid is the ultimate source of the disorders experienced by people with Alzheimer’s • Beta-amyloid is “stickier” than the other fragments formed by APP • The pieces first form small clusters called oligomers, then chains of clusters called fibrils, then sheets of fibrils called beta-sheets • Plaques are formed by clumps of beta-sheets and other substances

7. Beta-Amyloid and Alzheimer’s • In a healthy brain, amyloid protein fragments are broken down and eliminated • In Alzheimer's disease, amyloid fragments accumulate to form hard insoluble plaques • According to the amyloid hypothesis, beta-amyloid aggregation disrupt brain cells by clogging points of cell-to-cell communication and activating immune cells that trigger inflammation

8. Beta-Amyloid and Alzheimer’s (Cont’d) • By analyzing hundreds of extended families worldwide, scientists have identified rare genes that practically guarantee the development of Alzheimer’s, and these genes are also associated to an increase in production or accumulation of beta-amyloid Figure 4. Amyloid plaques in the Alzheimer’s brain. Retrieved from: http://www.ahaf.org/alzheimers/about/understanding/plaques-and-tangles.html

9. Beta-Amyloid and Down Syndrome • individuals with Down Syndrome have an extra copy of chromosome 21 • APP gene is located on chromosome 21 • Development of amyloid plaques is very common among these individuals and symptoms of Alzheimer’s disease usually occur by middle age Figure 5. The location of APP gene on chromosome 21. Retrieved from: http://ghr.nlm.nih.gov/gene/APP#normalfunction

10. Aβ in Prevention of Oxidative Stress • Has been the subject of many research studies • Mechanism not yet fully understood • Aβ has been known to act as a pro-oxidant, however Aβ has also been found to provide antioxidant protection under many circumstances • For example, low concentrations of Aβ 1-40 strongly inhibit the auto-oxidation of CSF and plasma lipoproteins

11. Other Studies • One study showed that inhibition of metal-induced peroxidation of lipids by Aβ led to reduced numbers of cultured neuron death • In vivo studies performed in the rat cerebral cortex through injection of Aβ reduced the neurotoxicity of iron and copper • Most likely to be due to the high binding affinity that Aβ has for metals • Post-mortem (after death) observations were made in Alzheimer’s disease brains • Showed markers of neuronal oxidation were inversely correlated with the frequency of Aβ plaques • Degree of resistance of lipoproteins in CSF is directly correlated with amounts of Aβ present in the CSF

12. Role in Facilitation of Neural Growth • Early research deduced that Aβ’s may have neurotropic properties • Research shows that addition of small concentrations of Aβ to primary neuronal cultures leads to increases in phospolipids and proteins responsible for neuron outgrowth • Membrane phospholipids, Tau and growth-associated protein (GAP) • Addition of pure fibrillarAβ drops onto cell surface led to rapid growth of retinal ganglion cells • Further studies would increase our understanding

13. Role in Neuronal Survival • Many studies suggest Aβ plays a role in neuronal survival • It has been shown that viability of primary cortical neuronal cultures decreases when AB production is inhibited (through γ and β secretases inhibition) • However viability was restored with addition of Aβ • Neuronal survival after exposure to toxic metal ions also improved with addition of Aβ • Conclusion: Cell and animal models indicate Aβ promotes neuron outgrowth and axonal sprouting and presence of Aβ increases survival of neurons under a variety of conditions

14. Beta-Amyloids as Antimicrobial Peptides (AMPs) • Beta-amyloids might be unrecognized AMPs which normally function in the innate immune system • One family of mammalian AMPs are the cathelicidins • LL-37 peptide: widely expressed AMP, the only member of the cathelicidin family that has been identified in humans • Low levels of LL-37 Increased risk for serious infections • Found that beta amyloids are very similar to LL-37 • For example, they both have a propensity to form small clusters called oligomers

15. Beta-Amyloids as Antimicrobial Peptides (Cont’d) • Antimicrobial activity against a particular microorganism is measured in vitro by a peptide’s minimal inhibitory concentration (MIC) • The MICs of synthetic LL-37, Aβ40, and Aβ42 were compared against selected microorganisms

16. Aβ peptides possess antimicrobial activity Table 1. (Soscia et al., 2010)

17. Results • Aβactive against at least eight common and clinically relevant microorganisms • The in vitro antimicrobial activity of Aβ either matched or exceeded the antimicrobial activity of LL-37 • Shows that Aβ is an AMP with potencies that are similar to, or, in some cases surpassing those of LL-37

18. Additional Findings • The researchers of this study wanted to see if the observed antimicrobial activity for synthetic peptides in vitro could be identified in the temporal lobe and cerebellum from the human brain • Tissue samples were taken from the temporal lobe of those with Alzheimer’s disease • Compared to tissue samples taken from aged-matched subjects with no Alzheimer’s, the tissue samples taken from those WITH Alzheimer’s contained more antimicrobial activity

19. Conclusions and Implications • Aβdisplays antimicrobial activity and is able to function as an antimicrobial peptide in vivo. It thus acts as an effector molecule of innate immunity • These findings have important implications for treating Alzheimer’s • If the normal function of Aβ is to function as an AMP, then we cannot treat Alzheimer’s by simply eliminating all of it from the brain • complete absence of beta amyloid greater susceptibility to infection

20. Normal Functions of APP and Aβ • APP and Aβ have a normal physiological function in the body as regulatory proteins • They control cellular cholesterol levels, which maintains homeostasis • Cholesterol is a type of steroid that is key to maintaining the fluidity of cell membranes, and is a precursor for steroid hormones, bile acids, and vitamin D

21. Cholesterol Trafficking • There is constant efflux and influx of cholesterol from the cerebral neurons via certain proteinsA) Lipoproteins-Normally, healthy individuals will have low levels of low-density lipoprotein (LDL), and moderate levels of high-density lipoprotein (HDL)-LDL=influxbinds cholesterol: brings it into neuronal cells for processing (to go into the cell membrane, or to become hormones)-HDL=effluxbinds cholesterol, in the extracellular space, and move it towards the liver at the liver, HDL influxes cholesterol into hepatocytescholesterol is then modified for elimination from the bodyB) apoE-apoE is a lipid and cholesterol transport protein that effluxes cholesterol from neuronal cells

22. Normal Conditions: Low LDL, Moderate HDL • APP is activated and cleaved by an enzyme called α-secretase, to produce specific Aβ peptides:Aβ17-40, Aβ1-40, (the numbers denote the amino acid length of the peptide)These peptides modulate the efflux and influx proteins of cholesterol

23. The Aβ Peptides • Aβ17-40-present in much greater extent than Aβ1-40-prevents binding of cholesterol within the neuronal cell to apoE-this decreases cholesterol efflux-so intracellular levels of cholesterol increaseAβ1-40-present in smaller amounts-it has no effect on apoE-rather, it prevents cholesterol from binding to LDL-this decreases influx of cholesterol into neurons-so extracellular levels of cholesterol increaseNote: Aβ1-42 is also present in the brain, but is produced from other secretase enzymes-it inhibits both apoE and LDL, which stops efflux and influx of cholesterolSo Aβis needed to help balance cholesterol levels in the extracellular space of the brain versus within neuronal cells, and does so by inhibiting certain cholesterol transport proteins (apoE or LDL)

24. Figure 6. (Yao & Papdopoulos, 2002).

25. When Things Go Wrong • An overproduction of APP and Aβ will lead to negative consequences in the body • This can occur due to:a) a genetic mutation within APP b) high cholesterol levels and LDLHigh LDL Levels (the bad lipoprotein) • Levels rise with a bad diet, and can lead to neurotoxicity • As LDL rises, it induces an increase in:a) production of APP, b) formation of Aβ peptides, andc) apoE protein

26. High LDL Levels (the bad lipoprotein) • -LDL will block the α-secretase enzyme from binding and cleaving APPless Aβ17-40 will be produced-BUT, other secretases can now bind APP more efficiently, creating other Aβ peptideslevels of Aβ1-40 will increase to greater than normal levels-Since there is more apoE present, and it is no longer inhibited (due to a lack of Aβ17-40), there is more efflux of cholesterol out of neurons into the extracellular space of the brain-This extracellular cholesterol builds up fast, because high Aβ1-40 prevents cholesterol from binding LDL, stopping influx into the neuronal cells-This huge increase in extracellular cholesterol leads to neuronal dysfunction, and plays a significant role in Alzheimer’s Disease (AD)

27. Figure 7. (Yao & Papdopoulos, 2002).

28. A Healthy Diet is Crucial to Preventing Alzheimer’s Disease (AD) • -HDL (the good lipoprotein) is unaffected by Aβ-it has a greater affinity for Aβ than apoE-so when apoE becomes over-activated with higher LDL or a genetic mutation in APP, HDL can take over - it will bind cholesterol in the extracellular space of the brain, and transport it towards the liver for processing and elimination from the body-thus a diet high in HDL and low in LDL is important for preventing the onset of AD

29. Summary Slide-Beta Amyloids • In the formation of beta-amyloids, beta-secretases make a cut in the portion of the amyloid precursor protein chain that is outside the cell membrane and gamma-secretases make a cut in the portion that is inside the cell membrane. • According to the Amyloid-Cascade Hypothesis, beta-amyloid are the ultimate source of the disorders experienced by people with Alzheimer’s. • Accumulation of amyloid plaques is considered a hallmark of the Alzheimer brain and is also commonly found in individuals with Down Syndrome • Cell and animal models indicate Aβ promotes neuron outgrowth and axonal sprouting and presence of Aβ increases survival of neurons under a variety of conditions • Aβ has been found to provide antioxidant protection under a variety of conditions, specifically in the detoxification of toxic metal ions and oxidized lipids • Aβ displays antimicrobial activity and one of its normal functions may be to act as an effector molecule of innate immunity • Aβ is normally good to keep cholesterol trafficking balanced in the body. Once Aβ is overproduced, it leads to neurotoxicity.

30. References Alzheimer’s Association. (2007, August). Experimental Alzheimer Drugs Targeting Beta-Amyloid and the “Amyloid Hypothesis”. Retrieved from http://www.bu.edu/alzresearch/files/pdf/AmyloidHypothandExperimentalDrugsAlzAssoc3.pdf American Health Assistance Foundation. (2012, April 10). Plaques and Tangles. Retrieved from http://www.ahaf.org/alzheimers/about/understanding/plaques-and-tangles.html Canadian Institute of Health Research. (n.d.). Beta-Amyloid Protein. Retrieved from http://thebrain.mcgill.ca/flash/d/d_08/d_08_m/d_08_m_alz/d_08_m_alz.html Institute for Complex Adaptive Matter. (n.d.). Celebrity Boxing: Aβ42 vs. Aβ40. Retrieved from http://www.emergentuniverse.org/#/evidence National Library of Medicine. (2012, November 5). APP. Retrieved from http://ghr.nlm.nih.gov/gene/APP#normalfunction Pearson, H.A., & Peers, C. (2006). Physiological roles for amyloidβ peptides. J Physiol, 575(1), 5-10. Soscia, S., Kirby, J.E., Washicosky, K.J., Tucker, S.M., Ingelsson, M., Hyman, B., Burton, M.A., Goldstein, L.E., Duong, S., Tanzi, R.E., & Moir, R.D. (2010). The Alzheimer’s Disease-Associated Amyloidβ-protein Is an Antimicrobial Peptide. PLoS ONE, 5(3), 1-10. Yao, Z & Papadopoulos, V. (2002). Function of β-amyloid in cholesterol transport: a lead to neurotoxicity. The FASEB Journal, 16(12), 1677-1679.