Macromolecules for The Demented and methods for their study

2. Macromolecules for The Dementedand methods for their study Help from Keunok Yu, Jirun Sun, Bethany Lyles, George Newkome and LSU’s Alz-Hammer’s Research Team Krispy Kreme Donut Day, September 2003 Supported by National Institutes of Health-AG, NSF-DMR and NSF-IGERT • How Alzheimer’s happens • Attempts to prevent or reverse it • Characterization challenges • Alzheimer’s model systems with materials implications

3. Amyloid Diseases • Several diseases are caused by the misfolding of proteins into self-associating structures (fibrils) w/ predominantly b-sheet secondary structure • Alzheimer’s Disease: Amyloid b-protein (Abin neurons/brain • Type II Diabetes: amylin in Islets of Langerhans • Mad Cow Disease (BSE): PrpSc in brain --transmittable by protein aggregates • Huntington’s Disease, triplet repeat expansion: (Gln)n

4. Positron emission tomography Age: 20 80 Normal 80 Alzheimer’s Postmortem Coronal Sections Normal Alzheimer’s

5. APP = Amyloid Precursor Protein • APP = the larger, lighter pink one • Transmembrane protein • Normal function not known • Educated guesses • May help stem cells develop identity • Or help relocate cells to final location • May “mature” cells into structural type • May protect brain cells from injury • Synaptic action • Copper homeostasis • Anyway, you need it. • Normal “clipping” of APP by a “secretase” enzyme (in red, and also assumed to be a transmembrane protein) is shown. • There are several secretases, also associated proteins, and they seem to mutate easily: there is a genetic link. • It is not exactly clear why things go awry with advanced age. http://www.bmb.leeds.ac.uk/staff/nmh/amy.html

6. Clipping APP the right & wrong ways NH2 terminus Incorrect Correct Feature article by Vernon M. IngramAmerican Scientist on-LineVol. 91, #4 July-August 2003http://www.americanscientist.org/template/IssueTOC/issue/394

7. Feature article by Vernon M. IngramAmerican Scientist on-LineVol. 91, #4 July-August 2003http://www.americanscientist.org/template/IssueTOC/issue/394 Exporting the dangerous fragments

8. Amyloid hypothesis: fibrils or protofibrils cause cell death, possibly as the body’s own defenses tries to clear such “foreign” matter. Peter Lansbury Group http://focus.hms.harvard.edu/1998/June4_1998/neuro.html Competing hypothesis: channel formation disrupts Ca+2 metabolism

9. Introduction of Varying Salts to Increase β-amyloid Aggregation, Ab10-35 NaCl NaNO3 NaF 10 mm x 10 mm scanning probe microscope images (on mica) of 300 mM Ab10–35 incubated for 8 days at room temperature in 15 mM phosphate buffer containing 50 mM salt.

10. Alzheimer research group, Team goal: Mediate or alter the Aggregation of Ab R = regular H = hydrophobic

11. Exemplify with Double-stick Tape

12. Peptide-based Mediation Requires a Specific Sequence • Hydrophobic KLVFF region is responsible for β-amyloid aggregation • Incorporation of such region for β-sheet breaking or capping H-(Lys)-Val-Leu-Phe-Phe-(Lys)6-NH2 A peptide construct incorporating the KLVFF region developed by Professor Regina Murphy at the University of Wisconsin-Madison

13. Peptide-based Inhibitors of Ab Fibrillogenesis

14. K L V F F MCP 1 K L V F F MCP 2 LSU Peptide-based Mediators AMY-1 x = 1, y = 6 AMY-2 x = 6, y = 1 AMY-3 x = 1, y = 1 Mediators Developed by Professor Robert Hammer & Professor Mark McLaughlin

15. Synthesis & Vetting of Peptidewith aaAA-Blocker MALDI-MS of purified peptide Calc’d for (M + Na) = 1731.3 HPLC of crude peptide

16. 10 mM 10 mM Determining Mediator Efficacy Using Transmission Electron Microscopy 50 mM Ab1-40 50 mM AMY-1 4.5 months 50 mM Ab1-40 5 mM AMY-1 4.5 months Control 50 mM Ab1-40 4.5 months 10 mM 1:1 Ab:Inhibitor 10:1 Ab:Inhibitor Control Even a sub-stoichiometric amount of AMY-1 inhibitor iseffective TEM image after 4.5 months at Room Temperature 50 mM phosphate buffer/ 150 mM NaCl pH 7.4

17. But such limited success is very after-the-fact. Can we use diffusion-based and other methods to determine the early stages of aggregation? Can we follow it in real-time in vitro? Two Possible choices: • Dynamic light scattering • Fluorescence photobleaching recovery

18. A series of dynamic light scattering runs can identify a peptide that has an effect on large fibrils.

19. That’s OK for simple screening, but there are problems with DLS 1) the size is only an apparent value, because of the single angle used for measurement; 2) the presence of small protofibrils, and the effect of inhibitors on them, is difficult to ascertain, especially in the presence of larger fibrils that dominate the scattering; 3) reversibility is not easily studied; and, 4) experimentally tedious for early stages of aggregation.

20. Modulation FPR Device Lanni & Ware, Rev. Sci. Instrum. 1982 SCOPE 5-10% bleach depth PA IF c * TA/PVD PMT D S * X * M DM RR OBJ * M ARGON ION LASER AOM * = computer link

21. Labeling β-Amyloid fragment 1-43 When fluorescein is attached, we call it L-Ab Fluorescein has about 7% the mass of Ab. Sticht, H.; Bayer, P.; Willbold, D.; Dames, S.; Hilbich, C.; Beyreuther, K.; Frank, R.; Rösch, P.Eur. J. Biochem. 1995, 233, 293-298.

22. Sodium Fluorescein Dye Theoretical/Experimental D0 for monomeric β-amyloid1-40 Diffusion Results – Great Reproducibility But Dye Shrinks it…and may stabilize against aggregation. 100 μM Mixture β-amyloid1-40 in phosphate buffer – pH 2.7, 6.9 and 11 Theory/Experimental result for monomeric Ab1-40 from: Massi, F.; Peng, J.W.; Lee, J.P.; Straub, J.E. Stimulation Study of the Structure and Dynamics of the Alzheimer’s Amyloid Peptide Congener in Solution. Biophysical Journal2001, 80, 31-44.

23. Back to DLS: L-Ab does not prevent formation of large fibrils when mixed with unlabeled material and fibrils increase in size with added salt. Mixed labeled & unlabled

24. Epifluorescence also shows L-Ab is actually incorporated into macrofibers. Bottom Line: we think L-Ab is OK to study.

25. Two FPR Contrast Decay Modes are Often Observed: Fast = small; Slow = large.

26. Cover slip Sample Pump PTFE spacer Dialysis membrane O-ring Exchange Fluid Doing More Experiments Faster with Less Precious Amyloid: Dialysis FPR

27. Evolution of protofibrils from labeled monomer after dialysis against a weak citrate buffer at pH 5.0. After one hour, large aggregates appear and represent ~ 18% of the signal.

28. Finding a convenient buffer for controlled self assembly. This run is at pH 4 Acetate Buffer. Adding calcium hastens aggregation. Amplitudes

29. Reversing Amyloid Aggregation…by pH Diffusion from in situ FPR of 5-carboxyfluorescein-Ab1-40 (25% mixed with unlabeled 75% Ab1-40) starting at pH 11, then alternately dialyzed between 50 mM phosphate (pH 2.7) and 50 mM phosphate (pH 7.4).

30. What has any of this got to do with Nanomaterials???

31. Ab = $400,000/gram Need cheaper model systems. They also have materials applications. Bolaform amphiphiles have a dumb-bell shape. hydrophilic hydrophilic hydrophobic

32. Arborol example: [9]-10-[9] 9 watery hydroxyl groups 10 oily methylene groups

33. q I Scattering envelope I q Toolbox Small angle X-ray scattering: analysis of the structure (inverse space) Basic idea: Detector Sample Synchrotron X-ray

35. Intensity q/nm Eleven runs Start time (30oC): 3:35 am End time (90oC): 5:27 am

36. z I(q) = S(q)P(qr) N-1 N+N-1 2N+(N-1) 3N+(N-1) (back) 5 N+5 2N+5 3N+5 (back) 3 N+3 2N+3 3N+3 (back) 1 N+1 2N+1 3N+1 (back) Origin(0,0,0) 4N+1 4N+2 2N+2 3N+2 (back) 2 N+2 2N+4 3N+4 (back) 4 N+4 a 2N+6 3N+6 (back) 6 N+6 3N 4N (back) N 2N x y b radius, r Stacked dumbbell model Based on molecular modeling, SAXS, FF-SEM, DSC, AFM, POM…and common sense.

37. Synthesis of Inhibitor [9]-6

38. Self-assembly of [9]-12-[9]Starting point is “extruded” fibers Rh from linear fit of gamma vs q2 of DLS data at five angles: 40, 50, 60, 70 and 90.

39. Scientific Conclusions • Promising inhibitors have been designed and constructed. Probably even more expensive than Ab itself. • DLS can screen promising ones. • Dialysis FPR can observe Ab deconstruction in real time. So far, only by pH, but dialysis experiments with precious inhibitor are coming. • Model systems to practice with can teach us better methods…and have some materials science applications. • Many things not shown: e.g., Ab slows diffusion of the lipids that make cell membranes. Is this important?

40. Broader Conclusions • Membrane proteins (or fragments) are hard to study. • Don’t expect a cure soon and you won’t be disappointed. • Take your statins once the doctor tells you to start, then hope for the best. • Science in the service of practical problems is increasingly multidisciplinary. • Scientists spend a lot more time scratching their heads and wondering what’s going on than it must seem from textbooks.

41. Discussion Points • We have spent $1.4 M for this research (so far). • Perhaps 10 papers will appear eventually. • About six Ph.D. students will be trained. • Could 100,000 teams like ours (that’s 2000 in every state of the United States!) cure Alzheimer’s, Mad Cow, Huntington’s and other related afflictions?

42. Lecture Checkup(do NOT write your name) • What is the single most important thing you learned from this lecture? • What is the ONE thing you wish you understood better about this lecture? • Action item: what will you do to understand better?