Characterization of Amyloid Aggregates and Aggregation Inhibition molecules using various techniques

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Macromolecules for The Dementedand methods for
their study
Help from Keunok Yu, Jirun Sun, Bethany Lyles,
George Newkome and LSUs Alz-Hammers Research
Team Krispy Kreme Donut Day, September
2003 Supported by National Institutes of
Health-AG, NSF-DMR and NSF-IGERT

• How Alzheimers happens
• Attempts to prevent or reverse it
• Characterization challenges
• Alzheimers model systems with materials
  implications

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Amyloid Diseases

• Several diseases are caused by the misfolding of
  proteins into self-associating structures
  (fibrils) w/ predominantly b-sheet secondary
  structure
• Alzheimers 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
• Huntingtons Disease, triplet repeat expansion
  (Gln)n

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Positron emission tomography Age 20 -- 80
Normal -- 80 AD
Postmortem Coronal Sections Normal Alzheimers
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• 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
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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/Iss
ueTOC/issue/394
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Feature article by Vernon M. IngramAmerican
Scientist on-LineVol. 91, 4 July-August
2003http//www.americanscientist.org/template/Iss
ueTOC/issue/394
Exporting the dangerous fragments
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Amyloid hypothesis fibrils or protofibrils cause
cell death, possibly as the bodys 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
Ca2 metabolism
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Introduction of Varying Salts to Increase
ß-amyloid Aggregation, Ab 10-35

NaCl NaNO3 NaF
10 mm x 10 mm scanning probe microscope images
(on mica) of 300 mM Ab1035 incubated for 8
days at room temperature in 15 mM
phosphate buffer containing 50 mM salt.
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Alz-Hammers Team goal Mediate the Aggregation
of Ab
R regular H hydrophobic
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Peptide-based Mediation Requires a Specific
Sequence

• Hydrophobic KLVFF region is responsible for
  ß-amyloid aggregation
• Incorporation of such region for ß-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
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Peptide-based Inhibitors of Ab Fibrillogenesis
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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
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Synthesis Vetting of Peptide with aaAA-Blocker

• MALDI-MS of purified peptide
• Calcd for (M Na) 1731.3

HPLC of crude peptide
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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
11 AbInhibitor
101 AbInhibitor
Control
Even a sub-stoichiometric amount of AMY-1
inhibitor is effective
TEM image after 4.5 months at Room Temperature 50
mM phosphate buffer/ 150 mM NaCl pH 7.4
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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

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

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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
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Cue The Movie
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Labeling ß-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.
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Diffusion Results Great Reproducibility But
Dye Shrinks itand may stabilize against
aggregation.
100 µM Mixture ß-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 Alzheimers Amyloid Peptide
Congener in Solution. Biophysical Journal 2001,
80, 31-44.
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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
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Epifluorescence also shows L-Ab is actually
incorporated into macrofibers.
Bottom Line we think L-Ab is OK to study.
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Two FPR Contrast Decay Modes are Often Observed
Fast small Slow large.
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Doing More Experiments Faster with Less Precious
Amyloid Dialysis FPR
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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.
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Finding a convenient buffer for controlled self
assembly. This run is at pH 4 Acetate Buffer.
Adding calcium hastens aggregation.
Amplitudes
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Reversing Amyloid Aggregationby 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).
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Ab 400,000/gram Need cheaper model
systems. They also have materials applications.
Bolaform amphiphiles have a dumb-bell shape.
hydrophilic
hydrophilic
hydrophobic
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Arborol example 9-10-9
9 watery hydroxyl groups
10 oily methylene groups
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Stacked dumbbell model
Based on molecular modeling, SAXS, FF-SEM, DSC,
AFM, POMand common sense.
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Synthesis of Inhibitor 9-6
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Self-assembly of 9-12-9Starting point is
extruded fibers
Rh from linear fit of gamma vs q2 of DLS data at
five angles 40, 50, 60, 70 and 90.
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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 methodsand have some materials science
  applications.
• Many things not shown e.g., Ab slows diffusion
  of the lipids that make cell membranes. Is this
  important?

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Broader Conclusions

• Membrane proteins (or fragments) are hard to
  study.
• Dont expect a cure soon and you wont 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 whats going on than it must
  seem from textbooks.

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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 (thats 2000 in
  every state!) cure Alzheimers, Mad Cow,
  Huntingtons and other related afflictions?

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Thank youWhat one thing did you learn?What
one thing do you wish you understood
better?Action Item Find REU or similar for
Summer 2005
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Synthesis ( illustrated for 9-10-9 )
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APPs normal function

• No one knows for sure
• May help stem cells develop identity
• Or relocate cells from birth site to normal
  location
• May mature cells into structural type rather
  than neuronal
• May protect brain cells from injury
• Synaptic action
• Copper homeostasis

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http//www.bmb.leeds.ac.uk/staff/nmh/amy.html
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XXXXThe title I promisedNadia Sun
PhotosAlzheimers Group Photo
Paul S. Russo Nadia J. Edwin, U.S. Virgin
Islands Jirun Sun, China
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How does FPR work?
Diffusion
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Major Goal of this Research
To determine the kinetics of ß-Amyloid
aggregation in aqueous buffer systems (in vitro
environment) for accurate studies of aggregation
inhibition by synthetic peptides in such
environments.
Techniques
Fluorescence Photobleaching Recovery
(FPR) Dynamic and Static Light Scattering
(DLS/SLS) Diffusion Ordered Spectroscopy
(DOSY) Small Angle X-ray Scattering
(SAXS) Analytical Ultracentrifugation (AU)
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Outline
The role of ß-Amyloid in the onset of
Alzheimers Disease The ß-Amyloid peptide
sequence Sample Handling Issues -
Preventing non-amyloid nucleation -
Sample preparation Results Summary Future Work
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What is Amyloid?
1853 Rudolf Virchow named cerebral deposits as
amyloid Amyloid proteinaceous aggregates
associated with diseases (Alzheimers,
Parkinsons, spongiform encephalopathies). ß-Amyl
oid peptide (39- 42 amino acids) ß-Amyloid
Precursor Protein ( 695 amino acids)
ß-APP an inhibitory molecule
that regulates the activity of
proteases
Dobson, C.M. Protein misfolding, evolution and
disease. Trends Biochem.Sci. 1999, 24, 329-332.
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APP- The Cause of Alzheimers Disease?
APP
Enzymes
Over-active protease producing excess b-amyloid?
or
Alzheimers patients simply cannot rid the brain
of the accumulating by-product?
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Amyloid Hypothesis
Neurodegeneration in Alzheimers disease (AD) may
be caused by deposition of amyloid ß-peptide (Aß)
in plaques in brain tissue. Current studies
probe effects of physical conditions (differing
pH, temperature, salt concentration) on Aß
aggregation.
Hardy, J. Selkoe, D.J. Science, 2002,
297, 353-356.
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Importance of Sample Preparation

• Impurities within the sample can initiate
  aggregation.
• -autoclave pipet tips,
  microcentrifuge tubes
• -pre-wet filters
• Introduction of salt induces aggregation.
• -use pure materials to eliminate
  heavy metals
• Proper sample containment is important for
  creating
• stable stock solutions.

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Preparation of Stock Solutions

• Dissolve Ab in filtered 10 mM KOH at high pH
• Add phosphate buffered saline to filtrate
• Filter Ab stock solution to remove dust using
• 0.02 mm syringe filter
• Place stock solutions within 1.5 ml
  microcentrifuge
• tubes

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Sample Preparation for Kinetic Studies

• Mix Aliquots of filtered Ab (both labeled and
  unlabeled)
• and buffer with appropriate ionic strength for
  desired
• sample ratio in microcentrifuge tubes
• Vortex sample
• Check pH with Micro-pH electrode from
  Microelectrodes,
• INC.
• Load samples in 0.2-mm-path-length rectangular
• microslide (VitroCom Inc.) by capillary, and
  flame-seal for
• FPR analysis.

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Future Work

• Effect of pH on fibril formation
• FPR/DLS Dialysis study of FABAB1-40
• Test of ß-Amyloid peptide in the presence of
  inhibitors
• Temperature study
• Pressure study
• Instrumental studies DOSY, AUC, KDLS

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DLS Diffusion Results
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ß-amyloid Aggregation

• Particulate
• Metal Ion
• Surface Effect

ß-amyloid Aggregation Mediation
X
M
M
Non-amyloid seed Beta-strand Fibril Mediator
M
M
M
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LSU USF Fibril Forming Cyclic Peptides
K L V F F
MCP 1
K L V F F
MCP 2
Peptides Designed by Professor Mark McLaughlin
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CD again shows labeled doesnt prevent unlabeled
from aggregating
100 ?M ?-amyloid1-40 in Phosphate Buffer (pH 7)
Day 3
Day 1
L-Ab is random coil, stays that way Normal Ab
alone makes beta sheet Mixture suggests mixed
conformations
Note Mixture 50/50 FABAB
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Arborol self-assembly issues materials science
rationale/agenda

• Can we control self-assembly?
• Can we make an inhibitor?
• Use for methods development related to
  Alzheimers disease fibril formation?
• Self-assembled structures that form lyotropic
  LCs?
• Removable templates for porous media?
• Removable stationary phase in separations?

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f/y Space of Peptides and Proteins