Alzheimer

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Alzheimers Understanding The Role of Membranes
in Amyloid Aggregation
Presentation Based on Correlation of ß-Amyloid
Aggregate Size and Hydrophobicity with Decreased
Bilayer Fluidity of Model Membranes John J.
Kremer, Monica M. Pallitto, Daniel J. Sklansky,
and Regina M. Murphy. Biochemistry 2000, 39,
10309-10318.

Nadia J. Edwin Macromolecular Seminar December 5,
2003
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OUTLINE

• Background
• - Amyloid
• - Membranes
• 2. Previous Studies
• Goal of Reference Paper
• Experimental Techniques Used
• - DPH Anisotropy
• - Dynamic Light Scattering
• - Static Light Scattering
• 5. Conclusion

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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) Amyloid
aggregates in brain cells are thought to play a
causative role in the onset of Alzheimers
Disease
Dobson, C.M. Protein misfolding, evolution and
disease. Trends Biochem.Sci. 1999, 24, 329-332.
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Origins of Amyloid?
ß-Amyloid peptide (39- 42 amino acids) is a
protein fragment cleaved
from a much
larger protein ß-Amyloid Precursor Protein
( 695 amino acids) ß-APP an
inhibitory molecule that
regulates the activity of proteases
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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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Plasma Membrane
Regulate transport of nutrients into and waste
out of the cell Provide a site for chemical
reactions not likely to occur in an aqueous
environment
Alberts et al. Molecular Biology of the Cell,
Garland Publishing, N.Y. Third edition, 1994.
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Membrane Fluidity
 
Fluidity of a lipid bilayer depends on its
composition and temperature The greater the
concentration of unsaturated fatty acid residues,
the more fluid the bilayer At body
temperature, the phospholipid bilayer has
consistency of olive oil Fluidity of the
phospholipid bilayer allows cells to be
pliable           
Alberts et al. Molecular Biology of the Cell,
Garland Publishing, N.Y. Third edition, 1994.
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Membrane Composition
Unsaturated fats one or more double bonds in
tail kinks the tail so cannot pack closely
enough to solidify at room temperature most
plant fats
Saturated fats no double bonds between carbons
in the tail saturated with hydrogen solid at
room temperature most animal fats, bacon
grease, lard, butter
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Effect of Temperature on membrane fluidity
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Phospholipids
POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine POPE 1-palmitoyl-2-oleoyl-sn-gl
ycero-3- phosphoethanolamine POPG
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-
glycerol) POPS 1-palmitoyl-2-oleoyl-sn-glycer
o-3-phospho-1-serine
Alberts et al. Molecular Biology of the Cell,
Garland Publishing, N.Y. Third edition, 1994.
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Cholesterol
Phospholipids, cholesterol, gangliosides
Alberts et al. Molecular Biology of the Cell,
Garland Publishing, N.Y. Third edition, 1994.
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Previous Studies Side 1
Aß associates with cells via membrane-bound
receptors

1. Aß binds to Serpin-enzyme Complex Receptor (SEC)
2. Aß binds to class A scavenger receptor (SR)
3. Aß binds to Receptor for advanced glycation end
   products (RAGE)
4. Aß binds to a hydroxysteroid dehydrogenase enzyme
   (ERAB)

Joslin, G. et al J.Biol.Chem.1991, 266,
21897-21902. El Khoury, J. et al. Nature, 1996,
382, 716-719. Yan, S.D. et al. Nature, 1996,
382, 685-691. Yan, S.D. et al. Nature, 1997,
389, 689-695.
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Side 2 of the Debate
Aß aggregates are toxic via nonspecific
association with cell membranes

• Membrane components promoted changes in Aß
  secondary structure and/or aggregation
  propensity.
• Aß or its fragments caused
• - formation of large ion channels in
  phospholipid bilayers
• - leakage of encapsulated dyes from
  phospholipid vesicles
• - fusion of small unilamellar vesicles
• 3. Loss of impermeability in lysosomal and
  endosomal membranes

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Goal of Paper

• Determine whether changes in membrane physical
  properties were correlated with Aß
  aggregates
• Relations of any such effects with biological
  membrane with specific membrane components
• Does changes depend on Aß aggregation state

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ANISOTROPY
PTI Spectrofluorometer with manual
polarizers Excitation wavelength 360
nm Emission wavelength 430 nm
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1,6-Diphenyl-1,3,5-hexatriene (DPH)
Absorption 350nm Emission 452nm
Absorption and emission spectra of DPH in hexane
at 25, polarization spectrum of DPH in frozen
(propylene glycol at -50)
Partition itself in the hydrophobic region of the
bilayer at or near the ends of the acyl
chains Detect changes in the order of the acyl
chains
Lentz, B.R. Barenholz, Y. Thompson, T.E.
Biochemistry 1976, 15, 4529-4537. Shinitzky, M.
Barenholzz, Y. J.of Biological Chemistry 1974,
25, 2652-2657.
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Dynamic Light Scattering
Lens
Sample Cell
LASER
?
Photomultiplier Detector
Computer
Amplifier
Correlator
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DLS Results
    Figure 1 Growth kinetics of Aß
aggregates at physiological pH. Aß was dissolved
in DMSO and then diluted 20-fold into PBS, pH
7.4, to a final concentration of 0.5 mg/mL. The
average apparent hydrodynamic diameter, dsph, was
determined from cumulants analysis of dynamic
light-scattering data taken at 90º scattering
angle.
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Static Light Scattering
Figure 2 Change in Aß aggregate molecular weight
and size with time. Static light-scattering data
taken at 24 (), 30 (), 44 (), 52 (), 69 (), and
93 h () after initiation of aggregation are shown
as Kratky plots. Lines indicate nonlinear
regression fit of semiflexible chain (24-52 h) or
semiflexible star (69-93 h) models to the data.
The increase in the y-axis intercept is
indicative of an increase in average molecular
weight, whereas the appearance of a maximum in
the curves at intermediate values of q is
characteristic of branched structures.
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SLS Results Contd
Figure 3 Growth of Aß aggregates at
physiological pH. Weight-averaged molecular
weight ltMgtw ( ) and average fibril length Lc ( )
were determined by nonlinear regression fit of
model equations to the light-scattering data of
Figure 2, as described in more detail in the
text. Error bars represent 95 confidence
intervals for fitted parameters.
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Microscopy Shows
Figure 4 Electron micrographs of Aß aggregated
for 2 days at neutral and acidic pH. (A) pH 7
fibrillar aggregates, scale bar 50 nm (B) pH 6
agglomerated aggregates scale bar 200 nm.
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Fluorescence Results
Figure 5 Effect of Aß aggregation on bis-ANS
fluorescence. PBS ( , n 18), freshly diluted
Aß ( , n 4), and Aß aggregated for 2 days in
PBS at pH 7 ( , n 6) or pH 6 ( , n 6)
were added to PBS containing the dye bis-ANS.
Fluorescence spectra were collected from 450 to
550 nm, with excitation at 360 nm. Results shown
are averaged scans from 4-18 samples the error
bars signify one standard deviation. Two other
data sets were taken with samples prepared on
different days with similar results (data not
shown). Binding of bis-ANS to exposed hydrophobic
sites is signaled by an increase in fluorescence
intensity and blue-shifting of the peak.
Fluorescence intensity of Aß aggregated at pH 6
and 7 was statistically different (p lt 0.01).
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Aggregation Results
Figure 6 Sketch of Aß aggregation at (A) neutral
and (B) acidic pH. (A) At pH 7, Aß steadily
increases from an average fibril length of 960
nm at 1 day to 4000 nm at 4 days. These fibrils
possess hydrophobic patches as shown by bis-ANS
binding (Figure 5). Fibril-fibril entanglement is
detectable as "branching" at 3 days and increases
with time. Precipitation occurs around 5 days,
accompanied by a loss of bis-ANS binding when
tested at 7 days. Together these results suggest
that at the later stages of Aß aggregation at
neutral pH, fibril-fibril association mediated by
hydrophobic interaction occurs, reducing
solvent-exposed hydrophobic patches but
generating macroscopic fibril bundles. (B) At pH
6, Aß instantaneously forms large, amorphous
aggregates that precipitate in less than 24 h.
These aggregates contain many highly hydrophobic
solvent-exposed patches, which are present even
at 7 days. This suggests that Aß aggregation at
pH 6 does not occur through orderly
self-association via burial of hydrophobic
interactions and that precipitation occurs due to
poor aggregate solubility near the isoelectric
point.
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DPH Anisotropy Results
Figure 7 Effect of Aß Aggregation at pH 7 on DPH
anisotropy. Freshly diluted ( ) and 2 day-aged (
) Aß samples were added to (A) POPC and (B) POPG
liposomes with embedded DPH. Data are
compilation of 2-4 replicate experiments at each
condition.
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DPH Anisotropy
Figure 8 Effect of Aß Aggregation at pH 6 on DPH
anisotropy. Freshly diluted ( ) and 2 day-aged (
) Aß samples were added to (A) POPC and (B) POPG
liposomes with embedded DPH. Data are
compilation of 2-4 replicate experiments at each
condition.
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DPH Anisotropy
Figure 9 DPH anisotropy with (A) type I and (B)
type 2 vesicles at pH 7 ( , )
and pH 6 ( , ) upon addition of
freshly diluted ( , ) and 2
day-aged ( , ) Aß. Aß
induces a significantly larger anisotropy
increase in vesicles containing gangliosides at
both pH 6 and pH 7.
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Conclusion
Observed decreases in membrane fluidity, detected
as an increase in DPH anisotropy. Changes in
membrane fluidity are not solely dependent on
binding of Aß to the bilayer surface.
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Acknowledgements

• NSF-IGERT
• Dr. Paul Russo
• Russo Group Members

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Fluidizing action of Aß
Mason, R.P. Jacob, R.F. Walter, M.F. Mason,
P.E. Avdulov, N.A. Chochina, S.V. Igbavboa,
U. Wood, W.G. J.Biol.Chem. 1999, 274,
18801-18807.
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Figure 5.10 The synthesis and structure of a
fat, or triacylglycerol
Carboxyl group has acid properties Hydrocarbon
chain, 16-18 carbons Nonpolar C-H bonds,
hydrophobic
(Condensation Reaction)
(bond between hydroxyl group and a carboxyl group)
Fats hydrophobic, not water soluble variation
due to fatty acid composition fatty acids
can be the same or different fatty acids
can vary in length fatty acids can vary in
the number and location of double bonds
(saturation)
A triglyceride
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PALMITIC ACID Palmitate. Fatty Acids. From fats,
oils (see Fatty Acids) mixed with stearic acid
(see). Occurs in many animal fats and plant oils.
In shampoos, shaving soaps, creams. Alternatives
palm oil and other vegetable sources. OLEIC
ACID Oleth-2, -3, -20, etc. Oleyl Alcohol.
Oleamine. Oleyl Betaine. Obtained from various
animal and vegetable fats and oils. Is usually
obtained commercially from inedible tallow (see).
In foods, soft soaps, bar soaps, permanent wave
solutions, shampoos, creams, nail polish, lips
ticks, liquid makeups, many other skin
preparations. Alternatives coconut oil see
alternatives for Animal Oils and Fats. STEARIC
ACID Tallow (see). Stearamide. Stearate.
Quaternium 27. Stearin. Fat from cows, sheep,
etc. (could be dogs and cats from shelters). Most
often refers to a fatty substance taken from the
stomachs of pigs. Can be harsh, irritating. Used
in cosmetics, soaps, lubricants, candles,
hairsprays, conditioners, deodorants, creams.
Alternatives can be found in many vegetable
fats, e.g., coconut. STEROID Sterol. From
various animal glands or from plant tissues.
Steroids include sterols. Sterols are alcohols
from animals or plants (e.g., cholesterol). Used
in hormone preparations. In creams, lotions, hair
conditioners, fragrances, etc. Alternatives
plant tissues, synthetics. STEARYL ALCOHOL
Stenol. A mixture of solid alcohols can be
prepared from sperm whale oil. In medicines,
creams, rinses, shampoos, etc. (Federal
regulations currently prohibit the use of
ingredients derived from marine mammals.)
Alternatives plant tissues, synthetics.
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Figure 5.12 The structure of a phospholipid
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5.3 Phospholipid structure
Figure 5-27a
Figure 5-28
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Cholesterol, polar steroid with acyl chain
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Membrane Contd
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Ganglioside
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Fluidity depends on temperature and
compositionTemp phase transitionComposition
acyl chain length saturation, cholesterol

• Short or kinksgtfewer van der Waals interactions
• Cholesterol has opposing effects is tightly
  regulated
• Polar head group restricts phospholipid head
  group movement-gt decreases fluidity
• Planar steroid separates phospholipid acyl
  tails-gtincreases fluidity

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Fluorescence polarization and intensity were
obtained by a si- multaneous measurement of 11
I/II and 11, where II 1 and II are
the fluorescence intensit,ies detected through a
polarizer oriented parallel and perpendicular to
the direct,ion of polarization of the excitation
beam. The lil/Il and the 11 values relate to the
de- gree of fluorescence polarization, P, to the
fluorescence anisotropy, r, and to the total
fluorescence intensity, F, by the
following equations - P 4, 1, w.L - 1 z?- 4,
1, Ill/I, 1 I,, - I, I,,/I, - 1 r------ I,,
21, I,,/I, 2 0) F I,, 21, Il(Z,,/I, f 2)