Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin...

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Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation of β-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.
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Page 1: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Alzheimer’s Understanding The Role of

Membranes in Amyloid Aggregation

Nadia J. EdwinMacromolecular Seminar

December 5, 2003

Presentation Based on

Correlation of β-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.

Page 2: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

OUTLINE1. Background - Amyloid - Membranes

2. Previous Studies

3. Goal of Reference Paper

4. Experimental Techniques Used

- DPH Anisotropy

- Dynamic Light Scattering - Static Light Scattering

5. Conclusion

Page 3: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

What is Amyloid?

1853 – Rudolf Virchow named cerebral deposits as amyloid

Amyloid –proteinaceous aggregates associated with diseases (Alzheimer’s, Parkinson’s, spongiform encephalopathies)

Amyloid aggregates in brain cells are thought to play a causative role in the onset of Alzheimer’s Disease

Dobson, C.M. Protein misfolding, evolution and disease. Trends Biochem.Sci. 1999, 24, 329-332.

Page 4: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Origins of Amyloid?β-Amyloid peptide : (39- 42 amino acids) is a protein fragment cleaved

from a much larger protein

β-Amyloid Precursor Protein : (~ 695 amino acids)

β-APP – an inhibitory molecule that regulates the activity of proteases

Page 5: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Amyloid Hypothesis

Neurodegeneration in Alzheimer’s disease (AD) may be caused by deposition of amyloid β-peptide (Aβ) in plaques in brain tissue

Current studies probe effects of physical conditions (differing pH, temperature, salt concentration) on Aβ aggregation

Hardy, J.; Selkoe, D.J. Science, 2002, 297, 353-356.

Page 6: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Plasma Membrane

Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.

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

Page 7: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Membrane Fluidity 

Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.

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

          

Page 8: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Membrane Composition

Saturated fats no double bonds between carbons in the tail saturated with hydrogen solid at room temperature most animal fats, bacon grease, lard, butter

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

Page 9: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Effect of Temperature on membrane fluidity

Page 10: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Phospholipids

Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.

POPC 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine

POPE 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine

POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-1-serine]

Page 11: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Cholesterol

Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.

Phospholipids, cholesterol, gangliosides

Page 12: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Previous Studies: Side 1

Aβ associates with cells via membrane-bound receptors

1. Aβ binds to Serpin-enzyme Complex Receptor (SEC)

2. Aβ binds to class A scavenger receptor (SR)

3. Aβ binds to Receptor for advanced glycation end products (RAGE)

4. Aβ 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.

Page 13: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Side 2 of the Debate Aβ aggregates are toxic via nonspecific association

with cell membranes

1. Membrane components promoted changes in Aβ secondary structure and/or aggregation propensity.

2. Aβ 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

Page 14: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Goal of Paper

• Determine whether changes in membrane physical properties were correlated with Aβ aggregates

• Relations of any such effects with biological membrane with specific membrane components

• Does changes depend on Aβ aggregation state

Page 15: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

ANISOTROPY

r =

perpar

perpar

gII

gII

2

PTI Spectrofluorometer with manual polarizers

Excitation wavelength – 360 nm

Emission wavelength – 430 nm

Page 16: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

CH

CH

CH

CH

CH

CH

1,6-Diphenyl-1,3,5-hexatriene (DPH)

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

Absorption – 350nm

Emission – 452nm

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.

Absorption and emission spectra of DPH in hexane at 25°, polarization spectrum of DPH in frozen (propylene glycol at -50°)

Page 17: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Dynamic Light Scattering

LASER

Lens Sample Cell

AmplifierCorrelatorComputer

Photomultiplier Detector

θ

Page 18: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

DLS Results

    Figure 1: Growth kinetics of Aβ aggregates at physiological pH. Aβ 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.

Page 19: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Static Light Scattering

Figure 2: Change in Aβ 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.

Page 20: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

SLS Results Cont’d

Figure 3: Growth of Aβ aggregates at physiological pH. Weight-averaged molecular weight <M>w ( ) 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.

Page 21: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Microscopy Shows

Figure 4: Electron micrographs of Aβ 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.

Page 22: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Fluorescence Results

Figure 5: Effect of Aβ aggregation on bis-ANS fluorescence. PBS ( , n = 18), freshly diluted Aβ ( , n = 4), and Aβ 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 Aβ aggregated at pH 6 and 7 was statistically different (p < 0.01).

Page 23: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Aggregation Results

Figure 6: Sketch of Aβ aggregation at (A) neutral and (B) acidic pH. (A) At pH 7, Aβ 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 Aβ 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, Aβ 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 Aβ 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.

Page 24: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

DPH Anisotropy Results

Figure 7: Effect of Aβ Aggregation at pH 7 on DPH anisotropy. Freshly diluted ( ) and 2 day-aged ( ) Aβ samples were added to (A) POPC and (B) POPG liposomes with embedded DPH. Data are compilation of 2-4 replicate experiments at each condition.

Page 25: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

DPH Anisotropy

Figure 8: Effect of Aβ Aggregation at pH 6 on DPH anisotropy. Freshly diluted ( ) and 2 day-aged ( ) Aβ samples were added to (A) POPC and (B) POPG liposomes with embedded DPH. Data are compilation of 2-4 replicate experiments at each condition.

Page 26: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

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

( , ) Aβ. Aβ induces a significantly larger anisotropy increase in vesicles containing gangliosides at both pH 6 and pH 7.

Page 27: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Conclusion

Observed decreases in membrane fluidity, detected as an increase in DPH anisotropy.

Changes in membrane fluidity are not solely dependent on binding of Aβ to the bilayer surface.

Page 28: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Acknowledgements

• NSF-IGERT

• Dr. Paul Russo

• Russo Group Members

Page 29: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Fluidizing action of Aβ

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.

Page 30: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Figure 5.10 The synthesis and structure of a fat, or triacylglycerol

Carboxyl group has acid propertiesHydrocarbon chain, 16-18 carbonsNonpolar C-H bonds, hydrophobic

(Condensation Reaction)

(bond between hydroxyl group and a carboxyl group)

A triglyceride

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)

Page 31: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Lipids: Diverse Hydrophobic Molecules

Lipids = Diverse group of organic compounds that are insoluble in water,but will dissolve in nonpolar solvents (e.g., ether chloroform, benzene).Important groups are fats, phospholipids, and steroids.

Fats store large amounts of energy

Fats = Macromolecules are constructed from:

Glycerol, a three-carbon alcohol

Fatty acid (carboxylic acid) = Composed of a carboxyl group atone end and an attached hydrocarbon chain (“tail”)

Page 32: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

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.

Page 33: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Figure 5.12 The structure of a phospholipid

Page 34: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

5.3 Phospholipid structure

Figure 5-27a Figure 5-28

Page 35: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Cholesterol, polar steroid with acyl chain

Page 36: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Membrane Cont’d

Page 37: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Ganglioside

Page 38: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Fluidity depends on temperature and compositionTemp: phase transitionComposition: acyl chain length & saturation, cholesterolShort or kinks=>fewer van der Waals interactionsCholesterol has opposing effects & is tightly regulated:•Polar head group restricts phospholipid head group movement-> decreases fluidity•Planar steroid separates phospholipid acyl tails->increases fluidity

Page 39: Alzheimer’s Understanding The Role of Membranes in Amyloid Aggregation Nadia J. Edwin Macromolecular Seminar December 5, 2003 Presentation Based on Correlation.

Fluorescence polarization and intensity were obtained by a si-multaneous measurement of 11 I/II and 11, where II 1 and II are thefluorescence intensit,ies detected through a polarizer orientedparallel and perpendicular to the direct,ion of polarization of theexcitation 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 followingequations:- P= 4, 1, w.L - 1 z?-4, + 1, Ill/I, + 1I,, - I, I,,/I, - 1 r=-----=-I,, + 21, I,,/I, + 20)F = I,, + 21, = Il(Z,,/I, f 2)