Quartz Crystal Microbalance Analysis of Growth Kinetics for Aggregation Intermediates of the Amyloid-β Protein

Transcription

1 Quartz Crystal Microbalance Analysis of Growth Kinetics for Aggregation Intermediates of the Amyloid-β Protein Joseph A. Kotarek, Kathryn C. Johnson, and Melissa A. Moss Department of Chemical Engineering

2 Alzheimer s Pathology Alzheimer s Brain Pathology Amyloid Plaques Courtesy of D. Dickson, Mayo Clinic, Jacksonville. Aβ Fibrils Neurofibrillary Tangles Nichols et al. (2002) Biochemistry, 41:

3 How Does Aβ Assemble Into Fibrils? nucleus monomer soluble aggregation intermediates mature fibril Nichols et al. (2002) Biochemistry, 41:

4 Pathogenic Role for Aggregation Intermediates Capable of inducing physiological response Impaired hippocampal long-term potentiation Synaptic loss Neurotoxicity Altered memory function Active growth has been observed to induce toxicity Human cortical neurons Cerebrovascular smooth muscle cells

5 Key Research Questions What kinetics govern Aβ aggregation intermediate growth Is growth of Aβ aggregation intermediates influenced by varying solution conditions

6 Isolation of Aβ Species Monomer 100 Aβ µm mau (280 nm) 2-10 mm NaCl Mixture 0 Void Protofibril 30 Volume, ml 20 Aβ µm 20 Monomer 10 mau (280nm) Volume, ml

7 Quartz Crystal Microbalance Decrease in resonance frequency is related to an increase in bound mass 2 f f = A( µρ ) 0. 5 q 2 m = C f m

8 Protofibril Immobilization via Avidin-Biotin Avidin was covalently coupled Biotinylated Aβ monomer bound avidin Unlabeled aggregation intermediates were immobilized as monomer was incorporated

9 Experimental Setup RQCM QCM Flow Cell Buffer Pump Aβ aggregation intermediate or monomer Waste Data Acquisition

10 Aβ aggregation intermediates are selectively immobilized onto the crystal surface. aggregate monomer

11 m (pmol cm 2 ) A aggregate + monomer monomer + monomer f (Hz) Addition of Aβ monomer onto immobilized aggregation intermediates is successfully detected time (min)

12 Growth of immobilized aggregation intermediates is reversible and non-saturable.

13 100 pmol/cm 2 AGG 10 µm monomer 780 pmol/cm 2 AGG 250 nm monomer Growth can be detected with high sensitivity.

14 Growth is linearly dependent upon monomer

15 and immobilized aggregation intermediate density.

16 Growth is modeled via a first-order kinetic model. k 1 [ AGG ] + [ M ] [ AGG] k -1 d[ M ] dt = k1[ AGG][ M ] k 1[ AGG] k k 1 1 = = µm 1 min 1 min 1 K d k = 1 = k nm

17 Addition of Aβ monomer onto immobilized aggregation intermediates increases with increasing solution ionic strength.

18 Addition of Aβ monomer onto immobilized aggregation intermediates increases as solution ph becomes more acidic.

19 Summary Aβ aggregation intermediates can be selectively immobilized onto the QCM quartz crystal surface using avidin-biotin chemistry. QCM can be used to observe the growth of aggregation intermediates via monomer addition. Growth of Aβ aggregation intermediates can be described using a first-order kinetic model. Growth of Aβ aggregation intermediates is modulated by solution ionic strength and ph.

20 Acknowledgements National Science Foundation CAREER Award American Heart Association, Mid-Atlantic Affiliate Beginning Grant-In-Aid Alzheimer s Association New Investigator Research Grant Postdoctoral Fellow Francisco Gonzalez Graduate Students Adriana Reyes Barcelo Joseph Kotarek Deborah Soto Chen Suo Undergraduate Students Fahmin Basher Gopal Chakrabarti Tim Davis Dee Dunagan Kathryn Johnson Sarah Holton Christie Long Chris Stewart

21 Quartz Crystal Microbalance Analysis of Growth Kinetics for Aggregation Intermediates of the Amyloid-β Protein Joseph A. Kotarek, Kathryn C. Johnson, and Melissa A. Moss Department of Chemical Engineering

22 A PEG-modified crystal surface significantly reduces nonspecific binding of aggregation intermediates

23 Growth of Mature Aβ Fibrils First-order kinetics Single growth phase Non-saturable and reversible growth Dock-and-lock model Two distinct growth phases locked monomer dissociates slower than docked monomer

24 Mechanisms of Growth for Aggregation Intermediates intermediates elongation association protofibrils elongation association Nichols et al. (2002) Biochemistry, 41: