Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Chemistry

Date of Defense

5-3-2013

Graduate Advisor

Michael R. Nichols, PhD

Committee

Michael R. Nichols

Wesley R. Harris

Cynthia M. Dupureur

Chung F. Wong

Abstract

Paranjape, Geeta S.,Ph.D.,University of Missouri-Saint Louis, May 2012. Aβ(1-42) Protofibrils But Not Fibrils Activate Microglia. Major Professor: Michael R. Nichols. One of the hallmark features of the Alzheimer’s disease (AD) brain is the extracellular deposition of amyloid-β protein (Aβ) in both fibrillar (senile plaques) and diffuse forms. Significant proinflammatory markers including activated microglia and cytokines have been detected surrounding the plaques but are absent in diffuse areas suggesting that microglial activation is sensitive to Aβ structure. Since Aβ displays structural polymorphism in vitro, we sought to determine the relationship between Aβ aggregation state and microglial proinflammatory response. Size exclusion chromatography (SEC) purification of freshly reconstituted Aβ(1-42) in NaOH/F12 cell culture medium isolated classical 100 nm long curvilinear protofibrils which stimulated a robust production of microglial TNFα. The Aβ(1-42) protofibrils produced a concentration-dependent response in the low micromolar range. In contrast to Aβ(1-42), Aβ(1-40) required a pre-incubation of 24h at 25⁰C in order to produce protofibrils. Although both preparations were similar in morphology, Aβ(1-42) protofibrils were a much better stimulator of microglia than Aβ(1-40) protofibrils. Aβ(1-40) containing the Arctic mutation (E22G) formed protofibrils as soon as 3h after reconstitution , yet they were largely ineffective in stimulating microglia. None of the Aβ protofibril preparations were toxic to microglia suggesting that Aβ(1-42) protofibrils activate microglia in a manner independent of toxicity. As expected, freshly-purified Aβ(1-42) or Aβ(1-40) monomer were not effective in stimulating microglia, but surprisingly, neither were Aβ(1-42) fibrils even though they exhibited extensive Thioflavin-T fluorescence compared to protofibrils. These findings suggest that Aβ(1-42) protofibrils are the most effective inducers of a proinflammatory response in mouse microglia.

OCLC Number

862966587

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