Potential Anti-Dementia Chemicals: Preventing APOE and NF-kB Release in Astrocyte Culture

Date of Award

6-2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Harold Komiskey, PhD

Second Advisor

Richard E White, PhD

Third Advisor

Xinyu Wang, PhD

Fourth Advisor

Brian M Matayoshi, PhD

Abstract

Alzheimer's disease is a neurodegenerative disorder that primarily affects the hippocampus and cortical areas of the brain. Dysfunction in Aβ clearance and neuroflammation are two key pathological issues in the development and progression of the disease. Astrocytes are highly implicated in the etiology of AD because they are the main producers of apolipoprotein E, a major contributor to Aβ clearance (or its dysfunction) in the CNS. Astrocytes are also important contributors to neuroinflammation because they possess Toll-like receptor 4, an important receptor in the production of NF-kB, a transcription factor that regulates the production of proinflammatory cytokines. Currently, no effective preventive options are available for AD. Effective options would target ApoE and NF-kB in astrocytes, as they are highly implicated in the pathogenesis of AD. Dietary changes, nutritional supplements, and other natural products may prove helpful in preventing this brain pathology. To assess this hypothesis, cell cultures were pretreated with the 3 µM curcumin, 0.3 µM withaferin A, or 1 µM CAPE to reverse the effects in NF-kB and ApoE production after stimulation of TLR4 with lipopolysaccharide (LPS) or monophosphoryllipid A (MPLA). After 24 hours, the amount of ApoE and NF-kB produced by the astrocytes was measured using a sandwiched Enzyme-Linked Immunosorbent Assay (ELISA). At the above concentrations and incubation time, ELISA analysis indicated that exposure to curcumin significantly lowered NF-kB levels, while withaferin A and CAPE significantly increased them. At these concentrations, none of the natural products had any significant effect on ApoE levels. A CCK-8 assay using a water-soluble tetrazolium-8 salt showed an significant increase in dehydrogenase activity for astrocytes treated with the above concentrations of curcumin or withaferin A, demonstrating an increase in cell viability. However, CAPE significantly decreased dehydrogenase activity, demonstrating a toxic effect on astrocytes. Additionally, a time course experiment was performed to investigate the optimal times of release and optimal concentrations for stimulation of TLR4 by LPS and MPLA. The experimental results suggest that curcumin may be able to attenuate the pathology caused by NF-kB and ApoE in astrocytes.

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