Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Neurosciences

Language

English (en)

Date of Award

12-31-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Randall J Bateman

Abstract

Alzheimer's disease: AD) is the most common dementia, currently afflicting 30 million people worldwide, with prevalence steadily increasing. Over 99% of AD cases are classified as sporadic, in which the major risk factors are age: greater than 65 years) and the ApoE-ε4 allele: in a gene dose dependent manner). The minority of AD cases: less than 1%) are caused by autosomal dominant inheritance of a genetic mutation in one of three genes: Amyloid Precursor Protein: APP), Presenilin-1: PSEN1), or Presenilin-2: PSEN2). Mutation carriers will generally notice cognitive decline starting at a relatively young age, anytime between their 30s-50s, and eventually succumb to this early-onset AD. Early-onset AD may also be the result of an extra copy of the APP gene, as manifested in Down Syndrome: DS) patients, who universally develop AD.

Alzheimer's disease pathophysiology appears to revolve around APP. This transmembrane protein may be processed through one of two pathways. If APP is processed down the amyloidogenic: β-secretase) pathway, soluble APPβ: sAPPβ) will be released first, and with a subsequent cleavage, β-amyloid: Aβ), will be released into the brain where it could sequester into amyloid plaques, which are a major hallmark of AD. The other processing pathway is caused by cleavage of APP by α-secretase. This results in the release of soluble APPα: sAPPα), which precludes the release of Aβ, thus making this pathway non-amyloidogenic.

Several studies have focused on measuring the products of APP cleavages in an effort to elucidate the pathophysiology of AD. These studies give a static view of APP because they measure absolute amounts of protein in single samples. However, proteins are constantly being produced, cleared, and aggregated which may result in protein concentrations fluctuating over time. Recently it was reported that Aβ exhibits a diurnal pattern, the definitive cause of which is still unknown.

The general goal of this thesis is to determine the interplay of the α- and β;-secretase APP processing pathways in both physiological and pathophysiological settings in humans, to establish the human metabolism rates of total sAPP, sAPPα, and sAPPβ, and to determine whether drug intervention of one of the two APP pathways, would have an effect the other pathway. The aims of this thesis are:

1) To determine the extent of interdependence of the α-secretase and the β-secretase APP processing pathways in humans and whether β diurnal patterns were being driven by APP. Human cerebrospinal fluid: CSF) was collected hourly for 36 h, and concentrations of sAPPα, sAPPβ, Aβ40, and Aβ42 were measured by four metabolite-specific ELISAs. Parameters associated with diurnal patterns were compared among metabolites. Further, correlation analyses were used to determine if any correlation among metabolites at a single time-point was evident. Samples from Alzheimer's patients, age-matched controls, and young healthy controls were analyzed.

2) To determine the physiological and pathophysiological metabolism of sAPPα and sAPPβ in humans. Total sAPP was isolated by immunoprecipitation from CSF of participants undergoing stable isotope labeling kinetics studies. Isolated sAPP underwent proteolytic cleavage by trypsin and turnover rates of total sAPP were determined by LC-MS and compared to turnover rates of Aβ. Isolated total sAPP that underwent proteolytic cleavage by Arginine-C produced some preliminary results that could lead to eventual determination of sAPPα or sAPPβ specific turnover rates in humans.

3) To determine the physiological metabolism of sAPPα and sAPPβ in rhesus macaques in comparison to Aβ metabolism and to determine if drug intervention of the β-secretase pathway by a BACE1 inhibitor would affect the α-secretase pathway. Soluble APPβ, sAPPα and Aβ were isolated from rhesus macaque CSF by serial immunoprecipitation. Monkeys were undergoing stable isotope labeling kinetics studies. Isolated APP metabolites underwent proteolytic cleavage by LysN, and then the turnover rates of these metabolites were measured by LC-MS to determine if the BACE1 inhibitor was hitting its target, and if it was having any effect of the α-secretase pathway.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K7125QNB

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