Date of Award

1-1-2016

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

Biological Sciences

First Advisor

David Patterson, Ph.D.

Second Advisor

Daniel Linseman

Third Advisor

Keith Miller

Fourth Advisor

Nancy Lorenzon

Fifth Advisor

Cedric Asensio

Keywords

Aging, Down syndrome, Metabolomics, mTOR, Purine biosynthesis, Ts65Dn

Abstract

Aging is often associated with impaired cognition and a progressive loss of organ function over time accompanied by an increased susceptibility for many disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), heart disease, osteoporosis, type II diabetes, and many forms of cancer. With a rapidly aging population, the negative impacts of aging and age-related disorders is a major cause of increased human suffering both for affected individuals and for families and caregivers. Metabolic changes are also apparent in normal aging, but may increase in magnitude or nature with accompanying disease states or with accelerated aging. Thus, studying aging in a disease state, or in a disorder characterized by accelerated aging, will facilitate identification of these changes. Trisomy of chromosome 21 (HSA21), or Down syndrome (DS), is an intellectual disability characterized by premature aging. We hypothesize that trisomy causes disruption of the metabolome leading to an accelerated aging phenotype. In the Ts65Dn mouse model of DS, a premature aging phenotype is also observed along with other common comorbidities associated with human DS. Here, we report changes in the both global and targeted metabolomics (the study of small molecules) in the brains of the Ts65Dn mouse. We also report that long-term treatment with microencapsulated dietary rapamycin changes the metabolomic profiles in a manner consistent with increases in healthspan.

Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes can lead to developmental anomalies in lower vertebrates. Alterations in PAICS (phosphoribosyl- aminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase) expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyl-transferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, renal stones, combined immunodeficiency, and autistic features. The pathogenetic mechanism is unknown for any of these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique, we detected accumulation of 5-Aminoimidazole riboside (AIR) in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of Succinylaminoimidazole carboxamide riboside (SAICAR) and adenylosuccinate (SAMP) and, somewhat unexpectedly, accumulation of AIR. The use of HPLC coupled electrochemical detection in combination with cellular assay methods have great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Nathan Gonzales Duval

Provenance

Recieved from ProQuest

File Format

application/pdf

Language

en

File Size

208 p.

Discipline

Molecular Biology, Aging, Biology

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