Novel methodology to study the relationship between cholesterol biosynthesis, prenylation and autophyagy in aging models

Abstract

As we get older, the likelihood of developing a chronic ailment increases which can change the quality of life we experience. Age is the biggest risk factor for many common diseases including Alzheimer’s disease, atherosclerosis, and cancer. Cellular aging is a complex and heterogeneous process that is contributed to by multiple factors. One important factor that has been identified is proteostasis and the autophagy process. Autophagy is a lysosomal cellular degradation system that is responsible for the breakdown of organelles and proteins. The autophagy process is known to decline with age leading to a buildup of deleterious material in the cell. A decrease in autophagy function has been associated with a decrease in the mevalonate pathway. The mevalonate pathway is responsible for the synthesis of cholesterol, which is needed to maintain cell membrane structure, and isoprenoids used for prenylation. Prenylation is a post-translational modification of proteins where an isoprenoid is covalently attached to proteins to facilitate localization to membranes and cell signaling. Abnormalities in prenylation have been linked to multiple age-related diseases, including Alzheimer’s and Progeria, and the modification occurs on some essential proteins in the autophagy pathway. The goal of this thesis is to develop methods to understand the response of the cell to a decline in autophagy with a focus on the mevalonate pathway. Better understanding of these cellular processes with age can potentially aid in the generation of therapies to extend healthspan and alleviate age-related disease burden. In order to understand how the intricate cell environment changes with age, new methodologies are needed that measure multiple cell types and cellular processes simultaneously. Single-cell analysis methods can provide a wealth of information in heterogenous cell samples that is not attainable by bulk cell analysis methods. Mass cytometry is a powerful single-cell analysis technique that enables the detection of over 45 parameters simultaneously, but currently lacks methods for tracking global changes in post-translational modifications. In this work, a novel method to measure prenylation, protein abundance and phenotypic markers in single-cells simultaneously by mass cytometry was established. This method uses a prenylation analog, C15AlkOPP, with a reporter, Tb-DOTA-azide, that is attached through copper catalyzed click chemistry to measure levels of prenylation. The technique was compatible with metal labeled antibody staining and performed in primary cells from mouse liver tissue. In the future, this method can be employed to study animal models of aging to understand the relationship between prenylation, autophagy and the mevalonate pathway with age at the single-cell level. In addition to the development of single-cell analysis methods, two cell models of reduced expression of an essential autophagy protein, ATG7, leading to a reduction in autophagy function were investigated to understand the relationship between reduced autophagy function, the mevalonate pathway and prenylation. Investigation of these cell models led to two proposed models of regulation for the mevalonate pathway with autophagy function decline, one for when ATG7 mRNA expression is reduced and one for when there is no functional ATG7 protein present. These cell models predict the response of the mevalonate pathway and its products to a decline in autophagy function. In addition to the cell models, inhibition of the mevalonate pathway by pharmacological treatment, lovastatin, resulted in an increase in autophagy proteins. Overall, the models of regulation and novel methods produced in this thesis will help to elucidate the relationship between autophagy and the mevalonate pathway in the context of aging which could inform novel treatments to extend healthspan.