Understanding cholesterol homeostasis and its impact on GPCR signalling in aging

Abstract

Full text embargo One year up to July 2024 Aging is defined as the time-related deterioration of physiological functions necessary for survival of organisms. Despite significant progress in the extension of human lifespan due to modern medicine, improved sanitation and nutrition, the undesirable effects associated with aging have not been alleviated. There is increasing evidence that aging occurs at the cellular level and cellular senescence is a major contributing factor to the process of aging. Accumulation of cellular damage over the years results in tissue dysfunction leading to organismal aging. Thus, it is important to understand the molecular basis of aging and identify possible therapeutic intervention approaches and targets. Senescence is associated with high inflammation as well as high oxidative and nitrosative stress which causes persistent damage to cellular components such as DNA, lipids and proteins. In this study, I have examined the effect of altered cholesterol homeostasis on CXCR4 signalling during cellular senescence.

The chemokine receptor CXCR4 is upregulated in senescent cells and however the signalling upon stimulation is impaired. However, during senescence the CXCR4 mediated calcium oscillations are disrupted and signalling is altered. To investigate the mechanism of altered signalling, I studied various steps in the signalling cascade, starting at the membrane level. Plasma membrane composition plays an important role in receptor organisation, dynamics and signalling and senescent cells show changes in several membrane properties. Cholesterol is the most abundant lipid species found in eukaryotic membranes which when oxidised disrupts the membrane dynamics. The oxidation of cholesterol to form oxysterols during aging led to altered CXCR4 signalling, which was recorded as delayed or disrupted calcium oscillations after ligand stimulation. Senescent cells behave like a cholesterol depleted and oxysterol rich environment. CXCR4 receptor signalling through the Gi G-protein, however the presence of oxysterols switches the affinity towards Gs leading to an anti-inflammatory arm of signalling.

The cholesterol homeostasis pathway is very tightly regulated leading to balanced free cholesterol levels inside the cell. The total cholesterol levels were found to be very high in senescent cells, at the same time the cell size is also larger than non-senescent cells. On analysis of per unit area cholesterol, the senescent cells had lower cholesterol per unit area and higher oxysterols. To understand this dysregulation in the homeostasis pathway, I analysed the proteins which are involved in the cholesterol biosynthesis and its efflux from cells. The cholesterol biosynthesis genes like HMGCS were found to be much lower in senescent cells, while the efflux genes like ABCA1 were high. This indicated that senescent cells were constantly eliminating cholesterol and oxysterols from cells.

Oxysterols are toxic to the cells in high amounts and need to be eliminated upon generation. The cells maintain a fine balance between the cholesterol and oxysterol levels, which is disrupted during senescence. I investigated the effect of cholesterol biosynthesis and efflux inhibition on the viability of senescent cells. Senescent cells died when treated with biosynthesis inhibitor Simvastatin, or efflux inhibitor GSK2033. This led to the discovery of a a novel mechanism for the maintenance of senescent cell viability and identified targets for senolytic development. Targeting the cholesterol homeostasis pathway for development of novel senolytics and effects on health-span of acceler-aged mice were also explored. Simvastatin, which is a novel potential senolytic, was used to study aging signatures in radiation induced aging mouse model. Simvastatin reduced liver injury and inflammation in irradiated mice. The present study has implications in cancer co-therapies, age-related disease prevention as well as rejuvenation therapies. Cholesterol homeostasis pathway can be targeted to develop novel compounds and therapies that reduce age related injury and systemic inflammation.