Madeo, Marianna

• Ph.D., Molecular Biology and Biochemistry, Universita' della Calabria, Italy

• B.S., Pharmaceutical Chemistry & Technology, Universita' della Calabria, Italy

My laboratory will focus on unexplored aspects of microvesicle biology and possible impacts on cholesterol synthesis disorders. This will allow me to leverage my expertise in isolation and characterization of microvesicles and exosomes (4). Although microvesicles and exosomes are structurally similar, they differ in size, lipid composition, content, and cellular origin. Microvesicles are larger, and are secreted differently from exosomes. MVs generally range from 200 nm to several microns in diameter, whereas exosomes are smaller, and range from 50 to 100 nm in diameter (4). Exosomes, the best characterized of the EV subtypes, are formed first as intraluminal vesicles within a mutlivesicular body (MVB), which are released into the extracellular space upon MVB fusion with the plasma membrane. MVs, are formed by outward blebbing of plasma membrane and subsequent fission of plasma membrane blebs. Microvesicle biology has not been previously explored with cholesterol synthesis disorders. We aim to identify microvesicles marker, i.e. flotillin-2, ARF6 and CD40 to use for imaging. Fluorescence Activated Cell Sorting (FACS) or western blot analysis. Further we propose to analyze the microvesicle content by GC-MS. This would have direct relevance to the rare disease associated with cholesterol aberrant metabolism. We will use widely used drugs to target specific enzyme in the sterols synthesis and analyze the effect on microvesicles, and we will also look at microvesicles content from the mouse genetic models for Smith-Lemli-Opitz syndrome or iPSCs from the Francis lab.

Thus we aim to validate the following hypothesis:

1) Cholesterol inhibition alters MVs production and alters the molecular profile of MVs. We will isolate MVs from different cell lines, embryonic kidney (HEK293T), keratinocytes (HaCaT), and microglia (HMC3) at different growth conditions to mimic inhibition of cholesterol. Cells will be cultured under cholesterol deplete conditions in 7.5% delipidated serum (LPDS) with small molecule inhibitors AY9944 (DHCR7 inhibitor), Atorvastatin or Simvastatin. We will look at MVs content in the different cell lines and quantifying them by FACS or Nanosight analysis. MVs will be characterized by FACS, imaging and Western Blot anaylsis using microvesicles marker, i.e. flotillin-2, ARF6 and CD40.

2) How is lipid content within cholesterol-inhibited MVs impacted? Is MVs cargo changed following cholesterol inhibition? Cargo of macrovesicles isolated from the different cell lines will be determined using GC-MS and FACS analysis.

3) Does cholesterol synthesis inhibition affect MV biology in other disease models. We will work with different cell lines with different inborn error of cholesterol synthesis. We will collect and analyze MVs secreted from fibroblasts  isolated from patient or mouse model in comparation with a related control and compare the release of MVs and their cargo.