Madeo, Marianna

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

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

Cholesterol inhibition alters the production and the molecular profile of microvescicles

Altered cholesterol homeostasis contributes to multiple human diseases, ranging from common disorders such as atherosclerotic cardiovascular disease and stroke to rare genetic syndromes. Cholesterol homeostasis is essential for normal growth and embryonic development (1). Due to the blood-brain barrier, brain tissue cannot utilize dietary or peripherally produced cholesterol (1). Genetic enzyme mutation of cholesterol synthesis can lead to a deficiency of cholesterol and increased levels of potentially bioactive or toxic precursor sterols. A number of human malformation syndromes are due to inborn errors of cholesterol synthesis, including Smith- Lemli-Opitz syndrome (SLOS) (1). This syndrome is associated with a deficiency of 7-dehydrocholesterol (7- DHC) but the cellular mechanisms that result from altered sterol biochemistry are poorly defined.

. The SLOL phenotypic spectrum is extremely broad. Severe phenotypes often die in utero or right after birth, mild cases show characteristic facial feature (microcephaly, ptosis, midface hypoplasia, small upturned nose) and learning and behavioral impairment. In addition, patients show cleft palate and rare vision-impairing cataracts. Syndactyly of the second and third toe is one the common feature reported (1).

Extracellular vesicles (EVs) comprise a varied and heterogeneous group of particles released from cells originating largely from endosomes and/or the plasma membrane (4). Initially described as the disposal mechanisms cells use to discard unwanted material, EVs are now considered mediators of intercellular communication in normal physiology and pathophysiology. Recent studies have shown that EVs contain various proteins, lipids, glycolipids, glycoproteins and nucleic acids including DNA, mRNA and noncoding RNAs (4). Tumor released exosomes mediate axonogenesis in cancer and that this innervation is sensory in nature (6). Consequentially, EVs have the potential to deliver multiplexed information to surrounding tissues and through the body.

My laboratory will focus on unexplored aspects of microvesicles (MVs) biology and possible impacts on cholesterol synthesis and disorders associated with it. 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. In addition of being larger, MVs 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). MVs, are formed by outward blebbing of the plasma membrane and subsequent fission of plasma membrane blebs. Microvesicle biology has not been previously explored within 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 microvescicle content by Gas Chromatography-Mass Spectrometry (GC-MS). This would have direct relevance to the rare disease associated with cholesterol aberrant metabolism. We aim to show the uptake and release of those vesicle in the cells explaining the trafficking of the cholesterol inside and outside the cell. 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 fibroblast obtained from patient.

References

1)  Porter FD, Herman GE. Malformation syndromes caused by disorders of cholesterol synthesis. J Lipid Res. 2011 Jan; 52(1):6-34 Epub 2010 Oct 7. PMID: 20929975; PMCID: PMC2999931.

2)      Irons M., Elias E. R., Salen G., Tint G. S., Batta A. K. 1993. Defective cholesterol biosynthesis in Smith- Lemli-Opitz syndrome. Lancet. 341: 1414 PMID: 7684480.

3)      Tint GS, Irons M, Elias ER, Batta AK, Frieden R, Chen TS, Salen G. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. N Engl J Med. 1994 Jan 13;330(2):107-13. PMID: 8259166.

4)      Irons M., Elias E. R., Salen G., Tint G. S., Batta A. K. 1993. Defective cholesterol biosynthesis in Smith- Lemli-Opitz syndrome. Lancet. 341: 1414 PMID: 7684480.

5)  Madeo M, Colbert PL, Vermeer DW, Lucido CT, Cain JT, Vichaya EG, Grossberg AJ, Muirhead D, Rickel AP, Hong Z, Zhao J, Weimer JM, Spanos WC, Lee JH, Dantzer R, Vermeer PD. Cancer exosomes induce tumor innervation. Nat Commun. 2018 Oct 16;9(1):4284. doi: 10.1038/s41467-018-06640-0. PMID: 30327461; PMCID: PMC6191452.