Comparison of Lipid and Cholesterol-Biosynthesis Gene Products in H- and K-mt-DNA haplogroup cybrids

Abstract

Purpose : Cholesterol has been associated in the pathogenesis of neurodegenerative and systemic pathologies, and certain populations have higher incidence of cholesterol-linked familial diseases. The purpose of this experiment was to determine if mitochondrial DNA (mt-DNA) haplogroup H and K transmitochondrial cybrids regulate the cholesterol and lipid biosynthesis pathways differently. Using transmitochondrial cybrids, cell lines with identical nuclei but either H or K mtDNA, we looked at the relationship between the gene expression levels of cholesterol-related genes and neutral lipid deposits in H and K cybrids.

Methods : Transmitochondrial cybrids were created by fusing mitochondria (mt) isolated from human platelets and cells lacking mtDNA; thus these cybrids have identical nuclei but either H or K mtDNA. RNA from H and K cybrids were analyzed using GeneChip arrays (n=3). Specific cholesterol-related genes FDFT1, SQLE, LSS, CYP51A1, EBP, and DHCR24 were analyzed using Q-PCR (H, n=7; K, n=9). Corresponding reference genes were used as housekeepers for each of the six genes studied. H and K cybrids (n=10) were seeded at 10⁴/well and stained with HCS LipidTox Green Neutral Lipid Stain. Statistical analyses were performed using student’s t-test.

Results : Q-PCR showed insignificant differences of the cholesterol biosynthesis genes in H vs. K; however, one K cybrid (CY#13-57) showed elevated expression of 4 out of 6 cholesterol biosynthesis genes compared to H cybrids; (17.4-fold increase in FDFT1 expression, a 31-fold increase in SQLE expression, a 10.8-fold increase in CYP51A1 expression, and a 5.4-fold increase in EBP). LipidTox staining showed cytoplasmic distribution of the lipids with significantly elevated intensity in the CY#13-57 compared to H cybrids or other K cybrids (p<0.001).

Conclusions : Due to all cybrids differing only in their mtDNA content, our findings suggest that H- and K-haplogroup mt-DNA do not necessarily affects genes involved in cholesterol biosynthesis. However, the increased expression of cholesterol-related genes and larger and more vibrant lipid deposits seen in CY#13-57 suggest the mt-DNA in that specific K cybrid is different than other K cybrids and is mediating cholesterol production and lipid transport through some mechanism.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.