Abstract
The premature aging disorder, Hutchinson-Gilford progeria syndrome (HGPS), is caused by mutant lamin A, which affects the nuclear scaffolding. The phenotypic hallmark of HGPS is nuclear blebbing. Interestingly, similar nuclear blebbing has also been observed in aged cells from healthy individuals. Recent work has shown that treatment with rapamycin, an inhibitor of the mTOR pathway, reduced nuclear blebbing in HGPS fibroblasts. However, the extent of blebbing varies considerably within each cell population, which makes manual blind counting challenging and subjective. Here, we show a novel, automated and high throughput nuclear shape analysis that quantitatively measures curvature, area, perimeter, eccentricity and additional metrics of nuclear morphology for large populations of cells. We examined HGPS fibroblast cells treated with rapamycin and RAD001 (an analog to rapamycin). Our analysis shows that treatment with RAD001 and rapamycin reduces nuclear blebbing, consistent with blind counting controls. In addition, we find that rapamycin treatment reduces the area of the nucleus, but leaves the eccentricity unchanged. Our nuclear shape analysis provides an unbiased, multidimensional "fingerprint" for a population of cells, which can be used to quantify treatment efficacy and analyze cellular aging.
Highlights
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that occurs in approximately 1 out of 4 million live births [1]
In order to test our automatic analysis of nuclear shape, we first cultured fibroblasts from two HGPS fibroblast cell lines (HGADFN155-p15 and HGADFN167-p12, HGPS1 and HGPS2 respectively) and from one normal control (HGFDFN168-p14, normal)
A blebbed cell, such as the cell shown in the top of Figure 1b, will have www.impactaging.com boundary regions that are dark red and dark blue, whereas a non-blebbed cell, such as the cell shown in the bottom of Figure 1b, will have boundary regions that are mostly blue and green with almost no red
Summary
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that occurs in approximately 1 out of 4 million live births [1]. The genetic mutation that leads to HGPS occurs in exon 11 of the human LMNA gene, which plays a role in nuclear scaffolding [5, 6] This HGPS mutation is a de novo single nucleotide substitution (1824 C => T), which does not change the amino acid coding sequence [GGC (glycine) => GGT (glycine)]. This mutation partially activates a cryptic splice donor site, which causes a 150-nucleotide sequence to be spliced out of exon 11 and leads to the production of the mutant protein progerin, known as LAΔ50 [7]. As progerin continues to build up inside prematurely aged cells, the nuclear blebbing www.impactaging.com
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