Noga Kozer 2 Fernanda Cerqueira 1 Orian Shirhai 1
1National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Beer-Sheva, Israel
2INCPM, Whole Institute for Drug Discovery, Weizmann Institute of Science, Rehovot, Israel

Normal physiology relies on maintaining mitochondrial mass and function by a continuous balance between mitochondrial biogenesis and disposal, termed `mitochondrial turnover`. Impaired mitochondrial turnover is increasingly acknowledged to be central in aging and in the etiology of several age-associated diseases (including Parkinson, Huntington, Alzheimer). Recent evidence indicate that in type-2 diabetes (T2D) mitochondrial turnover is suppressed as well, leading to the accumulation of damaged mitochondria, which results in beta-cell dysfunction and apoptosis.

The tools used so far to follow mitochondrial turnover are expensive and not suitable for a high-throughput screening. Recently, ‘Mitotimer’, a green fluorescent protein which emission shifts to red within about 24 hours after its translation, emerged as a reliable tool to follow mitochondrial turnover. The ratio between red (older mitochondria) and green (new mitochondria) reflects changes in mitochondrial turnover due to mitochondrial synthesis (biogenesis) or clearance (autophagy).

We aimed to establish ‘Mitotimer’ as a unique tool to search for compounds which increase mitochondrial turnover in pancreatic beta-cells, in a high content screen (HCS). The discovery of drugs which improve mitochondrial turnover in beta-cells is potentially a new approach for treating T2D.

HCS assay was developed at the Drug Discovery Unit, INCPM, Weizmann Institute of Science. The assay includes plating ‘Mitotimer’ expressing INS-1 cells in 384-well plates, exposing the cells to 10µM compounds over 18h and image them in two wave lengths (FITC and TRITC) to quantify the mitochondria age reported by ‘Mitotimer’ in individual cells. Image analysis protocol that calculates Red/Green intensity ratio for every cell was written in MetaXpress using Custom Module Editor extension. Due to low z’ values (0.2) it was decided to screen in duplicates.

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