Abstract
Background: Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1. This encodes a ubiquitous protein involved in mitochondrial dynamics, hence tissue specificity is not understood. Dysregulated mitophagy (mitochondria recycling) is implicated in ADOA, being increased in OPA1 patient fibroblasts. Furthermore, autophagy may be increased in retinal ganglion cells (RGCs) of the OPA1Q285STOP mouse model.Aims: We developed a mouse model for studying mitochondrial dynamics in order to investigate mitophagy in ADOA.Methods: We crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse, harboring red fluorescent mitochondria and green fluorescent autophagosomes. Colocalization between mitochondria and autophagosomes, the hallmark of mitophagy, was quantified in fluorescently labeled organelles in primary cell cultures, using two high throughput imaging methods Imagestream (Amnis) and IN Cell Analyzer 1000 (GE Healthcare Life Sciences). We studied colocalization between mitochondria and autophagosomes in fixed sections using confocal microscopy.Results: We validated our imaging methods for RedMIT/GFP-LC3 mouse cells, showing that colocalization of red fluorescent mitochondria and green fluorescent autophagosomes is a useful indicator of mitophagy. We showed that colocalization increases when lysosomal processing is impaired. Further, colocalization of mitochondrial fragments and autophagosomes is increased in cultures from the OPA1Q285STOP/RedMIT/GFP-LC3 mice compared to RedMIT/GFP-LC3 control mouse cells that were wild type for OPA1. This was apparent in both mouse embryonic fibroblasts (MEFs) using IN Cell 1000 and in splenocytes using ImageStream imaging flow cytometer (Amnis). We confirmed that this represents increased mitophagic flux using lysosomal inhibitors. We also used microscopy to investigate the level of mitophagy in the retina from the OPA1Q285STOP/RedMIT/GFP-LC3 mice and the RedMIT/GFP-LC3 control mice. However, the expression levels of fluorescent proteins and the image signal-to-background ratios precluded the detection of colocalization so we were unable to show any difference in colocalization between these mice.Conclusions: We show that colocalization of fluorescent mitochondria and autophagosomes in cell cultures, but not fixed tissues from the RedMIT/GFP-LC3, can be used to detect mitophagy. We used this model to confirm that mitophagy is increased in a mouse model of ADOA. It will be useful for cell based studies of diseases caused by impaired mitochondrial dynamics.
Highlights
Mitochondria are important for cells, not just for generating energy, calcium regulation and key biosynthetic processes including synthesis of iron Sulfur centers, and for apoptosis, signaling, and response to cellular stress (Suomalainen and Battersby, 2018)
We focussed on an earlier stage of mitophagy than the other two assays (Sun et al, 2015; McWilliams et al, 2016). We developed this as a readout for mitophagy driven by OPA1 knock down, in which we anticipated that excessive mitochondrial fragmentation, apparent in retinal ganglion cells (RGCs) of this mouse model (Williams et al, 2012), drives mitophagy (Liao et al, 2017)
In order to visualize mitochondrial fate, we first generated a mouse expressing monomeric red fluorescent protein (mRFP) fused to the COX VIII mitochondrial targeting sequence (Figure 1), engineered by random insertion into embryonic stem cells
Summary
Mitochondria are important for cells, not just for generating energy, calcium regulation and key biosynthetic processes including synthesis of iron Sulfur centers, and for apoptosis, signaling, and response to cellular stress (Suomalainen and Battersby, 2018). Mitochondrial location and transport are important in neurons, individual mitochondria moving along microtubules in the cytoplasm to synapses and other parts of the cell requiring energy (Li et al, 2004) These dynamics are under the control of molecular players such as Drp and Fis for fission and mitofusins (Legros et al, 2002) and OPA1 for fusion (Chen and Chan, 2006). The evidence for respiratory chain dysfunction in mitochondrial optic neuropathies may be very subtle (Yu-Wai-Man et al, 2002) These are important diseases that impair vision, resulting in lifelong disability. Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1 This encodes a ubiquitous protein involved in mitochondrial dynamics, tissue specificity is not understood. Autophagy may be increased in retinal ganglion cells (RGCs) of the OPA1Q285STOP mouse model
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