Abstract
Purpose: :
Mitochondria are highly dynamic organelles whose morphology, distribution and density can be regulated by fusion and fission. Mitochondrial fusion is necessary for normal respiratory function in cells and perturbations in fusion lead to developmental defects and neurodegenerative diseases. Mitochondrial fission leads to increased fragmentation and is normally associated with proapoptotic cells. We investigated the alterations in mitochondrial mass, fusion and fission in RPE cells from normal and AMD subjects.
Methods: :
Mitochondrial mass in cultured RPE cells from different topographical locations, different age and AMD level were determined using MitoTracker green stain and confocal microscopy together with MetaMorph image analysis to calculate mitochondrial mass per cell. To study mitochondria fusion and fission, macular RPE cells from normal and AMD donors was transfected with mitochondrial targeted GFP and mitochondrial targeted dsRed respectively. Two groups of cells expressing either mitochondrial targeted GFP or dsRed were fused and imaged. The images were analyzed using the MetaMorph image analysis system. Protein expression was determined by Western blotting.
Results: :
There was an age-related decline in mitochondrial mass in macular RPE cells from young (20-30 yr) to old (70-90yr) donors. The mitochondrial mass in macular RPE cells from AMD patients was 30% less than that in macular RPE cells from normal age-matched donors and this decrease was greatest in advanced AMD (p<0.05). Mitochondria in RPE cells from young maculas had a 20% higher capacity for remodelling compared with RPE cells from aged maculas and this capacity was least in RPE cells from donors eyes with AMD (p<0.05). Significantly greater mitochondrial fission, reduced fusion and greater mitochondrial fragmentation was observed in RPE cells from AMD donors compared with their normal aged matched counterparts and RPE cells from young donors (p<0.05). These alterations correlated with changes in expression of the mitochondrial fusion and fission proteins Mfn1, Mfn2, Drp1 and Fis1.
Conclusions: :
The decline in mitochondria mass and changes in mitochondria dynamics may be responsible for mitochondrial dysfunction observed in RPE cells. These alterations may contribute to AMD pathogenesis and represent a pharmacological target.
Keywords: age-related macular degeneration • mitochondria • retinal pigment epithelium