Figure 2 | Cell Death & Disease

Figure 2

From: HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion

Figure 2

Mitochondrial respiration is increased in HtrA2-deficient cells. (a) NADH autofluorescence was monitored in WT midbrain neurons by confocal microscopy. Addition of the uncoupler FCCP (1 μM) maximises mitochondrial respiration and thereby minimises mitochondrial NADH. NaCN (1 mM) was then added to block mitochondrial respiration and therefore maximise mitochondrial NADH. Redox index (the initial redox level expressed as a percentage of the range) and mitochondrial NADH level (the difference in absolute values between the minimum and maximum NADH autofluorescence) are described graphically. Data are represented as the mean of several cells on a single coverslip±S.E.M. (b) A representative trace is shown for a single HtrA2-KO midbrain neuron, demonstrating the smaller response to FCCP and larger response to NaCN in this cell compared with the WT (Figure 2a). (c) NADH redox index was calculated for a variety of primary cell types by calculating the initial NADH autofluorescence when the minimum NADH autofluorescence is normalised to 0% and the maximum to 100%. NADH redox index was reduced in all HtrA2-KO cell types but particularly in midbrain neurons. Data are represented as mean±S.E.M. (d) Mitochondrial NADH level was calculated as the difference in arbitrary units between the maximum and minimum NADH autofluorescence. Data were normalised to midbrain astrocytes (100%) and represented as mean±S.E.M. (e and f) FAD autofluorescence was monitored by confocal microscopy in WT (e) and HtrA2 KO (f) midbrain neurons. Addition of FCCP (1 μM) maximised respiration and therefore increased FAD autofluorescence to maximal levels. Addition of NaCN (1 mM) then inhibited respiration and reduced FAD autofluorescence to a minimum. Data shown are representative traces from single midbrain neurons. (g) FAD redox index was calculated for a range of primary cell types by normalising the FCCP response to 100% and the NaCN response to 0%. Data are represented as mean±S.E.M. In all cases, * indicates P<0.05 and ** indicates P<0.01 compared with WT values

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