Figure 5.
Calpain I activation contributes to OGD-induced AIF release in neurons. A, Left, Neurons were transfected with empty plasmid (lane 1), calpain I scramble shRNA sequence (Calp-Is; lanes 2, 3), or calpain I targeting shRNA sequence (Calp-It) for 12 d and then immunoblotted against calpain I. **p < 0.01 versus plasmid controls; n = 3. Right, Neurons were transfected with GFP expression plasmid alone, both GFP and Calp-Is, or both GFP and Calp-It for 12 d. Transfected neurons were collected by GFP cell sorting and then immunoblotted against calpain I and GFP. ***p < 0.001 versus GFP alone; n = 3. B, Transfected neurons were subjected to OGD for 60 min, and, at 6 h after OGD, nuclear (Nuc), mitochondrial (Mito), or total-cell extracts were immunoblotted against AIF. The graph at the bottom illustrates the semiquantitative results of nuclear AIF from three experiments. *p < 0.05 versus non-OGD controls; #p < 0.05 versus empty plasmid-transfected OGD neurons. C, D, Knockdown of calpain I attenuated OGD-induced neuronal cell death. Cell viability and cell death were measured at 24 h after OGD (60 min) using Alamar blue fluorescence (C) and LDH release (D), respectively. Data are mean ± SE; n = 12 per experimental condition from three independent experiments. *p < 0.05 versus empty plasmid-transfected neurons. E, Western blot detects endogenous calpain I in brain mitochondria. Purified brain mitochondria were subfractionated in the presence of digitonin at either 0.4% (outer membrane broken but no membrane protein dissolving) or 1.0% (resulting in membrane protein dissolving) and then immunoblotted against calpain I and AIF and fraction marker proteins COX IV (IM), VDAC (OM), and AK2 (IMS). Note that, under 0.4% digitonin, calpain I is present exclusively in the IMS, whereas AIF is associated with the IM; under 1.0% digitonin, both AIF and VDAC are dissolved from membranes and appear in all fractions, whereas calpain I remains in the IMS. For calpain I and AIF, the recombinant proteins (Rp) served as markers. The blots are representatives of three experiments. F, Translocation of calpain I and AIF within mitochondria after OGD. Neurons were subjected to OGD (60 min); 2 h later, mitochondria were purified and subfractionated under 0.4% digitonin. Note that calpain I is present in IM, IMS, and, to a lesser extent, in OM fractions after OGD; AIF appears in the IMS fraction at the size of 57 kDa after OGD. G, Semiquantitative analysis of calpain I and AIF levels in total or subfractions of mitochondria after OGD, based on three independent experiments described in F. Levels are expressed as fold increase over non-OGD neurons. *p < 0.05, **p < 0.01 versus non-OGD controls. H, Calpain activity measured in isolated whole mitochondria (30 μg protein/reaction) before or 2 h after OGD from neurons transfected with empty plasmid, Calp-Is, or Calp-It. #p < 0.05 versus non-OGD controls (con); *p < 0.05 versus empty plasmid-transfected neurons, from three experiments. I, Calpain activity measured in subfractions (IMS, 30 μg protein/reaction; OM or IM, 20 μg protein/reaction) of isolated mitochondria before or 2 h after OGD. *p < 0.05 versus non-OGD controls, from three experiments. J, Active calpain I is capable of entering mitochondria. Isolated brain mitochondria were incubated for 20 min with either inactive calpain I (1 U) or active calpain I (1 U) [preincubated with calcium (10 μm)], and subfractions (0.4% digitonin) were immunoblotted against calpain I and AIF. Note that calcium-activated calpain I translocates to IM and OM fractions, whereas the IM-bound AIF is truncated (57 kDa) and released to the IMS. The blots are representatives of three experiments.