Bim links ER stress and apoptosis in cells expressing mutant SOD1 associated with amyotrophic lateral sclerosis

Abstract

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.

Figure 1. Frequencies of SOD1-inclusions and nuclear morphology changes after Bim knockdown.

(A) Neuro2a cells were transfected with siRNA targeting Bim (siBim) or with non-specific control RNA (siC). After 24 h transfection of siRNA, cells were transfected with WT or SOD1 A4V-EGFP vectors for a further 48 h, or not further transfected (Untr). Cell lysates were subjected to Western immunoblotting and tested for binding of anti-Bim antibody and anti-GFP antibody. Western blot with β-actin antibody was used for control of equivalent protein loading. (B) Protein content of Bim corresponding to bands in the Western blots of panel A was quantified, first normalized to β-actin band intensity for the relevant lane, and then expressed as percentage of Bim measured in cells expressing control siC. (C) Quantification of prevalence of SOD1-inclusions. In each replicate experiment, 200 cells were scored for each population. (D) Quantified data for cells with apoptotic nuclei. For the populations containing untransfected cells (Untr) or cells expressing dispersed SOD1 (either WT or A4V), 200 cells were scored in each replicate experiment. For inclusion-positive cells (incl), more than 60 cells were scored in each of three replicate experiments. For panels B, C and D, results are expressed as mean ± SD, n = 3. * p<0.05; ** p<0.0001.

Figure 2. Impact on Bim depletion on mobilization of CHOP to nucleus in cells.

Cells were initially transfected with Bim siRNA (siBim) or non-specific control RNA (siC) as indicated, and secondarily transfected with different forms of SOD1-EGFP or not (Untr). (A) First column shows EGFP fluorescence in green channel. Cells were fixed and tested for immunoreactivity with antibodies recognizing CHOP (second column), and also stained with DAPI (third column). Merge (fourth column) indicates overlays of the confocal images of EGFP fluorescence, CHOP and DAPI. DIC images are shown in fifth column. Scale bar, 10 µm, applies to all fields. For cells transfected with mSOD1 A4V, fields displaying inclusion-positive cells (incl) are shown. White arrow indicates apoptotic nucleus; in this case DAPI staining is very weak, characteristic of many apoptotic nuclei in cells bearing inclusions, as reported elsewhere . (B) Quantified data for cells displaying CHOP mobilization to nucleus and/or apoptotic nuclei. Cells were classified as having CHOP either predominantly in cytoplasm or in nucleus. Numbers of cells scored for populations with dispersed SOD1 (disp) or those with inclusion-positive cells (incl), and expression of results are exactly as for Figure 1. * p<0.0001.

Figure 3. Impact of Bim depletion on co-localization of ATF6 and mSOD1 inclusions in cells.

Cells were initially transfected with Bim siRNA (siBim) or non-specific control RNA (siC) as indicated, and secondarily transfected with different forms of SOD1-EGFP or not (Untr). (A) First column shows EGFP fluorescence in green channel. Cells were fixed and tested for immunoreactivity with antibodies recognizing ATF6 (second column), and also stained with DAPI (third column). Merge (fourth column) indicates overlays of the confocal images of EGFP fluorescence, ATF6 and DAPI. DIC images are shown in fifth column. Scale bar, 10 µm, applies to all fields. For cells transfected with mSOD1 A4V, fields displaying inclusion-positive cells (incl) are shown. White arrow indicates apoptotic nucleus, in this case highly condensed and fragmented revealed by DAPI staining. (B) Quantified data for cells displaying co-localization of ATF6 and mSOD1 inclusions and/or apoptotic nuclei. Numbers of cells scored for populations with dispersed SOD1 (disp) or those with inclusion-positive cells (incl), and expression of results are exactly as for Figure 1. * p<0.001; ** p<0.0001.

Figure 4. Impact of Bim depletion on recruitment of Bax to mitochondria in cells.

Cells were initially transfected with Bim siRNA (siBim) or non-specific control RNA (siC) as indicated, and secondarily transfected with different forms of SOD1-EGFP or not (Untr). (A) First column shows EGFP fluorescence in green channel. Cells were fixed and tested for immunoreactivity with antibodies recognizing Bax (second column), and also stained with DAPI (third column). Merge (fourth column) indicates overlays of the confocal images of EGFP fluorescence, Bax and DAPI. DIC images are shown in fifth column. Scale bar, 10 µm, applies to all fields. For cells transfected with mSOD1 A4V, fields displaying inclusion-positive cells (incl) are shown. White arrow indicates apoptotic nucleus, with little DAPI staining (cf. Figure 2A). (B) Quantified data for cells displaying Bax recruitment and/or apoptotic nuclei. Numbers of cells scored for populations with dispersed SOD1 (disp) or those with inclusion-positive cells (incl), and expression of results are exactly as for Figure 1. * p<0.001; ** p<0.0001.

Figure 5. Impact of Bim depletion on redistribution of cyt c from mitochondria in cells.

Cells were initially transfected with Bim siRNA (siBim) or non-specific control RNA (siC) as indicated, and secondarily transfected with different forms of SOD1-EGFP or not (Untr). (A) First column shows EGFP fluorescence in green channel. Cells were fixed and tested for immunoreactivity with antibodies recognizing cyt c (second column), and also stained with DAPI (third column). Merge (fourth column) indicates overlays of the confocal images of EGFP fluorescence, cyt c and DAPI. DIC images are shown in fifth column. Scale bar, 10 µm, applies to all fields. For cells transfected with mSOD1 A4V, fields displaying inclusion-positive cells (incl) are shown. White arrows indicates apoptotic nucleus, in this case highly condensed and fragmented revealed by DAPI staining. (B) Quantified data for cells displaying cyt c redistribution and/or apoptotic nuclei. Numbers of cells scored for populations with dispersed SOD1 (disp) or those with inclusion-positive cells (incl), and expression of results are exactly as for Figure 1. * p<0.001; ** p<0.0001.

Figure 6. Impact of CHOP depletion on cyt c redistribution from mitochondria and caspase-3 cleavage in cells.

(A) Neuro2a cells were transfected with siRNA targeting CHOP (siCHOP) or with non-specific control RNA (siC). After 24 h transfection of siRNA, cells were transfected with WT or SOD1 A4V-EGFP vectors for a further 48 h, or not further transfected (Untr). Cell lysates were subjected to Western immunoblotting and tested for binding of anti-CHOP antibody and anti-GFP antibody. Western blot with β-actin antibody was used for control of equivalent protein loading. (B) Protein content of CHOP corresponding to bands in the Western blots of panel A was quantified, first normalized to β-actin band intensity for the relevant lane, and then expressed as percentage of CHOP measured in cells expressing control siC. (C) Quantified data for cells displaying cyt c redistribution and/or apoptotic nuclei. (C) Quantified data for cells displaying cleaved caspase-3. Numbers of cells scored for populations with dispersed SOD1 (disp) or those with inclusion-positive cells (incl), and expressing of results are exactly as for Figure 1. * p<0.05; ** p<0.001; *** p<0.0001.

Figure 7. Inter-relationship between cellular responses associated with the formation of mSOD1 inclusions, ER stress and apoptosis.

The primary response is thought to be elicited by the accumulation of a toxic species derived from mSOD1. One consequence is the formation of mSOD1 inclusions, via the aggresome. In parallel, ER stress is thought to be invoked if the toxic species are not destroyed by the ubiquitin-proteasome system, resulting in upregulation of CHOP. As demonstrated here the BH3-only protein, Bim, links ER stress to apoptosis. Specifically, this involves activation of apoptotic signalling pathways that sequentially include Bax recruitment to mitochondria, cyt c redistribution, caspase-3 activation and apoptosis.