Identification of Bax-interacting proteins in oligodendrocyte progenitors during glutamate excitotoxicity and perinatal hypoxia-ischemia

Abstract

OPC (oligodendrocyte progenitor cell) death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H-I (hypoxia-ischemia) in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL-Bax association in white matter following H-I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax-cofilin association was disrupted during glutamate-induced OPC apoptosis.

Figure 1. Bax movements in healthy cells and during apoptosis

In healthy cells, inactive Bax continuously cycles between mitochondria and the cytosol. Bax retrotranslocation requires interaction with an anti-apoptotic protein (Bcl-xL, Bcl-2, or Mcl1). Together, these two proteins leave the mitochondrial outer membrane (OM). Once in the cytosol, the complex immediately dissociates. The retrotranslocation process is stimulated by the anti-apoptotic proteins Bcl-xL, Bcl-2, or Mcl1 and is inhibited by vMIA, ABT-737, and BH3-only proteins. Upon induction of apoptosis, Bax is directly stimulated by activating BH3-only proteins (e.g., Bid, Bim, or Puma; blue arrow) to expose its C-terminal domain and insert in the mitochondrial OM. During this process, Bax exposes a novel N-terminal epitope (6A7), triggering the formation of foci and release of cytochrome c. Neutralizing BH3-only proteins (or small molecule inhibitors; green rectangle) can indirectly activate Bax by binding and inactivating antiapoptotic proteins. Consequently, Bax accumulates on the mitochondrial OM, where it acquires its active conformation. Figure and legend reprinted from, Cell 145(1), M.E. Soriano and L. Scorrano, Traveling Bax and Forth from Mitochondria to Control Apoptosis, Pages 15–17, (2011), with permission from Elsevier.

Figure 2. Detection of active Bax in OPCs exposed to glutamate

Late OPCs were treated with glutamate (500 μM)±IGF-I (20 ng/ml) for 24 h. (A) Immunostaining for conformation-specific Bax (6A7; upper panels) or total Bax (N20; lower panels). Bax was detected using an AF488-conjugated secondary antibody (green); DAPI-stained nuclei are shown as blue. (B) Active Bax was detected by immunoprecipitation from glutamate-treated OPCs using conformation-specific Bax antibody (6A7) followed by Western blotting with total Bax N20 antibody (21 kDa). I, IGF-I; G, glutamate; I+G, IGF-I plus glutamate. Data are representative of two independent experiments at 24 h. Similar results were obtained at 18 h. Line in gel in (B) indicates that a blank lane was removed from the gel for publication purposes.

Figure 3. Bax associates with mitochondria by 18 h in OPCs exposed to glutamate

Late OPCs were treated with glutamate (500 μM)±IGF-I (20 ng/ml) for 18 h. (A and B) Quantification of cells with Bax-CoxIV co-localization using the Amnis ImageStream System. (A) Shift of peak to the right vs controls indicates increased co-localization in glutamate-treated OPCs. (B) Images show examples of Bax (N20)-CoxIV co-localized after glutamate stimulation (Glut) and reduced co-localization upon addition of IGF-I (IGF-I). (C) Histogram showing fold increase of Bax association with CoxIV in two independent experiments. For each experiment, an average of 1500 individual cells were evaluated for each condition. An increase in Bax association with CoxIV was also observed at 24 h.

Figure 4. Bcl-2 family regulation in OPCs during glutamate excitotoxicity

(A) Mitochondrial (M) and cytoplasmic (C) fractions were analyzed for Bax, Bid and Bcl-xL in OPCs treated with glutamate (500 μM) or IGF-I (20 ng/ml) for 24 h. The mitochondrial protein VDAC was used to confirm fractionation. (B and C) Isolated protein was used to immunoprecipitate total Bax (N20) or Bid from OPCs treated for 24 h with glutamate (G), IGF-I (I) or IGF-I+glutamate (I+G). Bid and tBid were detected by Western immunoblotting (B). Bcl-xL was seen associated with Bax and Bid in IGF-I-treated OPCs and reduced in glutamate-treated OPCs (C). Subcellular fractionation data are representative of two independent experiments. IPs are representative of two independent experiments at 24 h; similar results were obtained at 18 h for detection of Bcl-xL associated with Bax or Bid.

Figure 5. Identification of cofilin as a Bax-binding partner in IGF-I-treated OPCs

(A) Bax was immunoprecipitated from OPCs treated for 24 h with IGF-I. The protein band corresponding to ~20 kDa was excised from the gel and subjected to ESI–LC–MS/MS analysis. Scaffold-generated MS/MS spectrum of a doubly charged ion corresponds to Y⁸²ALYDATYETK⁹² peptide from cofilin-2 (NCBI Reference Sequence NP_001102452.2). The observed y- and b-ion series confirmed the peptide sequence. (B) Isolated protein from OPCs treated for 24 h with glutamate or IGF-I was used to immunoprecipitate total Bax (N20). Cofilin was detected by Western immunoblotting in IGF-I-treated OPCs. Association of cofilin with Bax was barely detectable in OPCs treated with glutamate at 24 h. Data are representative of two experiments; similar results were obtained at 18 h. (C) Analysis of cofilin in mitochondrial (M) and cytoplasmic (C) fractions 24 h following exposure of OPCs to glutamate or IGF-I. (D) Detection of p-cofilin (Ser⁹) in OPCs treated with glutamate (G) but not in OPCs treated with IGF-I (I) at 18 h and 24 h. (E) Immunodetection of Bax (N20; AF488-green) and cofilin (AF546-red) in OPCs treated with either glutamate (Glut) or IGF-I (IGF). DAPI (blue) was used to detect nuclei. Data are representative of at least two independent experiments.

Figure 6. Bcl-2 family regulation in immature white matter after H–I

H–I was produced in P6 Wistar rat pups by cauterizing the common carotid artery followed by systemic hypoxia for 75 min. Subcortical white matter (WM) was dissected at 24 h recovery from ipsilateral (IL), contralateral (CL) and Sham (Sh) operated brains and used for protein isolation (n=6 pooled samples). (A) Western blot of MAP-2 expression in cortex vs WM dissected regions. (B) Active caspase 3 increased in IL white matter (bottom panel). (B-C) Isolated protein was used to immunoprecipitate total Bax (N20). Bax-associated Bid (B) and Bcl-xL (C) were detected by Western immunoblotting. The data for the Bax–BclxL association are representative of three independent experiments and the data for the Bax–Bid association are from two independent experiments. (D) Detection of pBad(Ser¹³⁶) and total Bad by Western immunoblotting of protein from IL, CL and Sh white matter. Histogram shows ratio of p-Bad/total Bad in IL vs CL white matter. Values represent averages±S.D. from three independent experiments; P =  0.08).

Figure 7. Model of Bax regulation in OPCs

Schematic diagrams showing Bax binding partners, their associations and their locations in OPCs under survival conditions (A) or under apoptotic conditions following exposure to excess glutamate (B). Models are based predominantly on data obtained from the in vitro studies reported in this paper. Bax shuttling from mitochondria to cytoplasm by Bcl-xL is supported by published models (see Figure 1).