Volume 141, Issue 5, Pages 859-871 (May 2010) Caspase-3 Activation via Mitochondria Is Required for Long-Term Depression and AMPA Receptor Internalization  Zheng Li, Jihoon Jo, Jie-Min Jia, Shih-Ching Lo, Daniel J. Whitcomb, Song Jiao, Kwangwook Cho, Morgan Sheng  Cell  Volume 141, Issue 5, Pages 859-871 (May 2010) DOI: 10.1016/j.cell.2010.03.053 Copyright © 2010 Elsevier Inc. Terms and Conditions

Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Caspase Inhibitors DEVD- and LEHD-FMK Block LTD in CA1 Hippocampal Neurons Acute hippocampal slices (from 3- to 4-week-old rats) were incubated with caspase inhibitors for 2 hr before recording (DEVD-FMK, 2 μM; LEHD-FMK, 2 μM; FA-FMK, 10 μM; YVAD-FMK, 5 μM). Extracellular field EPSPs were evoked by stimulating Schaffer collateral CA1 synapses. (A and B) Effect of DEVD-FMK on LTD and LTP, induced by low frequency stimulation (LFS) and tetanic stimulation (Tet) respectively (control, n = 6; DEVD-FMK, n = 6). (C and D) Effect of LEHD-FMK on LTD and LTP (n = 6). (E and F) Effect of YVAD-FMK or negative control peptide FA-FMK on LTD and LTP (n = 6). Pooled data and example traces are shown in all panels. Symbols and error bars indicate mean ± SEM. See also Figure S1. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Overexpression of Bcl-xL and XIAP Constructs Blocks LTD in CA1 Neurons of Organotypic Hippocampal Slice Cultures CA1 neurons were biolistically transfected with Bcl-xL, XIAP-Bir1,2, XIAP-Bir3, or CrmA (with GFP cotransfected as a visual marker) and recorded in whole-cell patch-clamp mode. (A–C) Pairwise analysis of the effects of Bcl-xL (11 pairs of transfected and neighboring untransfected cells), XIAP-Bir1,2 (11 pairs) or CrmA transfection (10 pairs) on basal AMPA-EPSC amplitude (left graph, recorded at a holding potential of −70mV) and NMDA-EPSC amplitude (right graph, recorded at a holding potential of +40mV) versus nearby untransfected cells (pairs of transfected and neighboring untransfected cells are individually plotted; black circles). Red symbol and error bars indicate mean ± SEM. Bcl-xL and XIAP caused an increased basal EPSCAMPA (rightward shift on graph) but had no effect on the EPSCNMDA, in comparison with neighboring untransfected cells. (D) Induction of LTD was blocked in CA1 cells overexpressing XIAP-Bir1,2 (p > 0.05, n = 7), and Bcl-xL (p > 0.05, n = 8). LTD was intact in nontransfected CA1 neurons from organotypic hippocampal culture (p < 0.01, n = 6). (E) LTD induction was blocked by overexpression of XIAP-Bir3 (n = 8) but not CrmA (n = 8). (F) LTP was unaffected by overexpression of Bcl-xL (n = 7), XIAP-Bir1,2 (n = 7) or XIAP-Bir3 proteins (n = 7). Graphs show the mean ± SEM. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 LTD Is Abolished in Hippocampal Slices from Caspase-3 Knockout Mice (A) Input-output relationship in caspase-3−/− and wild-type mice (2–4 weeks age). fEPSP slope was plotted against amplitude of presynaptic volley at four different stimulus intensities (10 μA, 20 μA, 50 μA and 100 μA). Graph indicates mean ± SEM (p > 0.05, n = 16). (B) Low frequency stimulation (LFS, 1 Hz, 15 min) failed to induce LTD in caspase-3−/− slices (p < 0.01, n = 6). (C) Magnitude of LTP induced by tetanic stimulation (Tet) was not different between caspase-3 −/− and wild-type slices (p > 0.05, n = 6). See also Figure S2. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Caspase Inhibitors DEVD- and LEHD-FMK Block NMDA-Induced AMPA Receptor Internalization Antibody-feeding internalization assay for endogenous GluR2 (A) and GluR1 (B) in hippocampal neurons stimulated with NMDA (50 μM for 10 min). Neurons were pretreated with various caspase inhibitors (5 μM) as indicated. (A) and (B) show double-label immunostaining for internalized GluR (green, first row) and surface-remaining GluR (red, second row), and merge (in color, third row). Individual channels are shown in grayscale. Quantitation in (C) shows internalization index (integrated fluorescence intensity of internalized GluR / integrated fluorescence intensity of internalized GluR plus surface GluR) normalized to unstimulated untreated cells (Lee et al., 2002). For all quantitations, n = 15 neurons for each group. ∗p < 0.05, ∗∗∗p < 0.001, compared to NMDA treated cells transfected with β-gal. The graph shows the mean ± SEM. The scale bar represents 20 μm. See also Figure S3. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Antiapoptotic Proteins Inhibit AMPA Receptor Internalization Cultured hippocampal neurons (DIV14) were transfected with Bcl-xL, XIAP-Bir1,2, XIAP-Bir3, or CrmA, or control β-gal. At 2–4 days after transfection, neurons were treated with NMDA as indicated, and GluR2 (A) and GluR1 internalization (B) was measured by the antibody-feeding internalization assay, as in Figure 4. (A) and (B) show immunostaining for the transfected protein (upper row), internalized GluR (second row), surface-remaining GluR (third row), and merge of internalized and surface GluR (bottom row). All individual channels of this triple labeling experiment are shown in grayscale. Arrowheads mark the cell body of transfected cells. Note that the cell body of a Bcl-xL transfected neuron in (A) lies adjacent to an untransfected neuron. Histograms show internalization index for GluR normalized to unstimulated control-transfected neurons (C). n = 15-30 neurons for each group. ∗∗∗p < 0.001 (compared to NMDA stimulated β-gal control). The graph shows mean ± SEM. The scale bar represents 20 μm. See also Figure S4. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Transient Activation of Caspase-3 in Neurons by NMDA (A, D, and E) Immunoblots showing cleaved (active) caspase-3 and caspase-9, and cytosolic cytochrome c in cortical neuronal cultures (DIV18) at various times after treatment with NMDA (50 μM for 10 min) or staurosporine (STS, 1 μM throughout the experimental period). In (D), neurons were preincubated as indicated with 2-APB (30 μM), dantrolene (100 μM), DL-APV (100 μM) or EGTA (5 mM) for 15 min before NMDA treatment, and were immunoblotted 30 min after NMDA stimulation for active (cleaved) and total caspase-3,-9; and cytochrome c in the cytosolic and pellet fractions. (B, C, and F) Quantitation (mean ± SEM) of immunoblot band intensities normalized to values before treatment (NMDA was applied from −10 to 0 min; STS treatment began at 0 min); n = 3 for each time point. See also Figure S5. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Overexpression of Caspase-3-Resistant Triple Mutant of Akt1 Blocks LTD in CA1 Neurons of Organotypic Hippocampal Slice Cultures (A) Cleavage of wild-type or mutant Akt1 proteins by caspase-3 in vitro. In vitro translated WT Akt1, D108A/D119A/D462N triple mutant or Quadruple mutant were incubated with increasing amounts of recombinant active caspase-3 (10, 20, and 40 ng) at 37°C for 18 hr. The caspase-3 inhibitor DEVD-fmk was either omitted or included in the reactions containing 40 ng caspase-3. Position of full-length Akt1 is indicated. (B) Pairwise analysis of wild-type Akt1 (Akt1) transfected cells and nearby untransfected cells on basal AMPA receptor-mediated EPSCs (left panel, EPSCAMPA, recorded at a holding potential of −70mV) and NMDA receptor-mediated EPSCs (right panel, EPSCNMDA, recorded at +40mV). Red symbols show mean ± SEM. (C) Overexpression of WT Akt1 had no effect on LTD compared to neighboring untransfected cells (p < 0.01, n = 6). (D) Pairwise analysis of Akt1triple mutant (D108A/D119A/D462N)-transfected cells and nearby untransfected cells on basal AMPA receptor-mediated EPSCs and NMDA receptor-mediated EPSCs. (E) Induction of LTD was blocked in Akt1 triple mutant transfected cells (p > 0.05, n = 6) but LTD was intact in neighboring untransfected cells. (F) LTP was unaffected by overexpression of Akt1 triple mutant. Graphs show the mean ± SEM. See also Figure S6. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S1 Related to Figure 1, Caspase-1 Inhibitor WEHD-FMK and Calpain Inhibitor LLY-FMK No Not Inhibit LTD, and Lack of Effect of DEVD-FMK on Basal Synaptic Transmission (A–D) For extracellular field potential recordings, acute hippocampal slices were incubated with 200 nM WEHD-FMK or 5 μM LLY-FMK, as in Figure 1. (A and B) WEHD-FMK had no effect on LTD (75 ± 3%, n = 7) or LTP (130 ± 4, p < 0.001, n = 5). (C) Calpain-selective inhibitor LLY-FMK had no effect on LTD (77 ± 4%, n = 6). (D) 2 hr preincubation with 5 μM LLY blocked LTP (106 ± 8%, p > 0.05, comparison between control input and tetanus-stimulated input at 80 min point, n = 6). Filled circles indicate test-input and open circles indicate control input. (E) DEVD-FMK (2 μM) had no effect on basal field EPSPs in acute hippocampal slices during 2 hr incubation. Both Schaffer collateral-CA1 (solid circles) and subiculum-CA1 inputs (open circles) were measured (97 ± 2% of baseline in Schaffer collateral input, p > 0.05, comparison between 20 min and 140 min, n = 6; 96 ± 3% baseline in subiculum-CA1 input, p > 0.05, comparison between 20 min and 140 min, n = 6). Error bars indicate the mean ± SEM. LFS, low frequency stimulation; Tet, tetanic stimulation. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S2 Related to Figure 3. Expression of NMDA Receptors and AMPA Receptors Is Not Altered in the Hippocampus of Caspase-3 Knockout Mice Cell lysates were prepared from hippocampi of wild-type and caspse-3 knockout mice (P17) and analyzed by immunoblotting for the indicated proteins. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S3 Related to Figure 4, Caspase Inhibitors Do Not Affect Basal Internalization of GluR2 Antibody-feeding internalization assay for endogenous GluR2 in cultured hippocampal neurons. (A) Neurons were pretreated with various caspase inhibitors (5 μM), as indicated. Internalized GluR2 (first row), and surface remaining GluR2 (second row) were measured in the absence of NMDA stimulation. (B) Quantitation of data from (A). n = 15 neurons for each group. The scale bar represents 20 μm. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S4 Related to Figure 5, Antiapoptotic Proteins Do Not Affect Basal Internalization and Surface Expression of GluR2, and Loss of NMDA-Induced GluR2 Internalization in Caspse-3 Knockout Hippocampal Neurons (A–C) Cultured hippocampal neurons (DIV14) were transfected with Bcl-xL, XIAP-Bir1,2, XIAP-Bir3, CrmA, or β-gal. At 2–3 days after transfection, neurons were either double-labeled for internalized GluR2 (second row), and surface remaining GluR2 (third row) in the absence of NMDA stimulation (internalization index (integrated fluorescence intensity of internalized GluR / integrated fluorescence intensity of internalized GluR plus surface GluR) normalized to β-gal transfected cells quantified in [A]) or stained for surface GluR2 ([C], quantified in [B]). n = 15 neurons for each group in (A) and (B). (D and E) Cultured hippocampal neurons (DIV17) prepared from caspase-3 knockout mice or wild-type littermates were double-labeled for internalized GluR2 and surface remaining GluR2 after NMDA treatment (50 μM, 10 min). Internalization index is quantified in (E). n = 15 neurons for each group. (F) Expression of caspase-7 in the hippocampus, cortex and liver detected by immunoblotting. The scale bar represents 20 μm. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S5 Related to Figure 6, NMDA-induced activation of Caspase-3 in Neurons Measured by DEVD-Labeling and Immunocytochemistry Active caspase-3 in neurons was detected in situ with biotin-DEVD indirect fluorescence labeling (A and C) or with an antibody against active caspase-3 (E). Neurons were unstimulated, or treated with 10 min NMDA (cells fixed at 30 min after stimulation), as indicated. Untransfected neurons were stained for MAP2 ([A] left panels, red). Neurons transfected with Bcl-xL or XIAP-Bir3 were identified by immunostaining for the transfected protein ([C] left panels, red; the beaded pattern in dendrites reflects the subcellular distribution of Bcl-xL and XIAP-Bir3). The same cells were double-labeled for active caspase-3 (middle panels, green). Individual channels are shown in grayscale, merge in color. High magnification view of a single dendrite segment is shown at bottom of each panel. Scale bars, 20 μm (low magnification), 5 μm (high magnification). (B) Time course of caspase-3 activation in dendrites in response to NMDA treatment for 10 min, as measured by in situ biotin-DEVD labeling (integrated intensity of fluorescence per μm of dendrite), normalized to unstimulated cells. (D) Biotin-DEVD labeling for active caspase-3 in neurons transfected with indicated constructs and stimulated with NMDA (cells were fixed at 30 min after stimulation), normalized to unstimulated β-gal-transfected cells. n = 15 neurons for each time-point or group in (B) and (D). ∗∗∗p < 0.001. Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S6 Related to Figure 7, Identification of a Caspase-3-Resistant Kinase-Active Akt1 Mutant (A) Domain structure of human Akt1, showing locations of the four putative caspase-3 cleavage sites that were mutated individually or in combination. In addition to the aspartate substitutions, we constructed a mutant lacking the C-terminal region (amino acids 463–480). (B) Expression of the wild-type (WT) or mutant Akt1 proteins (as indicated) following transient transfection in HeLa cells, analyzed by immunoblotting with antibodies that recognize the N-terminus (N19) or C terminus (5G3) of Akt1, or phosphoantibodies against Akt1 phosphorylated at Thr-308 or Ser-473. The asterisk indicates an endogenous protein that cross-reacts with the antibodies. (C) Proteolysis of WT or mutant Akt1 proteins in apoptotic HeLa cells. HeLa cells were transfected with WT or mutant Akt1, as indicated, then incubated with 50 μg/ml cycloheximide for 4.5 hr before lysis. In addition, the cells were either left without further treatment, or treated with 2 μM staurosporine (STS) for 2 hr or 4 hr before lysis. (D) Quantification of data from (C). Histograms show mean ± SEM (n = 3). Statistical analysis was performed by t test. ∗p < 0.05, ∗∗p < 0.01, compared to the wild-type. (E) Cleavage of WT Akt1 and Akt1 mutants (1-462), D108A/D119A, D434A, D462N, and D108A/D119A/D434A by caspase-3 (40 ng). (F) Kinase activity of the WT and mutant Akt1 proteins in vitro. FLAG-tagged Akt1 proteins were transiently expressed in HeLa cells. In vitro kinase assay was performed as described in Extended Experimental Procedures. (G and H) Levels of endogenous Akt1 protein upon NMDA stimulation. Cultured rat hippocampal neurons at DIV16 were either untreated, or stimulated with 75 μM NMDA for indicated times. Bar graph shows mean ± SEM of Akt1 immunoblotting band intensity normalized to untreated samples (n = 4). Cell 2010 141, 859-871DOI: (10.1016/j.cell.2010.03.053) Copyright © 2010 Elsevier Inc. Terms and Conditions