Metalloproteinase Shedding of Fas Ligand Regulates β-Amyloid Neurotoxicity  Douglas W Ethell, Ross Kinloch, Douglas R Green  Current Biology  Volume 12, Issue 18, Pages 1595-1600 (September 2002) DOI: 10.1016/S0960-9822(02)01140-5

Figure 1 Fas Signaling Is Required for Aβ Neurotoxicity Cerebellar neuron cultures were treated for 5 days, fixed, and stained with Hoecsht 33258, and the nuclei were visualized with an inverted fluorescence video-microscope at 40×. (A) Control cultures show many large round nuclei, scored as viable cells. The scale bar represents 10 μM in all panels. (B) Treatment with 100 μg/ml Aβ1–40 for 5 days decreased the number of viable nuclei and increased the number of condensed, fragmented, and bright nuclei, which were scored as apoptotic. Small individual fragments were considered debris and were not used in the calculations. (C) Inclusion of FasFc (30 μg/ml) protected neurons from Aβ neurotoxicity. (D) Non-specific IgG had no effect on Aβ neurotoxicity. (E) Apoptotic and viable nuclei were scored from three separate images, and the percentages of three wells each in two separate experiments were pooled. Using one-way analysis of variance (ANOVA, with 95% confidence), we found the difference between Aβ and Aβ+FasFc conditions to be statistically significant (indicated by an asterisk). The error bars represent standard errors of the mean. (F) WT and FasL-defective (gld) neurons were treated with 100 μg/ml Aβ1–40 for 3–5 days, and nuclear morphology was scored as described. Using one-way ANOVA (95% confidence), we found the percentage of apoptotic nuclei in the WT 5-day Aβ-treated cultures to be statistically significant (indicated by an asterisk) when compared with WT 5-day control, gld 5-day control, or gld 5-day Aβ-treated cultures. The error bars represent standard errors of the mean. Current Biology 2002 12, 1595-1600DOI: (10.1016/S0960-9822(02)01140-5)

Figure 2 Aβ Treatment Alters FasL Expression (A) An anti-FasL immunoblot of whole-cell lysates from cerebellar neuron cultures treated with 100 μg/ml Aβ1–40 or media control for 2 days shows bands of the expected 31 kDa size for FasL. Blots were reprobed for actin to confirm equal loading. (B) Real-time PCR analysis of the FasL RNA message after 1, 12, 24, and 48 hr of Aβ treatment. Current Biology 2002 12, 1595-1600DOI: (10.1016/S0960-9822(02)01140-5)

Figure 3 Synergistic Effects of GM6001 on Aβ Neurotoxicity (A) Primary neurons were treated for 1–4 days with control media (open circle) or media containing Aβ1–40 (100 μg/ml; filled circle), GM6001 (22 μM; open triangle), or Aβ+GM6001 (filled triangle). Percentages of apoptotic nuclei were calculated from DAPI-stained cultures and were plotted. (B) An immunoblot of a whole-cell lysate from primary neurons treated for 2 days with control media, Aβ1–40 (100 μg/ml), Aβ+GM6001 (22 μg/ml), or Aβ+MMP-7 (1.5 U/ml) and probed with anti-FasL shows bands at the expected 31 kDa. A reprobe of the blot with an actin-specific antibody is shown below. Relative FasL expression was calculated from scans of the films with actin to normalize all lanes. (C) The effects of GM6001+Aβ1–40 were compared with WT and gld neuron cultures after 5 days. The percentage of apoptotic nuclei scored show the importance of FasL to this synergy. Using one-way ANOVA (95% confidence), we found a statistically significant (indicated by an asterisk) difference between Aβ+GM6001-treated WT and gld cultures. (D) The synergistic effects of GM6001+Aβ can be blocked by interfering with Fas-FasL engagement by using FasFc. The percentages of apoptotic nuclei after 3-day treatments are shown. Using one-way ANOVA (95% confidence), we determined that there is a statistically significant (indicated by an asterisk) difference between Aβ+GM6001 and Aβ+GM6001+FasFc conditions, but no significant difference between Aβ+GM6001 and Aβ+GM6001+IgG conditions. The error bars represent standard errors of the mean. (E) Real-time PCR analysis of TIMP RNA levels in neurons treated with Aβ1–40 (100 μg/ml) for 0, 24, or 48 hr. (F) Lack of synergistic effects between TIMPs and Aβ neurotoxicity. The percentages of apoptotic nuclei after 3-day treatments with Aβ1–40 (100 μg/ml) and TIMP-1 (500 ng/ml), TIMP-2 (500 ng/ml), or TIMP-3 (500 ng/ml) were compared to Aβ and GM6001 (25 μM). Using one-way ANOVA (95% confidence), we found statistically significant differences (indicated by an asterisk) between the percentage of apoptotic nuclei in Aβ+GM6001 treatment versus Aβ+TIMP-1, Aβ+TIMP-2, or Aβ+TIMP-3. The error bars represent standard errors of the mean. Current Biology 2002 12, 1595-1600DOI: (10.1016/S0960-9822(02)01140-5)

Figure 4 MMP-7 Protects Neurons from Aβ Neurotoxicity Paired Hoffman-optics and UV excitation images of neurons show cellular and DAPI-stained nuclear morphology at 20×. (A and B) Cultures treated for 5 days with control media showed very little background apoptosis. The scale bar represents 10 μM in all panels. (C and D) Aβ1–40 (100 μg/ml)-treated cultures showed substantial apoptosis. (E and F) The inclusion of recombinant human MMP-7 (1.5 U/ml) eliminated any detectable Aβ-mediated apoptosis. (G and H) Conversely, the inclusion of GM6001 (22 μM) exacerbated Aβ neurotoxicity, as almost every nucleus appeared apoptotic. Current Biology 2002 12, 1595-1600DOI: (10.1016/S0960-9822(02)01140-5)

Figure 5 Complementary Effects of Changing Metalloproteinase Activity on Aβ Neurotoxicity and sFasL Release (A) The percentages of apoptotic and nonapoptotic nuclei from six images in three wells were calculated. Repeated experiments gave similar results. Using one-way ANOVA, we found a statistically significant (indicated by an asterisk) difference between cultures treated with Aβ1−40 (100 μg/ml) alone and Aβ+MMP-7 (1.5 U/ml). (B) An anti-FasL immunoblot of concentrated media after 5-day treatments is shown in the top panel. High amounts of 26-kDa sFasL from samples containing recombinant MMP-7 show the extent of shedding that accompanied total rescue from Aβ neurotoxicity. Note that more sFasL was detected in the Aβ+MMP-7 cultures versus MMP-7 alone, due to the increased FasL expression triggered by Aβ. The Ponceau-stained blot prior to blocking is shown in the bottom panel. Current Biology 2002 12, 1595-1600DOI: (10.1016/S0960-9822(02)01140-5)