Photic injury promotes cleavage of p75NTR by TACE and nuclear trafficking of the p75 intracellular domain

www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 36 (2007) 449 – 461

Photic injury promotes cleavage of p75NTR by TACE and nuclear trafficking of the p75 intracellular domain Bhooma Srinivasan,a Zhaohui Wang,a Anne M. Brun-Zinkernagel,a Robert J. Collier,b Roy A. Black,c Stuart J. Frank,d Philip A. Barker,e and Rouel S. Roquea,⁎ a

Department of Cell Biology and Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA Alcon Research, Ltd., Fort Worth, TX 76134, USA c Amgen Inc., Seattle, WA 91320, USA d Departments of Medicine and Cell Biology, University of Alabama at Birmingham and Veterans Affairs Medical Center, Birmingham, AL 35294, USA e Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4 b

Received 9 March 2007; revised 20 July 2007; accepted 6 August 2007 Available online 15 August 2007 The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily that paradoxically mediates neuronal survival and differentiation or apoptotic cell death. Cleavage of p75NTR by a constitutively active metalloprotease could result in shedding of its extracellular domain (p75ECD) and generation of a proapoptotic intracellular domain (p75ICD). In this study, we established that exposure of a transgenic mouse photoreceptor cell line to intense light upregulated the expression of p75NTR and of the disintegrin metalloprotease tumor necrosis factor-converting enzyme (TACE) and resulted in apoptotic cell death. Light damage promoted TACE cleavage of p75NTR resulting in shedding of the soluble p75ECD and nuclear translocation of the p75ICD. Overexpression of TACE and p75NTRinduced p75NTR cleavage and secretion of p75ECD, but not nuclear transport of p75ICD. Light-induced cleavage of p75NTR, nuclear localization of p75ICD, and apoptosis were inhibited by IC-3, a metalloprotease inhibitor. Increased levels of p75NTR and TACE were observed in photoreceptor cells of animals with photic injury. Our findings support a role for TACE in the proteolytic cleavage of p75NTR and light-induced apoptosis. © 2007 Elsevier Inc. All rights reserved. Keywords: p75 neurotrophin receptor; p75ICD; p75NTR; Intracellular domain; Tumor necrosis factor-α converting enzyme; Photic injury; Apoptosis; Photoreceptor cells

Abbreviations: TNFR, Tumor necrosis factor receptor; p75NTR, p75 neurotrophin receptor; p75ECD, p75NTR extracellular domain; p75ICD, p75NTR intracellular domain; TACE, Tumor necrosis factor-converting enzyme; IC-3, Immunex compound-3; PI, Propidium iodide; H-33342, Hoechst 33342. ⁎ Corresponding author. Fax: +1 817 735 2610. E-mail address: [email protected] (R.S. Roque). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2007.08.005

Introduction p75NTR1 is a member of tumor necrosis factor receptor (TNFR) superfamily that includes CD27, CD30, CD40, OX40, Fas/CD95, DR3, DR4, DR5, and TNFR-1 and TNFR-2 (Baker and Reddy, 1996, 1998; Miller and Kaplan, 1998) and can induce cell death both in vitro and in vivo (Barrett and Bartlett, 1994; Casaccia-Bonnefil et al., 1996; Frade et al., 1996; Majdan et al., 1997; Miller and Kaplan, 1998; Frade and Barde, 1999; Coulson et al., 2000). p75NTR contains a cysteine-rich extracellular domain (p75ECD), and an intracellular domain (p75ICD) containing a type II death domain that does not aggregate or self-associate like the Fas/CD95 death domain nor bind other death domain-containing proteins in order to induce cell death (Liepinsh et al., 1997; Barker, 1998; Wang et al., 2001; Roux and Barker, 2002). Instead, studies show that overexpression of p75ICD, which is highly conserved among animal species (Heuer et al., 1990; Liepinsh et al., 1997), induces neuronal cell death within the central and peripheral nervous systems (Frade et al., 1996; Majdan et al., 1997). The full-length p75NTR has been reported to be cleaved by a constitutively active membrane-bound metalloprotease to generate a soluble p75ECD and a membranebound receptor fragment containing the p75ICD (DiStefano and Johnson, 1988; Zupan et al., 1989; Barker et al., 1991; DiStefano et al., 1993). Elevated levels of p75ICD such as those generated by cleavage of full-length p75NTR could thus promote cell death. Tumor necrosis factor-α-converting enzyme (TACE) is a member of A disintegrin and metalloprotease (ADAM) family (Schlondorff and Blobel, 1999) of transmembrane glycoproteins that contain both a disintegrin and a metalloprotease domain (Black and White, 1998). These glycoproteins have been implicated in various cellular processes such as matrix degradation, cell migration, cell–cell interaction, and shedding of

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Fig. 1. Light exposure induces photoreceptor apoptosis. 661 W cells were pulsed with 1400fc of light for 0–5 h and then chased for 20 h in the dark prior to assays. (A) Cells were labeled with 2.0 μM calcein AM (CA) and 4.0 μM ethidium homodimer (EH) for 45 min to test for cell survival. 661 W cells exposed to light for 0–1 h stained intensely for CA (green) but not with EH (red). Light exposure from 2 to 5 h resulted in decreasing amount of staining for CA and increased nuclear staining with EH. (B) 661 W cells were double-stained with 500 nM propidium iodide (PI) and 500 nM Hoechst 33342 for 10 min to test for apoptosis. 661 W cells exposed to light for 2–3 h, exhibited intense nuclear staining for PI (red) while cells exposed for 0–1 h were negative. (C) Ethidium bromide-labeled bands (arrows) in multiples of ∼180 bp were observed in 661 W cells exposed to light for 2–3 h. Genomic DNA from cells exposed to light for 0–1 h barely migrated in the agarose gels. M, molecular sizes.

cytokines and growth factors from membrane-bound precursors (Schlondorff and Blobel, 1999). TACE, which is expressed constitutively in many tissues (Black et al., 1997), is a major proteolytic enzyme for tumor necrosis factor-α (Black et al., 1997; Moss et al., 1997) and also promotes cleavage of a diverse group of transmembrane proteins including transforming growth factor-α (Peschon et al., 1998), L-selectin (Peschon et al., 1998), p75 TNFR (Peschon et al., 1998), growth hormone receptor (Zhang et al., 2000), amyloid precursor protein (APP) (Buxbaum et al., 1998), and prions (Vincent et al., 2001). Cleavage by TACE results in shedding of the soluble extracellular domain of

the affected substrates and, in the case of Notch, APP, and the ErbB-4 receptor, initiates the release and nuclear translocation of their intracellular domains in a process called regulated intramembrane proteolysis (Heldin and Ericsson, 2001). Due to its high homology to TNFR, p75NTR appears to be a suitable substrate for TACE-mediated cleavage that may both lead to shedding of a soluble p75ECD and generation of a pro-apoptotic p75ICD. In the following study, we investigated the effects of intense light exposure on expression of p75NTR and TACE; the ability of TACE to promote cleavage and nuclear translocation of p75ICD; and the role of p75ICD in light-induced apoptosis.

Fig. 2. Increased expression and cleavage of p75NTR in light-exposed photoreceptor cells. (A) 661 W cells were exposed to light for 0–3 h and labeled with antip75ICD antiserum at 1:200 dilution. After 3 h of exposure, cells were rounded and their nuclei (stained with DAPI) much smaller in size suggestive of cell death. Increased staining for p75NTR (green) was observed with increased light exposure and appeared to co-localize with DAPI (blue) in the nuclear areas. (B) Western blot of 661 W lysates using 1:2000 dilutions of the anti-p75ICD or the anti-p75ECD antisera both showed ∼75-kDa bands, while ∼50-kDa bands reacted only with the anti-p75ICD antiserum. β-Tubulin was used to normalize protein loading. (C) Subcellular fractionation followed by Western blots with both antisera showed increased intensity of the ∼75-kDa bands in both membrane and cytosolic fractions, but not in the nuclear extracts. Bands ∼50-kDa reactive for p75ICD, but not p75ECD, were absent in the membrane fractions but appeared in the cytosolic fractions as early as 1 h of light exposure and in the nuclear fractions after 2–3 h light exposure. The purity of the fractions was verified by probing for β-actin (membrane), splicing factor (nuclear), or Akt (cytosolic). Scale bar = 50 μm.

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Results Intense light exposure induces photoreceptor apoptosis A cell culture paradigm of light-induced apoptosis was established using a photoreceptor cell line (661 W) known to undergo photo-oxidative stress-induced apoptosis (Krishnamoorthy et al., 1999; Kanan et al., 2007). To test for photo-oxidative damage, 661 W cells were pulsed with 1400 foot candles (fc) of white light for 0–5 h followed by 20 h chase prior to assays using fluorescent probes calcein AM and ethidium homodimer. Cultures exposed for 0– 1 h stained intensely for calcein AM but did not exhibit nuclear labeling with ethidium homodimer, consistent with metabolically

active cells. Light exposure from 2 to 5 h resulted in gradual decrease of calcein AM labeling and increase in nuclear staining for ethidium homodimer, suggestive of cell death (Fig. 1A). To test for apoptosis, cells were double-stained with propidium iodide (PI) and Hoechst 33342 (H-33342) (Fig. 1B). Although both dyes bind DNA, PI is membrane impermeant and is normally excluded by viable cells unlike H-33342. 661 W cells exposed to light for 2–3 h exhibited graded increase in nuclear staining for PI unlike cells exposed for 0–1 h. The smaller sizes of the H-33342+ and PI+ nuclei of cells exposed for 2–3 h was consistent with nuclear condensation observed during apoptosis. Apoptotic cell death was further confirmed by DNA laddering showing genomic fragmentation in cells exposed to light for 2–3 h, but not in cells exposed for 0–1 h (Fig. 1C).

Fig. 3. Light exposure promotes increased expression of TACE. (A) 661 W cells exposed to light for 0–3 h were immunostained using rabbit polyclonal anti-TACE antiserum [AL45] at 1:200 dilution and 300 nM DAPI. TACE immunostaining (green) appeared to increase with light exposure but did not colocalize with DAPI (blue) in the cultured cells. (B, C) Protein levels of TACE were determined by Western blot and densitometric analyses of 661 W lysates using AL45 at 1:500 dilution and showed significant increase with longer light exposures. A single TACE-reactive band ∼ 90 kDa in size was observed in the untreated cells while multiple bands reactive for TACE ∼ 40–90 kDa were seen following light exposure. β-Tubulin was used to normalize protein loading. Scale bar = 50 μm.

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Expression, cleavage, and nuclear trafficking of p75NTR in light-exposed cells 661 W cells were exposed to light for 0–3 h and processed for immunocytochemistry or Western blots using the anti-p75ICD [9992] or the anti-p75ECD [9651] antisera. Light staining for p75NTR was distributed primarily in the cytoplasm of untreated cultures. With longer exposures of 2–3 h, increased staining was observed not only in the cytoplasm but also in the nucleus, as shown by colocalization of p75NTR with the nuclear stain DAPI (Fig. 2A). Densitometric analyses verified the elevated p75NTR levels in Western blots showing increased intensity of ~75 kDa bands reactive with either anti-p75ICD or anti-p75ECD antisera (Fig. 2B). The ratio of the density of the ~75-kDa band to that of the β-tubulin band in blots with either antisera increased from ~0.34 at 0 h to ~0.65 and ~0.96 after 2 h and 3 h of light exposure, respectively (data not shown). Bands ~50 kDa were also observed in blots probed with the anti-p75ICD, but not with the anti-p75ECD, suggesting that the ~50-kDa band contains the intracellular but not the extracellular domain. The ~50-kDa band could have resulted from molecular splicing, deficient glycosylation, and/or post-translational proteolysis of p75NTR. To verify the nuclear staining for p75NTR, subcellular fractionation was performed on light-damaged cells followed by Western blot analyses using anti-p75ICD or anti-p75ECD antisera. Antibodies against major proteins were used to assess the purity of each fraction as well as to normalize loading of samples. With longer light exposures, blots using either antiserum showed increased intensity of the ~75-kDa bands in both membrane and cytosolic fractions, but not in the nuclear extracts (Fig. 2C). The ~50-kDa putative p75ICD was absent in membrane fractions but appeared as early as 1 h of light exposure in the cytosolic fractions and 2 h in the nuclear fractions, indicative of nuclear trafficking of the ~50-kDa p75ICD. Bands at ~25 kDa were at times appreciated in blots probed with the antip75ICD antiserum. Light exposure promotes increased expression of TACE While the identity of the enzyme(s) responsible for cleavage of p75NTR has not been definitively established, the high homology of p75NTR to TNFR suggested that p75NTR might be a suitable substrate for TACE-induced proteolysis. TACE immunostaining appeared to increase with light exposure in cultured cells (Fig. 3A). Western blot (Fig. 3B) and densitometric analyses (Fig. 3C) of the TACE/β-tubulin ratio showed significant increase from ~0.85 at 0 h to ~1.23 and ~1.5 after 2 h and 3 h of light exposure, respectively. A single TACE-reactive band ~90 kDa in size was observed in the untreated cells while multiple bands ~40–90 kDa were observed following light exposure. The mature TACE migrates as a ~100-kDa protein under reducing conditions and generates multiple bands following cleavage (Schlondorff et al., 2000). Co-expression of TACE and p75NTR promotes p75NTR cleavage To establish TACE cleavage of p75NTR, 661 W cells were stably transfected with plasmids expressing the full-length p75NTR (lane 2), TACE (lane 3), both (lane 4), or the empty vector (lane 1) and processed by Western blot (Figs. 4A–D). TACE-stable transfectants exhibited multiple bands of ~40–90 kDa similar to those in lightdamaged cells, consistent with proteolytic cleavage of TACE (Fig. 4A, lanes 3 and 4). An intense ~50-kDa p75ICD band was observed only in lysates of cells co-transfected with both TACE and p75NTR

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(Fig. 4B, lane 4), while a ~50-kDa p75ECD band was observed only in the 24-h conditioned media of cells co-expressing both TACE and p75NTR (Fig. 4C, lane 4). These findings are consistent with TACE cleavage of the full-length p75NTR resulting in shedding of the ~50kDa p75ECD and generation of a ~50-kDa p75ICD. Subcellular fractionation followed by Western blot using antip75ICD antiserum showed ~75-kDa bands in the membrane and cytosolic fractions of p75NTR-overexpressing cells and a ~50-kDa band in the cytosolic fractions but not in the membrane nor nuclear fractions of cells co-transfected with p75NTR and TACE (Fig. 4D). Thus, while TACE promoted the cleavage of the full-length p75NTR and release of the p75ICD into the cytosol, this was insufficient to promote nuclear translocation of the p75ICD. These findings are consistent with the absence of a nuclear localization signal in p75NTR and suggest the involvement of carrier proteins, i.e. adaptor proteins or chaperones, that could facilitate nuclear transport of the ~50-kDa p75ICD. Moreover, activation of these putative adaptor proteins, i.e. by photo-oxidative stress during light exposure, might be required to promote nuclear transport of p75ICD. The presence of ~75-kDa band in the cytosolic fraction of both p75NTR overexpressing cells and light-damaged cells could be attributed to the release of endosomal p75NTR during subcellular fractionation or from endosomal contamination of the preparations. Internalization of p75NTR via clathrin-coated pits into early endosomes and vesicles has been reported in glia and PC12 cells (Zupan and Johnson, 1991; Kahle and Hertel, 1992; Bronfman et al., 2003). To further confirm that the ~50-kDa p75ICD is a cleavage product of p75NTR, cells transfected with the full-length rat p75NTR containing a C-terminus myc-tag (p75-c-myc) (Fig. 4E, lane 5) were exposed to light for 3 h, immunoprecipitated using anti-c-Myc antibody, and processed for Western blot. Bands reactive for p75ICD were observed at ~50 kDa and ~75 kDa, consistent with the sizes of truncated and full-length p75NTR, respectively, in p75-c-myc transfected cells (lane 5), but not in cells transfected with p75NTR alone (lane 2) or the empty vector (lane 1). This confirmed the identity of the ~50-kDa band as a cleavage product of p75NTR and not a novel spliced variant of p75NTR nor a nascent poorly glycosylated p75NTR. The ~40-kDa and ~60-kDa bands found in all three samples were probably non-specific. IC-3, a TACE inhibitor, blocks cleavage of p75NTR and inhibits light-induced cell death The effects of light damage on TACE activity were further tested by exposing the cells to light in the presence of 5 μM IC-3. Western blots of light-damaged cells treated with IC-3 showed increased expression of the ~75-kDa p75NTR as in untreated cultures (Fig. 5A); however, the ~50-kDa p75ICD was absent. Immunoblots of subcellular fractions showed a similar increase in the intensity of the ~75-kDa bands in membrane and cytosolic fractions, but not in the nuclear fractions, as well as the absence of the ~50-kDa p75ICD band in all fractions of IC3 treated cultures (Fig. 5B). Multiple TACE-reactive bands increased with light exposure and appeared similar in both IC-3-treated and untreated cultures (Fig. 5C). These findings suggested that IC-3 did not inhibit the stimulatory effects of light exposure on the expression of TACE or of the full-length p75NTR but suppressed TACE cleavage of p75NTR. The presence of multiple TACE-reactive bands in light exposed cells in the presence of IC-3 further suggests that TACE does not undergo autocleavage during light damage. The multiple TACE bands could have resulted from cleavage by other enzymes such as furin-related pro-protein convertases which have been shown to

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Fig. 4. Co-expression of TACE and p75NTR promotes p75NTR cleavage. 661 W cells stably transfected with empty vector (lane 1), full-length p75NTR (lane 2), fulllength TACE (lane 3), TACE and p75NTR together (lane 4), or p75NTR-c-myc (lane 5) were lysed and probed by Western blot using rabbit antisera against TACE [AL45] at 1:500 dilution or p75ICD [9992] and p75ECD [9651] at 1:2000 dilution. (A) Increased levels and presence of multiple TACE-reactive bands ∼40–90 kDa were seen in TACE-stable transfectants. (B) p75NTR-transfected cells showed increased expression of ∼75-kDa bands. A strong ∼50-kDa p75ICD-reactive band was also observed only in lysates of cells co-transfected with TACE and p75NTR. (C) A ∼50-kDa band reactive with the anti-p75ECD antiserum was identified in the 24-h conditioned media of cells co-expressing both TACE and p75NTR. (D) Subcellular fractionation followed by Western blot using anti-p75ICD antiserum at 1:2000 showed ∼75-kDa bands in the membrane and cytosolic fractions of p75NTR-overexpressing cells and a ∼50-kDa band in the cytosolic fractions but not in the membrane nor nuclear fractions of cells co-transfected with p75NTR and TACE. The fractions were tested for purity using β-actin (membrane), splicing factor (nuclear), or Akt (cytosolic). (E) ∼ 50-kDa and ∼ 75-kDa bands (arrows) reactive for p75ICD were observed in immmunoprecipitates from cells transfected with c-myc-tagged p75NTR (lane 5) and exposed to light for 3 h but not in light-exposed cells containing the full-length p75NTR alone (lane 2) or the empty vector (lane 1). Bands at ∼ 40 kDa and ∼ 60 kDa were probably non-specific since they were present in all the lanes.

promote maturation and activity of TACE (Peiretti et al., 2003; Srour et al., 2003). Additional studies are needed to identify the enzymes responsible for the cleavage of TACE during light damage as well as their role in TACE-mediated shedding. Light-exposed cells treated with IC-3 were also tested for survival or apoptosis using MTS assay or FACS analysis, respectively. IC-3, at concentrations ranging from 1 to 10 μM, protected 661 W cells from cell death due to 2–3 h light exposure (Fig. 5D). In the presence of 5 μM IC-3, the amounts of cell death after 2–3 h light exposure were not significantly different ( p N 0.05) from cultures exposed to light for 0–1 h. In FACS analyses, the subG1 population of apoptotic cells, indicative of DNA fragmentation, increased from 0.95% in control cultures to 22.43% and 34.62% in cells exposed to light for 2 h and 3 h, respectively (Fig. 5E). In the presence of IC-3, the subG1 peak decreased to about 0.16% and 2.98% in cells exposed to light for 2 h and 3 h, respectively (Fig. 5F). Since IC-3 might also block the activity of other metalloproteases, the role of other IC-3 sensitive

metalloproteases and their substrates in the mechanisms of lightinduced cell death could not be ruled out. Increased expression of p75NTR and TACE in light-damaged rat retinas To establish the effects of light exposure on the expression of p75NTR and TACE in vivo, free-moving normal Sprague–Dawley rats were exposed to light for 6 h and their retinas were processed for immunostaining following 24 h or 5 days of recovery in the dark (Fig. 6). In normal retinas (control), immunoreactivity for p75NTR and TACE was negligible, with p75NTR mostly distributed in the inner retina around ganglion cells while TACE localized to the retinal pigment epithelium (RPE). Increased staining for both p75NTR and TACE in the retina was observed with light exposure and 24 h recovery. Although the most intense labeling was observed in the inner retina, p75NTR and TACE immunoreactivity was also observed

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Fig. 5. IC-3 blocks cleavage of p75NTR and inhibits light-induced cell death. (A, B) 661 W cells exposed to light for 0–3 h in the presence or absence of 5 μM IC-3 were lysed and probed with anti-p75ICD antiserum [9992] diluted 1:2000 or (C) with anti-TACE antiserum [AL45] diluted at 1:500. β-Tubulin was used to normalize protein loading. Immunoblots showed increased expression of the ∼ 75-kDa bands as in untreated cultures; however, the ∼ 50-kDa p75ICD-reactive band in light exposed cells was absent following IC-3 treatment. Immunoblots of subcellular fractions showed a similar increase in the intensity of the ∼ 75-kDa bands in membrane and cytosolic fractions, but not in the nuclear fractions, as well as the absence of the ∼ 50-kDa p75ICD-reactive band in all fractions of IC-3 treated cultures. Multiple TACE-reactive bands ∼40–90 kDa in size increased with light exposure and appeared similar in both IC-3-treated and untreated cultures. (D) To determine the effect of IC-3 on light-induced cell death, 661 W cells were exposed to light for 0–3 h in the presence of 0–10 μM IC-3 and assayed for cell survival by MTS assay. IC-3, at concentrations ranging from 1 to 10 μM, protected 661 W cells from cell death due to intense light exposure for 2–3 h. In the presence of 5 μM IC-3, the amounts of cell death after 2–3 h light exposure were not significantly different ( p N 0.05) from control cultures or cultures exposed to light for 1 h. Experiments were done in triplicates at least five times and subjected to ANOVA. Values represent cell numbers (×1000) ± SD for each treatment. (E, F) To establish that light-induced cell death was apoptotic, 661 W cells exposed to light for 0–3 h in the presence or absence of 5 μM IC-3, were stained with 40 μg/ml of PI and analyzed by flow cytometry. The subG1 population, reflective of apoptotic cells, increased from 0.95% in control cultures to 22.43% and 34.62% in cells exposed to light for 2 h and 3 h, respectively. In the presence of IC-3, the subG1 peak decreased to about 0.16% and 2.98% in cells exposed to light for 2 h and 3 h, respectively. Experiments were done in triplicates at least three times.

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Fig. 6. Increased expression of p75NTR and TACE in light-damaged rat retinas. To determine the effects of light exposure on the expression of p75NTR and TACE in vivo, free-moving normal Sprague–Dawley rats were exposed to 220 fc blue light for 6 h and their retinas were processed for immunostaining using rabbit antiserum against p75ICD or TACE following 24 h or 5 days of recovery in the dark. In normal rat retinas (control), immunolabeling for p75NTR (red) and TACE (red) was very low, with p75NTR mostly distributed in the inner retina around ganglion cells, while TACE localized to the retinal pigment epithelium (RPE). Increased staining for both p75NTR and TACE in the retina was observed with light exposure and 24 h recovery (6 hL/24 hR). p75NTR and TACE immunoreactivity was observed in numerous processes in the inner retina and photoreceptor outer segments (OS). With light exposure and longer recovery of 5 days (6 hL/5 dR), the photoreceptor cell layer appeared thinner and the p75NTR and TACE labeling was further increased especially in the inner retina and inner segments (IS). Nuclear layers were stained with DAPI (blue).

in the outer segments (OS) of photoreceptor cells. With light exposure and longer recovery of 5 days, the outer nuclear layer (ONL) appeared thinner as compared with age-matched control retinas that contained the normal 12–14 layers of photoreceptor cell bodies. The staining for p75NTR and TACE was also more intense especially in the inner retina. The increased expression of TACE and p75NTR in photoreceptor cells of animal retinas with photic injury was consistent with increased levels of both proteins in the light-damaged 661 W cells, suggestive of a role for TACE and p75NTR in light-induced photoreceptor cell death. The absence of nuclear staining for p75ICD, especially in photoreceptor cells, was consistent with previous

findings of difficulty of following the cellular distribution of cleaved products of p75NTR (Kanning et al., 2003). Consequently, the increased p75NTR immunoreactivity in the light-damaged retinas is most probably due to increased expression of the full-length p75NTR as observed in light-damaged 661 W cells. Discussion In this study, we report that light exposure stimulates increased expression of p75NTR and TACE in cultured photoreceptor cells. While p75NTR expression has been shown to be upregulated in

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various genetic and environmental conditions (Kokaia et al., 1998; Roux et al., 1999; Oh et al.,2000; Harada et al., 2000; Sheedlo et al., 2002), to our knowledge, this is the first report establishing a direct relationship between light-induced apoptosis and proteolytic cleavage of p75NTR. While overexpression of the p75ICD (Majdan et al., 1997; Coulson et al., 2000) has been reported to promote neuronal apoptosis, this study also suggests a role for p75ICD nuclear signaling and putative adapter proteins in apoptosis. This is also the first report of light-mediated upregulation of TACE expression. TACE is constitutively expressed in various tissues, and while the proteolytic activity and degradation of TACE are both stimulated by various molecules such as phorbol esters and protein kinase C (Heldin and Ericsson, 2001; Black, 2002), the regulation of TACE expression is unknown. Moreover, while TACE has been suggested to promote ectodomain shedding of several membranebound proteins including p75NTR (Black et al., 1997; Moss et al., 1997; Buxbaum et al., 1998; Peschon et al., 1998; Zhang et al., 2000; Vincent et al., 2001; Kanning et al., 2003; Jung et al., 2003), this is the first report to definitively demonstrate that p75NTR is a substrate of TACE. Our results are consistent with recent findings showing inhibition of TACE-mediated cleavage of p75NTR in tace−/− fibroblasts (Weskamp et al., 2004). Overexpression of the full-length p75NTR and TACE genes in photoreceptor cells showed cleavage of p75NTR and release of a ~50-kDa p75ECD into the culture medium. Soluble p75NTR ~45–77 kDa in sizes released in the conditioned media of cultured rat Schwann cells, PC12 cells, superior cervical ganglion neurons, and schwannoma cells, or A875 human melanoma cells have been identified as shed extracellular domains of p75NTR (DiStefano and Johnson, 1988; Zupan et al., 1989; Barker et al., 1991; DiStefano et al., 1993). Purification of soluble p75NTR from human infant urine and amniotic fluid yielded species with molecular masses of ~35–45 kDa (Zupan et al., 1989) while ~50–77-kDa species have been isolated from rat plasma or biological fluids (DiStefano and Johnson, 1988). The variability in the reported size of the soluble p75ECD is in accord with suggestions of multiple TACE cleavage sites in p75NTR (Weskamp et al., 2004) and the lack of substrate specificity and the variability in the sequence cleaved by TACE (Black, 2002). Variations in the amount of glycosylation, additional extracellular proteolytic events, or association with neurotrophins could generate p75ECD fragments of variable sizes. TACE, an α-secretase, has been shown to initiate regulated intramembrane proteolysis (RIP) of several molecules such as Notch, APP, and the ErbB-4 receptor by a cleavage within the extracellular domain (Heldin and Ericsson, 2001). This is often followed by cleavage of the membrane-bound fragment in its transmembrane domain by a γ-secretase, usually a presenilincontaining protease complex, to release the cytoplasmic domain that translocates to the nucleus. The membrane-bound p75NTR receptor fragment has been suggested to migrate at ~ 30 kDa and is rapidly cleaved by γ-secretases to the ~ 25-kDa p75ICD that undergoes nuclear translocation or proteosomal degradation (Kanning et al., 2003). Few reports, however, have convincingly demonstrated nuclear localization of γ-secretase products of p75NTR. γ-Secretase cleavage products, in general, rapidly undergo proteolytic degradation through proteasomal pathways (Oberg et al., 2001). Hence, visualization of the ~ 25-kDa and ~ 30-kDa fragments often require inclusion of γ-secretase inhibitors and/or proteosome inhibitors, respectively, in sample preparations (Jung et al., 2003). In lightdamaged 661 W cells, we never consistently observed the ~ 30-kDa or ~25-kDa fragments in spite of our use of γ-secretase inhibitors (compound E, DAPM) or proteosome inhibitors (ALLN, lactacys-

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tin) (data not shown). The short duration of exposure of blots in the photoimaging system utilized in our study could have also prevented development of minor bands containing cleaved products. Exposing the blots for longer durations, however, often resulted in numerous bands whose specificity were difficult to ascertain. In 661 W cells, cleavage of p75NTR by TACE resulted in the production of a ~ 50-kDa p75ICD-containing band. Our findings suggest that the ~ 50-kDa band is a cleavage product of full-length p75NTR and not a result of molecular splicing nor deficient glycosylation of an immature p75NTR. The larger size of the p75ICD could, theoretically, be explained by the presence of multiple putative TACE cleavage sites in p75NTR (Weskamp et al., 2004). The ~50-kDa fragment could have been immediately internalized by endocytosis instead of undergoing proteolytic cleavage by γ-secretases. Internalization of p75NTR via clathrin-coated pits into signaling endosomes and vesicles has been reported in glial cells and PC12 cells (Zupan and Johnson, 1991; Kahle and Hertel, 1992; Bronfman et al., 2003). Internalization of the ~50-kDa p75ICD could protect it from further proteolytic processing while allowing association with adapter proteins for signal transduction or nuclear transport (Bronfman et al., 2003). In addition, the membrane-bound receptor fragment in 661 W cells could have been spared from additional processing by γ-secretases following TACE cleavage. In primary mouse embryonic fibroblasts, p75NTR is cleaved by α-secretase but not by γ-secretases as compared with Schwann cells, suggesting that post-translational processing of p75NTR is highly dependent on cell type (Kanning et al., 2003). On the other hand, the large size of the ~ 50-kDa p75ICD could also have been generated by dimers of ~ 25 kDa γ-secretase products. We observed ~ 25-kDa bands in blots of light-damaged cells probed with the anti-p75ICD antiserum occasionally, but it was not consistent. Monomeric p75NTR is known to bind its ligands, even those that exist as biologically active dimers such as nerve growth factor (He and Garcia, 2004), however, p75NTR can also undergo dimerization (Wang et al., 2000; Ye et al., 1999) or trimerization (Yaar et al., 2002) and maintain biological activity. It is plausible that light damage promoted dimerization and cleavage of p75NTR by TACE followed by γ-secretases, resulting in the generation of p75ICD dimers. Clearly, additional in-depth studies are needed to fully explain the nature and function of the ~ 50-kDa p75ICD. The p75ICD was found in nuclei of light-exposed 661 W cells, but in the presence of IC-3, a metalloprotease inhibitor, nuclear translocation of p75ICD was inhibited, suggesting that prior processing of p75NTR by TACE was required for its nuclear trafficking. In cells overexpressing TACE and p75NTR, the products of TACE cleavage were present but the p75ICD did not translocate to the nucleus consistent with the absence of a nuclear localization signal in p75NTR. Preliminary studies in our laboratory showing increased protein levels and association of NRAGE, a p75NTRadaptor protein, with the p75ICD following intense light exposure, suggest a mechanism for the nuclear shuttling of p75ICD in lightinduced apoptosis. Association of p75NTR and MAGE proteins, necdin and NRAGE, within the signaling endosomes (Bronfman et al., 2003), could provide the vehicle to transport the protein complex to subcellular compartments such as the nucleus. Similarly, internalization of APP to recycling synaptic vesicles in primary cerebellar neurons promoted APP trafficking to the cell body (Marquez-Sterling et al., 1997). Nuclear trafficking of the p75ICDadapter protein complex would allow entry of the adapter proteins to the nucleus where they could perform various functions such as transcriptional activation of pro-apoptotic genes or regulation of the cell cycle (Barker and Salehi, 2002).

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Photic injury to the retina involves intense light exposure to retinal photoreceptor cells resulting in the activation of the phototransduction cascade leading to cell death via apoptosis (Noell et al., 1966). The extent of the retinal damage is highly dependent on the duration (Moriya et al., 1986) and intensity of light exposure (Organisciak et al., 1998) such that short exposure to bright light or continuous low light exposure for prolonged periods both lead to retinal damage with fewer photoreceptor cells, shorter outer segments, and damaged membranes (Penn et al., 1992). Outer segment changes are the earliest findings from continuous exposure to fluorescent light, whereas exposure to bright light induces changes in the photoreceptor inner segments and pigment epithelial cells before outer segment changes occur (Kuwabara and Gorn, 1968). While several mechanisms have been proposed to explain the development of apoptotic cell death from photic injury, recent reports showing increased expression of p75NTR in photoreceptor cells in animals with retinal dystrophy (Sheedlo et al., 2002; Srinivasan et al., 2004) and the anti-p75NTR antibody-mediated delay of light-induced photoreceptor apoptosis in albino Wistar rats (Harada et al., 2000), suggest that p75NTR may be involved in light-induced photoreceptor cell death. However, the lack of p75NTR expression in light-damaged mouse photoreceptor cells and conflicting reports on the effects of light damage on photoreceptor survival in p7NTR knock-out mice (Harada et al., 2000; Rohrer et al., 2003) suggest that lightinduced photoreceptor cell death might involve several apoptosis pathways. Whether p75NTR-mediated apoptosis involves specific photoreceptor cell types, i.e. cones vs. rods, also remains to be established. The 661 W cell line used in this study has been reported to express both rod and cone photoreceptor markers (Roque et al., 1999; Tuohy et al., 2002; Kanan et al., 2007), but the effects of light damage on p75NTR in cone cells has not been addressed in studies utilizing p75NTR knock-out mice. Moreover, while our study did not directly establish that TACE-mediated cleavage of p75NTR and nuclear translocation of p75ICD was responsible for light-induced photoreceptor apoptosis, the redistribution and nuclear localization of the proapoptotic p75ICD suggest a role for p75ICD in photoreceptor cell death. One could also speculate that TACE cleavage of membranebound growth factors that perform pro-survival functions via autocrine signaling could also contribute to cell death from intense light exposure. Clearly, additional studies are needed to determine the exact roles of TACE and p75NTR in photoreceptor cells.

Transfections Plasmids containing cDNA of the full-length murine TACE (Black et al., 1997), full-length human p75NTR, C-terminus myc epitope (c-Myc)tagged rat p75NTR, or empty vector were used for transfections. Cells were transfected with 2–5 μg of plasmid using Lipofectamine plus (Life Technologies, Inc., Carlsbad, CA) in serum-free medium. Transfected cells were selected with 800 μg/ml G418 (Life Technologies, Inc.) for 2 weeks and maintained in 200 μg/ml G418. Immunocytochemistry Cells were fixed in 2% paraformaldehyde for 20 min and were permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS; 10 mM phosphate [pH 7.4], 150 mM NaCl) containing 50 mM glycine for 15 min. Cells were incubated in PBS containing 0.1% Triton X-100, 5%BSA, and 5% normal goat serum for 1 h at room temperature (RT) followed by 1:2000 dilution of rabbit antisera against murine TACE cytoplasmic domain [AL45] (Zhang et al., 2000), murine p75ECD [9651] (Huber and Chao, 1995), or human p75NTR cytoplasmic domain [9992] (Huber and Chao, 1995) at 4 °C overnight. After several rinses, cells were incubated with 10 µg/ml anti-rabbit IgG Alexa 488 or 594 (Molecular Probes Inc., Eugene, OR) for 1 h, then counterstained with 300 nM DAPI (Molecular Probes Inc.). Images of representative fields were captured in Nikon Microphot FXA microscope with epifluorescent attachment. Cytotoxicity assays The effect of light exposure on photoreceptor cell death/cell survival was determined using fluorescent probes calcein AM and ethidium homodimer (Molecular Probes Inc.) as described (Rosales and Roque, 1997). 661 W cells plated at 10,000 cells/well on 24-well plates were incubated with 2 µM calcein AM and 4 µM ethidium homodimer at 37 °C for 45 min and viewed under Olympus inverted microscope with epifluorescent attachment. The number of surviving cells was determined in 661 W cells plated at 5000 cells/well on 96-well plates using Cell titer 96 assay (Promega Corp, Madison, WI) as described (Roque et al., 1999). Briefly, cells were incubated in 333 µg/ml MTS and 25 µM phenazine methosulfate and the absorbance measured at 490 nm at 15 min intervals for 1 h. A standard curve was prepared from cells plated at 0–50,000 cells/well. Experiments were done at least three times in triplicates. Values were expressed as cell counts and subjected to statistical analyses using ANOVA. Propidium iodide-Hoechst 33342 staining

Experimental methods Cell culture The 661 W photoreceptor cells line (a gift from Dr. Muayyad AlUbaidi, University of Oklahoma Health Sciences Center, Oklahoma City, OK) were obtained from transgenic mice retinas expressing SV40 T-antigen and found to maintain photoreceptor phenotypes (Al-Ubaidi et al., 1992; Roque et al., 1999) and photo-oxidative pathways (Krishnamoorthy et al., 1999; Kanan et al., 2007). 661 W cells were grown to 80% confluency in growth medium (Dulbecco's modified Eagles medium, 2 mM L-glutamine, 100 U/ml Penicillin, 100 g/ml Streptomycin, 15 mM HEPES buffer, and 10% fetal bovine serum) then transferred to serum-free medium for 18 h before exposure to 1400 foot-candles (fc) of light for 0–5 h. The culture medium was changed to fresh serum-free medium and the cultures were returned to the incubator for additional 20 h following light exposure. Cells maintained under similar conditions without light exposure were used as control. The effects of TACE on 661 W cells were verified using 0–50 M IC-3 (Immunex compound-3; Immunex Corporation, Seattle, WA), a metalloprotease inhibitor that blocks TACE activity and secretion of TNF-α (Black et al., 1997).

661 W cells were fixed with 2% paraformaldehyde and stained by incubating in PBS containing 500 nM Hoechst 33342 (H-33342) and 500 nM propidium iodide (PI) (Molecular Probes Inc.) at 37 °C for 10 min in the dark. Stained cells were viewed and the images were photographed with appropriate filters to visualize blue fluorescence (H-33342) and red fluorescence (PI). Flow cytometry (FACS) DNA content was determined by flow cytometry using PI. Following light exposure, 661 W cells were harvested by trypsinization, resuspended in PBS, fixed by addition of ice-cold ethanol while vortexing, and incubated in ethanol at −20 °C overnight. Cells were pelleted at 1500 rpm for 10 min and resuspended in PBS containing 40 μg/ml PI and 100 μg/ml RNase A. Cells were incubated at 37 °C for 30 min prior to flow cytometric analysis using a Coulter EPICS XL/XL-MCL flow cytometer (Beckman Coulter Inc., Fullerton, CA). DNA content was determined from histograms using WinMDI (Windows Multiple Documentation Interface, The Scripps Research Institute, La Jolla, CA) by gating on an area versus width in dot plot to exclude cell debris and cell aggregates. The percentage of degraded

B. Srinivasan et al. / Mol. Cell. Neurosci. 36 (2007) 449–461 DNA was determined by the number of cells with subdiploid DNA divided by the total number of cells examined under each experimental condition.

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for immunofluorescence using 1:200 dilution of rabbit antisera against TACE [AL45] or p75ICD [9992], followed by 10 µg/ml anti-rabbit IgG Alexa 488 and 300 nM DAPI (Molecular Probes Inc.).

DNA laddering To verify the induction of apoptosis, 661 W cells in 35 mm wells were processed for DNA laddering as described (Roque et al., 1999). Briefly, cells were lysed with 0.5 mg/ml proteinase K in 10 mM Tris–HCl, 100 mM NaCl, 10 mM EDTA, 0.5% SDS at 50 °C overnight. DNA was extracted with phenol:chloroform:isoamyl alcohol at 25:24:1 and centrifuged at 4 °C for 60 min at 12000×g. The aqueous phase was collected, treated with 1/ 10 vol of 3 M Na acetate (0.3 M final concentration) and precipitated with 2 volumes of ice-cold 100% ethanol overnight at −20 °C. DNA was resuspended in 10 mM Tris–HCl and 1 mM EDTA, pH 8.0, treated with 100 μg/ml DNAse-free RNAse A for 1 h at 37 °C, and electrophoresed in a 2.0% agarose gel.

Acknowledgments The authors wish to thank Dr. Moses V. Chao of New York University Medical Center, New York, NY for the use of his antip75NTR antibodies and Dr. Larry Oakford of the University of North Texas Health Science Center for technical contributions to this study. This work was taken in part from a dissertation (BS) submitted to the University of North Texas Health Science Center at Fort Worth in 2003 in partial fulfillment of the requirements for the degree Doctor of Philosophy. This work was supported by an ALCON Research Grant (RSR) and a VA Merit Review award (SJF).

Subcellular fractionation

References Cells in 100 mm dishes were scraped in PBS containing protease inhibitors (1 mM phenymethylsulfonylfluoride, 1 mM sodium orthovanadate and 1 mg/ml aprotinin). Cells were disrupted using Dounce homogenizer, and the lysate was centrifuged at 20,000×g for 30 min at 4 °C. The supernatant (S1) was separated from the pellet (P1) and centrifuged again at 180,000×g for 75 min at 4 °C. The resulting supernatant (S2) was collected as the cytosolic fraction. P1 was resuspended in PBS and centrifuged in 0–1 M sucrose gradient at 100,000×g for 1 h. The membrane fraction was collected at interface between 0.25 and 0.50 M gradients, while the nuclear fraction was collected from the pellet. The purity of each fraction was demonstrated in Western blots by probing for specific markers including splicing factor (1:100 dilution; Sigma Immunochemicals, St. Louis, MO), β-actin (1:200 dilution; Chemicon International Inc., Temecula, CA), and Akt (1 µg/ml; Cell Signaling Technology Inc., Beverly, MA). Western blot Cell lysates, cellular fractions, or immunoprecipitated proteins were separated by SDS–PAGE and processed for Western blot using 1:2000 dilution of rabbit antisera against TACE [AL45], p75ECD [9651], or p75ICD [9992], or with monoclonal antibody against c-Myc (clone 9E10, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500, overnight at 4 °C. Reactions were developed using SuperSignal West Pico Chemiluminescent substrate (Pierce Biotechnology Inc., Rockford, IL). Membranes were reprobed for β-tubulin (1 μg/ml; Santa Cruz Biotechnology) or β-actin (1:200 dilution; Chemicon International Inc.) to determine amounts of sample loaded. Bands were visualized with a photoimaging system using the shortest exposure time during development to allow visualization only of major bands. Animal model of photic injury Normal Sprague–Dawley rats were maintained on a 12-h light/dark cycle under broad band fluorescent light (Sylvania Cool White, 45 foot-candles [fc]) in cages. To induce photochemical lesions, free-moving rats were exposed to long wavelength blue light of 220fc (Philips fluorescent lamps F40/BB) for 6 h after 24 h-dark adaptation. Animals were maintained in clear polycarbonate cages with minimal bedding during light exposure. Animals were allowed to recover in the dark for 24 h or 5 days after light exposure prior to sacrifice using high doses of pentobarbital. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication 8523, revised 1996). Eyeballs were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned at 5 µM thickness. Paraffin sections were processed

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