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J. Biol. Chem., Vol. 277, Issue 37, 34295-34302, September 13, 2002

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Mechanisms of p75-mediated Death of Hippocampal Neurons

ROLE OF CASPASES^*

Carol M. Troy, Jonathan E. Friedman^§, and Wilma J. Friedman^¶

From the  Department of Pathology, Taub Institute for the Study of Alzheimer's Disease and the Aging Brain, and the Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, New York 10032 and ^§ D-Pharm Ltd., Kiryat Weizmann Science Park Building 16, Rehovot 76123, Israel

Received for publication, May 24, 2002, and in revised form, July 2, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Neurotrophins support neuronal survival and differentiation via Trk receptors, yet can also induce cell death via the p75 receptor. In these studies, we investigated signaling mechanisms governing p75-mediated death of hippocampal neurons, specifically the role of caspases. Although p75 is structurally a member of the Fas/TNFR1 receptor family, caspase-8 was not required for p75-mediated death, unlike other members of this receptor family. In contrast, p75-mediated neuronal death was associated with mitochondrial loss of cytochrome c and required Apaf-1 and caspase-9, -6, and -3. In particular, caspase-6 plays a central role in mediating neurotrophin-induced death, illuminating a novel role for this caspase. Inhibition of DIABLO/Smac, which blocks inhibitor of apoptosis proteins, protected cells from death, whereas simultaneous inhibition of both DIABLO/Smac and MIAP3 allowed trophin-induced death to proceed. In vivo, pilocarpine-induced seizures, previously shown to up-regulate p75 expression and increase neurotrophin production, caused activation of caspase-6 and -3 and cleavage of poly(ADP-ribose) polymerase in p75-expressing hippocampal neurons. In p75^/ mice, no activated caspase-3 was detected, and there was a marked reduction in the number of dying neurons after pilocarpine treatment compared with wild type mice. Neurotrophin-induced p75-mediated death is likely to play an important role in mediating neuronal loss consequent to brain injury.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The signaling pathways regulating neuronal death in development and after brain injury have been widely studied but are not fully elucidated. The neurotrophins nerve growth factor (NGF),¹ brain-derived neurotrophin factor (BDNF), neurotrophin-3, and neurotrophin-4 clearly play a role in determining developmental survival of neurons but also can cause neuronal death, depending on the receptors that are activated. Neurotrophin effects on survival and differentiation are mediated by activation of Trk receptors (1, 2), whereas effects on cell death are mediated by activation of the p75 receptor in the absence of Trk signaling (3-5). The pathways by which neurotrophins signal cell survival have been studied extensively, whereas little is known concerning the mechanisms by which neurotrophins signal neuronal death. It is increasingly apparent that neurotrophins play important roles in signaling neuronal death during development and after brain injury.

We have previously demonstrated that all neurotrophins can elicit death of hippocampal neurons that express p75 in the absence of the cognate Trk receptor (6). In this study, we have analyzed the mechanisms governing p75-mediated death of hippocampal neurons, specifically the role of caspases, a family of cysteine-dependent aspartate-specific proteases that are critical mediators of apoptosis. Caspases are synthesized as zymogens and can be activated by cleavage, by oligomerization, or by interacting with an adapter molecule to form an apoptosome (7, 8). Two different pathways of caspase activation leading to cell death have been identified, an intrinsic and an extrinsic pathway (9). The intrinsic death pathway involves mitochondrial release of cytochrome c, which interacts with Apaf-1, an adapter protein, to form an apoptosome that activates caspase-9 (10). Activated caspase-9 can then cleave and activate downstream effector caspases. This apoptotic pathway can be regulated at a variety of checkpoints. Activation of caspase-9 by cytochrome c/Apaf-1 can be prevented by cytosolic inhibitor of apoptosis proteins (IAPs). IAPs can themselves be inhibited by a recently identified protein released from the mitochondria, Smac (11), also called DIABLO (12). Thus, IAPs have antiapoptotic activity, whereas Smac/DIABLO facilitates apoptosis by inhibiting the IAPs.

The extrinsic pathway involves activation of death receptors, such as Fas, and recruitment of caspase-8 via interaction of adapter proteins with the receptor's death domain (9). Caspase-8 then activates effector caspases, such as caspase-3, -6, and -7. Caspase-8 can also activate the intrinsic pathway by cleavage of BID, which induces mitochondrial release of cytochrome c (13). Due to characteristic structural features, including the presence of a cytoplasmic death domain, p75 has been classified as a member of the Fas receptor family (14).

In these studies, we have investigated the role of specific caspases in p75-mediated death of hippocampal neurons in vitro and in vivo. By defining the caspase cascade activated in p75-mediated death, we will gain more insight into the mechanism of p75 signaling and how it compares with other tumor necrosis factor (TNF) receptor family members and gain a broader understanding of neurotrophin actions in the brain.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Neuronal Cultures-- Neuronal cultures were prepared as described previously (6, 15). Hippocampi were dissected from embryonic day 18 rat fetuses, dissociated by trituration in serum-free medium, plated on poly-D-lysine (0.1 mg/ml)-coated tissue culture wells or plastic Lab-Tek slide wells, and maintained in a serum-free environment. Medium consisted of a 1:1 mixture of Eagle's minimal essential medium and Ham's F-12 (Invitrogen) supplemented with glucose (6 mg/ml), putrescine (60 µM), progesterone (20 nM), transferrin (100 µg/ml), selenium (30 nM), penicillin (0.5 units/ml), and streptomycin (0.5 µg/ml) (Sigma). In all experiments, neurons were cultured for 4-5 days before treatment. Cultures contained <2% glial cells, confirmed by staining for glial markers. The absence of glia is critical, since astrocytes in culture produce high levels of NGF.

Neuronal Survival Assay-- Survival of cultured hippocampal neurons was assayed by a method we adapted (6, 15, 16), which has been used routinely to assess PC12 cell viability (17). After removal of the medium, cultured cells were lysed, and intact nuclei were counted using a hemacytometer. Nuclei of dead cells either disintegrate, or, if in the process of dying, appear pyknotic and irregularly shaped. In contrast, nuclei of healthy cells are phase-bright and have clearly defined limiting membranes. Cell counts were performed in triplicate wells. Statistical significance was determined by analysis of variance with Bonferroni's post hoc analysis.

Penetratin-linked Antisense Oligonucleotides-- Antisense oligonucleotides were synthesized with a thiol linker at the 5' terminus and purified by high pressure liquid chromatography. Oligonucleotides were resuspended in deionized water, treated with an equimolar mixture of tris(2-carboxyethyl)-phosphine hydrochloride buffer. An equimolar ratio of penetratin 1 (Oncor) was added, and the mixture was incubated at 37 °C for 1 h. The yield of the reaction was estimated by SDS-PAGE followed by Coomassie staining for the penetratin peptide.

Western Blot Analysis-- For antisense down-regulation studies, hippocampal cultures were treated with various antisense constructs for 5 h and harvested in sample buffer. Equal amounts of protein were separated by 15% PAGE, transferred to nitrocellulose, and immunostained as described (18). To ensure that there was no cross-reactivity of each antisense with other nontargeted caspase family members, the effect of each antisense construct (240 nM) on the other caspase family members was determined. Anti-caspase-1 was used at 1:1000 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-caspase-2 (19) at 1:330, anti-caspase-3 (Upstate Biotechnology) at 1:500, anti-caspase-6 (BD PharMingen) at 1:1000, anti-caspase-7 (R & D Systems) at 1:1000, anti-caspase-8 (Oncogene) at 1:500, and anti-caspase-9 (Medical and Biological Laboratories, Co., Ltd.) at 1:1000.

For analysis of caspase activation, cells were lysed in a buffer consisting of Tris-buffered saline with 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 mM sodium vanadate. Total protein was quantified by the Bradford assay (Bio-Rad). Equal amounts of protein were run on a 15% polyacrylamide gel and transferred electrophoretically to nitrocellulose membrane. The membranes were stained with Ponceau S to control for equal loading and transfer of samples. The filters were then probed with anti-caspase-6 (BD PharMingen) or anti-cleaved caspase-3 (Cell Signaling Technology) used at 1:1000 and visualized by enhanced chemiluminescence (Pierce). Films were scanned into Adobe Photoshop.

Pilocarpine-induced Seizures-- Male Wistar rats (250-275 g) were pretreated for 0.5 h with methyl-scopolamine (1 mg/kg, subcutaneously; Sigma) and then treated with pilocarpine hydrochloride (380 mg/kg, intraperitoneally; Sigma). After 1 h of status epilepticus, rats were treated with diazepam (10 mg/kg; Teva) and phenytoin (50 mg/kg; Sigma) to stop seizure activity. Additional diazepam was administered as necessary to prevent further seizures. Adult mice (24-30 g) were also pretreated for 0.5 h with methyl-scopolomine and in addition were pretreated with phenytoin (50 mg/kg; Sigma) to prevent mortality associated with tonic seizure, and then injected with 320 mg/kg pilocarpine and scored for generalized clonus with loss of righting reflex. The p75^/ mice are available on two different genetic backgrounds, the original 129/Balb/c mixed strain and those that have been backcrossed onto the C57Bl/6 background. Certain genetic mouse strains are more resistant to neuronal loss induced by seizure activity than others, with the C57BL/6 strain being among the most resistant (20, 21). Therefore, p75^/ mice on the 129/Balb/c background were used and compared with wild type 129 and Balb/c mice as controls. Five mice of each genetic background (129, Balb/c, p75^/) were injected with pilocarpine. Since mice are more resistant to neuronal loss after seizures than rats, status epilepticus was allowed to proceed for 2 h prior to treatment with diazepam (10 mg/kg; Teva). Additional diazepam was administered as necessary to prevent further seizures. Control animals received all the same treatments except they were injected with saline instead of pilocarpine. During recovery, the animals were treated with Hartman's solution (130 mM NaCl, 4 mM KCl, 3 mM CaCl, 28 mM lactate; 1 ml/100 g) injected subcutaneously twice daily until capable of eating and drinking freely. All animal studies were conducted using the National Institutes of Health guidelines for the ethical treatment of animals.

Immunocytochemistry-- Animals were anesthetized with ketamine/xylazine and perfused transcardially with saline followed by 4% paraformaldehyde. The brains were removed and postfixed in 4% paraformaldehyde for 2 h and cryoprotected in 30% sucrose overnight. Sections (12 µm) were cut on a cryostat (Leica) and mounted onto coated slides. Sections were blocked in PBS plus 5% goat serum and permeabilized with PBS plus 0.3% Triton X-100 and then exposed to anti-p75 (192 IgG; Chemicon; 1:500) and anti-cleaved caspase-3, anti-cleaved caspase-6, or anti-cleaved PARP (Cell Signaling Technology; 1:500) overnight at 4 °C in PBS plus 0.3% Triton. Slides were then washed three times in PBS, exposed for 1 h at room temp to secondary antibodies coupled to the Alexa 488 or 594 fluorophores (Molecular Probes, Inc., Eugene, OR), and washed again in PBS in the presence of Hoechst 33342 (1 µg/ml; Sigma) to identify apoptotic neurons. No immunostaining was seen in controls with omission of the primary antibodies. Sections were coverslipped with anti-fading medium (Biomeda) and analyzed by fluorescence microscopy (Zeiss). At least 15 sections were analyzed per animal. Cultured cells were fixed with 4% paraformaldehyde, exposed to primary antibodies overnight at 4 °C or at room temperature for 1.5 h, washed with PBS, exposed to the appropriate fluorescent secondary antibodies for 1 h at room temperature, and analyzed with a Perkin-Elmer Spinning Disc confocal imaging system mounted on a Nikon inverted microscope. Epifluorescent (Zeiss) or confocal (Nikon) images were captured digitally and assembled in Adobe Photoshop.

Fluoro-Jade B Labeling-- The number of dying neurons in wild type and p75^/ mice after pilocarpine-induced seizures was assessed by labeling with Fluoro-Jade B (22, 23) according to the published protocol (23). Labeled neurons were counted in three fields from each of three different sections in both the hippocampus and cortex. Epifluorescent (Nikon) images were captured digitally and assembled in Adobe Photoshop.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have previously demonstrated that neurotrophins elicit death of ~30% of cultured hippocampal neurons, which corresponds to the population expressing p75 without a Trk receptor (6). Since this death pathway may play a critical role in neuronal death during development and after injury, we investigated the mechanisms governing p75-mediated death of hippocampal neurons. To determine whether caspases were necessary for p75-mediated death, we examined whether inhibitors of caspase activity could protect the neurons from neurotrophin-induced death. Pseudosubstrate inhibitors have been widely used to block caspase activity. Although these inhibitors have different affinities for distinct caspases, they are not completely specific. However, at low concentrations they provide an indication of which class of caspases may be involved in the death pathway. The concentrations used for each inhibitor are those that have been found to distinguish among different families of caspases. These experiments demonstrated that DEVD-FMK at 10 µM, a concentration that blocks caspase-3-like caspases, partially protected the hippocampal neurons from neurotrophin-induced death, providing about 50% protection, whereas VEID-FMK (25 µM) and IETD-FMK (25 µM), inhibitors that block both caspase-6 and caspase-8, among other caspases (24-26), substantially prevented neuronal death, providing more than 80% protection (Fig. 1). In contrast, YVAD-FMK (25 µM), which blocks caspase-1-like family members, did not protect the neurons from NGF-induced death (not shown). Since VEID-FMK and IETD-FMK can block the activity of both caspase-6 and -8, these inhibitors do not permit discrimination between activation of these caspases.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Caspases are required for neurotrophin-induced neuronal death. Caspase inhibitors prevent neurotrophin-induced death. Hippocampal neurons were cultured for 5 days and treated overnight with vehicle, NGF (100 ng/ml), or BDNF (100 ng/ml) in the presence or absence of pseudosubstrate inhibitors. Neuronal death is reported as a percentage of untreated controls and presented as the mean ± S.E. The peptides IETD-FMK (25 µM) and VEID-FMK (25 µM) completely prevented neuronal loss, whereas DEVD-FMK (10 µM) gave partial protection. Each data point represents triplicate samples from four independent experiments (n = 12). *, significantly different from control, p < 0.001.

Distinct caspases are activated by different death-inducing stimuli (27, 28). To identify the specific caspases necessary for p75-mediated death, antisense oligonucleotides to individual caspases were used to determine whether down-regulation of specific caspases could prevent neurotrophin-induced neuronal death. The oligonucleotides were linked to penetratin-1 as a vector to facilitate entry into cells. We have previously demonstrated the specificity and efficacy of these constructs (16, 18, 19). Each oligonucleotide down-regulates the targeted caspase by 70-90%, shown for V-ACasp6 in Fig. 2b and does not down-regulate the nontargeted caspases (Fig. 2c). The p75 receptor is related to several known death receptors such as Fas and TNFR1. These receptors, when bound to ligand, directly initiate a cascade of caspase cleavages via interaction with adapter proteins. Caspase-8 is the initiator caspase activated by Fas (29). To assess whether this pathway mediates p75-induced death, cells were treated with antisense oligonucleotides to caspase-8 together with overnight exposure to NGF or BDNF. Down-regulation of caspase-8 did not prevent neurotrophin-induced death (Fig. 2a). However, down-regulation of caspase-6 (Fig. 2b) provided about 90% inhibition of p75-mediated death (Fig. 2a). In addition, down-regulation of caspase-3 partially protected the neurons from NGF and BDNF-induced death, providing about 50% protection, suggesting a role for caspase-3 as well as caspase-6 in this death pathway. In contrast, down-regulation of caspase-7, which protects caspase-2 null sympathetic neurons from trophic factor deprivation-induced death (18), had no protective effect in this paradigm. Antisense oligonucleotides provided to the cultures in the absence of neurotrophins had no effect on neuronal survival.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Down-regulation of caspase-6 and -3 protects hippocampal neurons from p75-mediated death. A, hippocampal neurons were cultured for 5 days and then treated overnight with NGF or BDNF and penetratin (vector)-linked antisense oligonucleotides (240 nM) directed against specific caspases. Down-regulation of caspase-6 (V-AC6) completely protected, whereas down-regulation of caspase-3 (V-AC3) partially protected, against p75-mediated neuronal death. Neuronal death is reported as a percentage of untreated controls and presented as the mean ± S.E. Each data point represents triplicate samples from nine independent experiments (n = 27). *, significantly different from control, p < 0.001. #, significantly different from neurotrophin alone, p < 0.01. **, significantly different from neurotrophin alone, p < 0.001. B, Western blot demonstrating down-regulation of caspase-6 protein by the antisense oligonucleotide (V-AC6). C, Western blots demonstrating that antisense oligonucleotides to caspase-6 do not down-regulate other caspases in hippocampal neurons. For B and C, hippocampal cultures were treated for 5 h with V-ACasp6 and harvested in sample buffer. Cell lysates containing equal amounts of protein were subjected to Western blotting using the indicated antisera. Actin staining confirmed equal loading (not shown).

The use of peptide inhibitors and antisense oligonucleotides suggested that caspase-6 and -3 were involved in mediating neurotrophin-induced death of hippocampal neurons. Both of these effector caspases require cleavage for activation. To determine whether these caspases were cleaved and activated in the hippocampal neurons, neurotrophin-treated or control cells were lysed and subjected to Western blot analysis for caspase cleavage. NGF and BDNF elicited an increase in the cleaved forms of caspase-6 and caspase-3 in the cultured hippocampal neurons (Fig. 3). There is also an increase in the caspase-6 zymogen after trophin treatment, suggesting increased caspase-6 synthesis in response to the death stimulus.

View larger version (76K): [in this window] [in a new window]   Fig. 3.   Western blots showing neurotrophin-induced cleavage of caspase-6 and -3 in hippocampal neurons treated with NGF or BDNF for 4 h. A, lysates were probed with an antibody recognizing the caspase-6 zymogen and cleavage products. The arrowheads indicate cleaved fragments seen after neurotrophin treatment. The nonspecific band above the middle cleaved fragment is seen in all lanes and indicates equal loading of samples. B, lysates were probed with an antibody that recognizes only the cleaved fragment of caspase-3.

Down-regulation of caspase-8 with antisense oligonucleotides did not prevent neurotrophin-induced death of hippocampal neurons, suggesting involvement of a pathway distinct from that of Fas-mediated death. An alternative signaling pathway leading to activation of caspase-3 and -6 involves the mitochondrial release of cytochrome c, which interacts with Apaf-1 to activate caspase-9. Caspase-9 then activates downstream effector caspases including caspase-3 and -6 (30, 31). To determine whether this pathway mediated p75-activated neuronal death, cultured hippocampal neurons were treated overnight with NGF or BDNF in the presence of antisense oligonucleotides to Apaf-1 or caspase-9. Down-regulation of either Apaf-1 or caspase-9 prevented neurotrophin-induced neuronal loss (Fig. 4), providing more than 80% protection from death.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Down-regulation of proteins with vector-linked antisense oligonucleotides elucidates a pathway for p75-mediated neuronal death. Down-regulation of caspase-9 (V-AC9), Apaf-1 (V-AAPAF), or Smac/DIABLO (V-ADIABLO) protects neurons from NGF- or BDNF-induced death. Down-regulation of MIAP-3 (V-AMIAP-3) together with Smac/DIABLO restores neurotrophin-induced death. Neuronal death is reported as a percentage of untreated controls and is presented as the mean ± S.E. Each data point represents triplicate samples from four independent experiments (n = 12). *, significantly different from control, p < 0.001.

The activity of caspase-9 and -3 can be inhibited by IAPs, which thereby suppress apoptosis. The inhibitory activity of IAPs is opposed by a protein released from the mitochondria, Smac/DIABLO, which therefore promotes apoptosis by disinhibiting caspases. Down-regulation of Smac/DIABLO may thus permit the IAPs to block activity of caspase-9 and -3 and protect the neurons from neurotrophin-induced death. To test this possibility, hippocampal neurons were treated with an antisense oligonucleotide to Smac/DIABLO and exposed to NGF or BDNF overnight. The antisense oligonucleotide to Smac/DIABLO prevented neurotrophin-induced neuronal loss by more than 80% (Fig. 4). Simultaneous down-regulation of Smac/DIABLO and MIAP-3, the rodent homologue of XIAP that blocks caspase-9, -3, and -7, restored the ability of NGF and BDNF to induce neuronal death (Fig. 4), whereas down-regulation of MIAP-3 alone had no effect.

The hippocampal cultures contain a heterogenous group of neurons, of which 30-40% express p75 in the absence of a Trk receptor (6). To determine whether the neurons showing activation of caspase-3 in response to neurotrophin treatment were those expressing p75, we used the antibody to activated caspase-3 together with anti-p75 for double label immunofluorescence. This caspase-3 antibody, used for Western blot analysis in Fig. 3, recognizes only the cleaved p18 fragment and not the p32 zymogen and can therefore be used for immunostaining to detect activation of caspase-3 in situ. Cultured hippocampal neurons were treated with NGF or BDNF for 5 h and then fixed and double-labeled for p75 and activated caspase-3. Analysis by confocal microscopy demonstrated an induction of activated caspase-3 in p75⁺ neurons after NGF or BDNF treatment (Fig. 5). Nearly 40% of the neurons showed activated caspase-3 after neurotrophin treatment, which corresponds to the percentage of p75⁺ neurons that lack a Trk receptor and die in response to neurotrophins, as we have previously shown (6). The labeling for activated caspase-3 was prevented by treatment with antisense oligonucleotides to caspase-6 but not by a control (scrambled) oligonucleotide (Fig. 5f), indicating the requirement for caspase-6 in the activation of caspase-3 in this apoptotic pathway.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   NGF and BDNF induce immunostaining for activated caspase-3 in cultured hippocampal neurons. Cells were cultured for 5 days and then treated for 5 h with vehicle (a), NGF (b), or BDNF (d). The presence of the caspase-6 antisense oligonucleotide largely prevented activation of caspase-3 by NGF (c) or BDNF (e). Cells were fixed and labeled with antibodies to p75 (red) and activated caspase-3 (green). Size bar, 100 µm; magnification is the same for a-e. f, quantitation of neurons with activated caspase-3 immunostaining after treatment. 100 cells from six different fields in two wells were counted from each treatment group, and the numbers with activated caspase-3 are shown in the graph. V-AC6, vector-linked-anti-caspase-6 oligonucleotide; V-SC6, vector-linked scrambled caspase-6 oligonucleotide.

The protective effects of the caspase-9 and Apaf-1 antisense oligonucleotides demonstrated that the activation of the intrinsic caspase pathway mediated neurotrophin-induced death of hippocampal neurons. To confirm the role of the mitochondrial pathway, hippocampal neurons were treated with NGF for 5 h and double-labeled with antibodies to cytochrome c and activated caspase-3. In untreated neurons, punctate labeling for cytochrome c was detected throughout the cells, consistent with mitochondrial labeling, and no immunostaining for activated caspase-3 was detected (Fig. 6a). When cytochrome c is released from the mitochondria, the protein is diffusely distributed in the cell and undetectable by immunostaining (32). After NGF treatment, all the neurons with activated caspase-3 immunostaining no longer showed the punctate cytochrome c labeling, whereas neurons that still showed punctate cytochrome c labeling did not have activated caspase-3 labeling (Fig. 6b), showing that loss of mitochondrial cytochrome c was associated with activation of caspase-3.

View larger version (7K): [in this window] [in a new window]   Fig. 6.   NGF elicits activated caspase-3 labeling in cells with loss of mitochondrial cytochrome c. Cells were cultured for 5 days and then treated for 5 h with vehicle (a) or NGF (b). Cells were labeled with antibodies to cytochrome c (red) and activated caspase-3 (green). Size bar, 100 µm; magnification is the same for all panels.

To determine whether caspases are involved in p75-mediated death of hippocampal neurons in vivo, rats were treated with pilocarpine to induce seizures leading to neuronal degeneration (33). A previous study demonstrated expression of p75 on apoptotic neurons in this paradigm (34). To assess whether caspases were activated in the p75⁺ apoptotic neurons, rats were analyzed by double label immunofluorescence for p75 and cleaved caspase-3 or cleaved caspase-6 1 day after pilocarpine-induced seizures. Sections through the hippocampus demonstrated that both caspase-6 and caspase-3 were activated in p75⁺ neurons (Fig. 7). No labeling for either p75 or activated caspase-3 or -6 was detected in the hippocampal neurons of control rats (shown for caspase-3). Additional sections demonstrated staining for cleaved PARP, a substrate for caspase-3, in p75⁺ hippocampal neurons (not shown), indicating that this pathway of neuronal death is activated in p75⁺ neurons in vivo as well as in culture. To confirm that expression of p75 was necessary for pilocarpine to induce caspase-3 activation in hippocampal neurons, p75^/ mice were compared with wild type mice. Since the C57Bl/6 strain of mice are extremely resistant to neuronal death induced by seizures (20), we used the original p75^/ mice produced on a mixed 129/Balb/c background and compared the knockout mice with both 129 and Balb/c wild type mice. All animals displayed generalized clonus with loss of righting reflex in response to pilocarpine. In mice, seizures were allowed to proceed for 2 h from the onset of clonus before diazepam was administered. In wild type mice of both strains, p75 expression was detected on scattered hippocampal neurons by 2 h after pilocarpine treatment; however, no activated caspase-3 was detected yet at this early time point (not shown). By 1 day after seizure, as in the rats, pilocarpine treatment induced caspase-3 activation and apoptosis in p75⁺ hippocampal neurons in both wild type strains (shown for the 129 mice; Fig. 8, a and b); however, no labeling for activated caspase-3 was detected in the p75^/ animals (Fig. 8c), confirming the role for p75 in caspase-3 activation by seizure activity in vivo. The cells double-labeled for p75 and activated caspase-3 show a membranous rim of p75 staining surrounding the cytoplasm (Fig. 8b). It is clear that in the cells positive for p75 and activated caspase-3, there is condensation of the nuclear chromatin as shown by the Hoechst staining (Fig. 8b), confirming that these neurons are dying.

View larger version (137K): [in this window] [in a new window]   Fig. 7.   Pilocarpine-induced seizures elicit activation of caspase-6 and caspase-3 in p75+ hippocampal neurons in vivo. Shown are sections through the hippocampus of adult rats 1 day after treatment with saline (a and b) or pilocarpine (c, d, e, and f) double-labeled with anti-p75 (a, c, and e), anti-activated caspase-3 (b and d), or anti-activated caspase-6 (f). Size bar, 100 µm; magnification is the same for all panels. The arrows indicate double-labeled cells.

View larger version (109K): [in this window] [in a new window]   Fig. 8.   p75 is required for activation of caspase-3 by pilocarpine-induced seizures. A, double label immunostaining for p75 and cleaved caspase-3 of wild type 129 mice 1 day after pilocarpine treatment. B, high magnification of a hippocampal pyramidal neuron expressing p75 and activated caspase-3 and showing condensed chromatin indicative of an apoptotic cell. C, the hippocampus of p75/ mice 1 day after pilocarpine treatment shows no p75 labeling (as expected) and no activation of caspase-3. Size bars in a and c, 50 µm; size bar in b, 25 µm. C3, activated caspase-3; H, Hoechst nuclear stain.

Wild type and p75^/ mice were also analyzed by Fluoro-Jade B labeling to assess whether there was a decrease in the number of dying neurons in the absence of p75 after pilocarpine treatment. Fluoro-Jade B is an anionic dye that specifically labels degenerating neurons (23, 35). Fluoro-Jade B labeling demonstrated a reduction in the number of degenerating neurons in the p75^/ mice to 20% in the hippocampus and 35% in the cortex relative to wild type (Fig. 9). Thus, neuronal loss induced by pilocarpine is clearly attenuated in the absence of p75.

View larger version (124K): [in this window] [in a new window]   Fig. 9.   Neuronal death is attenuated in the absence of p75. Sections through the hippocampus (a and b) and cortex (c and d) from wild type (a and c) or p75/ (b and d) mice were stained with fluoro-jade B to label dying neurons after pilocarpine treatment. In the p75/ mice, there was a marked reduction in the number of dying neurons to 20% in the hippocampus and 35% in the cortex compared with wild type. Size bar in a, 100 µm; magnification is the same for all panels.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Activation of the p75 receptor in the absence of Trk signaling leads to neuronal death (4-6, 36), whereas activation of Trk receptors leads to regulation of a variety of neuronal functions, including survival, differentiation, and synaptic efficacy (1, 2). Thus, the consequence of neurotrophin actions in the brain depends upon the receptor and signaling pathway activated. The p75 receptor is more widely expressed during development than in the adult (37, 38) and is also highly expressed after damage in many neuronal populations (39-41), specifically on apoptotic neurons (34), suggesting that neurotrophins induce death via a p75-mediated mechanism in these situations. In vivo studies have demonstrated induction of neuronal death via p75 in developing retinal neurons (5) and lesioned facial motoneurons (42), supporting the findings that activation of this receptor can lead to apoptosis. This contrasts with the role of neurotrophins acting via Trk receptors to prevent inappropriate developmental death (43) and to act as neuroprotective agents after injury (44). Thus, neurotrophins have opposing actions on neuronal viability depending on the receptor phenotype. We have previously demonstrated that hippocampal neurons expressing p75 but lacking a Trk receptor die after treatment with neurotrophins (6). In this study, we have identified specific caspase and caspase-regulatory molecules required for neurotrophin-induced cell death. In contrast to a recent study showing p75 up-regulation on nonapoptotic neurons after injury in the striatum (45), we show that in an in vivo model of injury in the hippocampus, p75 is induced in apoptotic neurons with activation of the same death pathway defined in vitro.

Overexpression of caspases induces apoptosis (28). In contrast, mice that have a null mutation of caspase-3 (46) or caspase-9 (47, 48) show profound developmental abnormalities of the nervous system. These mice have enlarged brains with an overabundance of neurons resulting from a lack of developmental cell death, demonstrating a major role for caspase-3 and -9 in mediating developmental neuronal death (49). Mice with a null mutation of Apaf-1 have a similar phenotype (50). In contrast, mice with null mutations of caspase-1 (51), caspase-2 (52), caspase-6 (53), caspase-11 (54), and caspase-12 (55) develop normally, although there may be roles for these caspases in different types of evoked cell death (16, 55).

In these studies, we demonstrated that pseudosubstrate inhibitors that block the actions of caspase-3-like and caspase-6-like caspases partially or completely prevented NGF and BDNF-induced neuronal death. However, these inhibitors are not sufficiently specific to implicate individual caspases. In particular, VEID-FMK and IETD-FMK can prevent the actions of caspase-8 as well as caspase-6-like caspases (24-26). To gain greater specificity, we used penetratin-linked antisense oligonucleotides to down-regulate individual caspases, to determine which caspases were necessary for death. This technique has been widely and successfully used for such purposes (55-58). These experiments demonstrated that down-regulation of caspase-6 completely prevented neurotrophin-induced death, and depletion of caspase-3 gave partial protection. We further demonstrated by Western blotting that caspase-6 and caspase-3 were cleaved by neurotrophin treatment in cultured hippocampal neurons. We also see an increase in the caspase-6 zymogen after trophin treatment. Many different studies have demonstrated that caspase zymogens can increase, decrease, or not change in various death paradigms. Cleavages of caspase-6 and -3 were detected in p75⁺ neurons after pilocarpine-induced seizures in vivo. Cleaved PARP, a substrate of caspase-3, was also detected in p75⁺ hippocampal neurons after pilocarpine-induced seizures, indicating that the cleaved caspase-3 was actively promoting a death signal. In mice lacking the p75 receptor, there was an overall reduction in the number of dying neurons in the hippocampus and cortex, and no cleaved caspase-3 was detected in hippocampal neurons after pilocarpine treatment, confirming the requirement for p75 activation to stimulate this death pathway. Caspase-3 has been implicated in many paradigms of neuronal cell death; however, the role of caspase-6 in neuronal death has not been well characterized, although it has been implicated in the processing of amyloid precursor protein to the neurotoxic -amyloid (59). In agreement with our data, a recent study has also implicated caspase-6, and not caspase-8, in p75-mediated death of a cell line derived from striatal neurons (60). Although caspase-3 has been shown to cleave caspase-6 in cell-free lysates (61), caspase-6 has been shown to cleave and activate caspase-3 in dying cells (62-64). In our studies, down-regulation of caspase-6 completely prevented neurotrophin-induced death and also largely prevented activation of caspase-3, suggesting that caspase-6 contributes to activation of caspase-3 and is a critical mediator of death in this pathway.

The p75 receptor has been characterized as a member of the Fas/TNFR1 family due to characteristic structural features including the presence of cysteine repeats in the ligand binding domain (65) and a cytoplasmic death domain (14). Fas and TNFR1 activate the extrinsic caspase pathway, recruiting caspase-8 via interaction of adapter proteins with the death domain of these receptors. However, investigation of the different domains of the p75 receptor contributing to death signaling indicated that the juxtamembrane domain, rather than the death domain, of p75 was critical for induction of cell death (66), suggesting that p75 may signal distinctly from other members of the Fas/TNFR family. In our study, down-regulation of caspase-8 did not protect hippocampal neurons from p75-mediated death, supporting the suggestion that p75 signaling is different from Fas. A previous study suggested that caspase-8 might play a role in p75-mediated death of Schwann cells transfected with CrmA (67). CrmA preferentially blocks caspase-8 and -1; however, it can also block other caspases, including caspase-9, especially when overexpressed (26). Our data are consistent with a previous study indicating that caspase-8 did not mediate NGF-induced death of oligodendrocytes (68) and a recent study using an immortalized cell line derived from striatal neurons demonstrating that caspase-6 and not caspase-8 mediated p75-activated cell death (60). Those studies, together with the data reported here, indicate that the p75-activated death pathway is not analogous to Fas signaling and does not induce apoptosis by recruitment of caspase-8. In contrast, activation of c-Jun N-terminal kinase plays a critical role in p75-mediated cell death (6, 69), and c-Jun N-terminal kinase signaling is necessary for mitochondrial release of cytochrome c during UV-induced apoptosis (70). In this study, we demonstrated that loss of mitochondrial cytochrome c labeling was associated with activation of caspase-3 in response to NGF treatment. Moreover, down-regulation of caspase-9 and Apaf-1 protected neurons from neurotrophin-induced death. These data suggest a mechanism for neurotrophin-induced death of hippocampal neurons, mediated by binding to p75, involving mitochondrial release of cytochrome c and Smac/DIABLO. Interaction of cytochrome c with Apaf-1 leads to activation of caspase-9, which is facilitated by Smac/ DIABLO inhibition of IAPs (Fig. 10). Caspase-9 activation leads to cleavage of caspase-6 and -3 and subsequent cleavage of cellular substrates, such as PARP, leading to apoptosis.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Schematic diagram illustrating the caspase pathway critical for p75-mediated death of hippocampal neurons.

Many types of injury, including pilocarpine-induced seizures (71), elicit increases in NGF and BDNF expression in hippocampal and cortical neurons. Moreover, inflammatory cytokines, which are highly expressed in the brain during damage and disease, increase NGF production in glial cells in culture (72, 73) and in vivo (74). Thus, neurotrophins are abundantly produced as a consequence of brain injury. The up-regulation of p75 on neurons after central nervous system injury, together with the elevated levels of neurotrophins, suggest that activation of this death pathway may serve to eliminate neurons that are compromised by damage. The complete lack of caspase-3 activation in the hippocampus of p75-null animals after pilocarpine demonstrates an absolute requirement for p75 in the activation of this death pathway in this model. Thus, neurotrophin actions in the brain influence neuronal survival or death, according to which receptor and signaling pathways are activated, with important consequences for the potential use of these factors as therapeutic agents in neurodegenerative disease.

	ACKNOWLEDGEMENTS

NGF was generously provided by Genentech. BDNF was a gift from C. F. Ibáñez. We thank Kelly Milton and Seonia Hutchinson for excellent technical assistance and L. A. Greene and C. F. Ibáñez for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by grants from the National Science Foundation (to W. J. F.), NINDS, National Institutes of Health (to W. J. F. and C. M. T.), and Muscular Dystrophy Association (to C. M. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biological Sciences, Rutgers University, 101 Warren St., Newark, New Jersey 07102. Tel.: 973-353-1160; Fax: 973-353-1007; E-mail: wilmaf@andromeda.rutgers.edu.

Published, JBC Papers in Press, July 3, 2002, DOI 10.1074/jbc.M205167200

	ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; IAP, inhibitor of apoptosis protein; PARP, poly(ADP-ribose) polymerase; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; FMK, fluoromethylketone.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES