A Role for the p75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress*

Background: The p75 neurotrophin receptor (p75NTR) promotes neurodegeneration during development and in response to cellular injury. Results: Reactive oxygen species promoted cleavage of p75NTR, leading to axonal degeneration and apoptosis. Conclusion: Oxidative stress activates intracellular p75NTR signaling to induce neurodegeneration. Significance: These results suggest a novel mechanism through which p75NTR contributes to neurodegeneration associated with cellular injury or pathological conditions. The p75 neurotrophin receptor (p75NTR) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75NTR has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75NTR signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75NTR−/− mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75NTR via a ligand-independent mechanism. Previous studies have established that proteolysis of p75NTR by the metalloprotease TNFα-converting enzyme and γ-secretase is necessary for p75NTR-mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease- and γ-secretase-dependent cleavage of p75NTR. Pharmacological blockade of p75NTR proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75NTR is necessary for oxidant-induced neurodegeneration. In vivo, p75NTR−/− mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligand-independent cleavage of p75NTR, resulting in axonal fragmentation and neuronal death.

The p75 neurotrophin receptor (p75 NTR ) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75 NTR has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75 NTR signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75 NTR؊/؊ mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75 NTR via a ligand-independent mechanism. Previous studies have established that proteolysis of p75 NTR by the metalloprotease TNF␣-converting enzyme and ␥-secretase is necessary for p75 NTR -mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease-and ␥-secretase-dependent cleavage of p75 NTR . Pharmacological blockade of p75 NTR proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75 NTR is necessary for oxidant-induced neurodegeneration. In vivo, p75 NTR؊/؊ mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligandindependent cleavage of p75 NTR , resulting in axonal fragmentation and neuronal death.
The p75 neurotrophin receptor (p75 NTR ) 2 is a multifunctional transmembrane protein originally identified by its ability to bind members of the neurotrophin family, which consists of NGF, BDNF, neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4). Although p75 NTR has been studied for over 20 years, its signaling mechanisms remain poorly understood, primarily because of the complexity of ligands, coreceptors, and cytosolic interactors that regulate p75 NTR in a cell-specific manner (1). However, one established mechanism of p75 NTR signaling occurs through regulated proteolysis of the receptor (2)(3)(4). Within this process, p75 NTR is first cleaved in its extracellular domain by the metalloprotease TNF-␣-converting enzyme (TACE, also known as ADAM17). Subsequently, the remainder of the membrane-bound receptor, termed the p75 NTR C-terminal fragment, is cleaved within its transmembrane region by the ␥-secretase complex, thereby releasing the cytosolic intracellular domain (p75 NTR ICD). These cleavage events promote a variety of downstream signals with differing cellular functions, including nuclear translocation of neurotrophin receptor-interacting factor (5) and prolonged activation of JNK (6) to promote apoptosis, activation of the small GTPase Rho to inhibit neurite outgrowth (7), and enhancement of Trk receptor signaling to promote cell survival (8).
Studies of p75 NTRϪ/Ϫ animals have revealed that the receptor is critical for naturally occurring developmental apoptosis within the retina, superior cervical ganglia, spinal cord, and basal forebrain (9 -12). In addition to promoting cellular apoptosis, more recent findings have demonstrated that p75 NTR also causes the breakdown of mislocalized axons because the receptor has been found to promote degeneration of aberrantly sprouting septal cholinergic axons, thereby preventing the fibers from growing into myelinated tracts (13), and to mediate developmental pruning of sympathetic axons projecting to the iris (14). Hence, p75 NTR functions as a regulator of neurodevelopment, ensuring the removal of unsuitable neurons or neuronal projections. Apart from these roles, however, numerous studies have indicated that p75 NTR also promotes neuronal apoptosis in response to cellular injuries or pathological conditions. The receptor is necessary for programmed cell death caused by seizures (15,16), corticospinal axotomy (17,18), and spinal cord injury (19). Additionally, p75 NTR has been linked to neurodegeneration occurring in models of Alzheimer disease (20), amyotrophic lateral sclerosis (21), and ischemia (22)(23)(24)(25). Therefore, p75 NTR appears to function as a stress-activated receptor that promotes degeneration in response to neuronal injury.
Of the numerous pathological conditions in which p75 NTR signaling has been implicated, nearly all are associated with oxidative stress (26 -31). Therefore, we speculated that reactive oxygen species (ROS) activate p75 NTR , leading to neurodegeneration. During conditions that promote oxidative stress, free radicals oxidize bioactive molecules such as proteins and nucleic acids, thereby disrupting enzymatic activities and signaling pathways, increasing protein degradation, and causing DNA damage. Additionally, unstable ROS attack polyunsaturated fatty acids within lipid membranes in a process termed lipid peroxidation, resulting in the production of more stable, yet still actively damaging molecules. A major end product of lipid peroxidation is 4-hydroxynonenal (HNE), a highly reactive ␣,␤-unsaturated aldehyde (32). HNE is widely regarded as a key mediator of neuronal apoptosis induced by oxidative stress and has been shown to induce apoptosis of neurons in response to a multitude of conditions associated with increases in ROS (32)(33)(34)(35). HNE directly associates with and modifies transmembrane receptors, cytosolic enzymes, and DNA, ultimately causing the activation of apoptotic signaling cascades (35). In addition, HNE can disrupt mitochondrial function or deplete cellular antioxidants, leading to further production of ROS (36 -39). In this study, we used sympathetic neurons exposed to HNE to model oxidative stress-induced neurite degeneration and apoptosis, evaluating whether p75 NTR has a functional role. Treatment of sympathetic neurons with HNE caused neurite degeneration and neuronal apoptosis, which was associated with proteolysis of p75 NTR . Importantly, deletion of p75 NTR or inhibition of receptor cleavage attenuated neurite degeneration and death. These events were not associated with increased neurotrophin production and did not require neurotrophin binding, therefore suggesting a novel, ligand-independent mechanism of p75 NTR activation occurring in response to oxidative stress.

EXPERIMENTAL PROCEDURES
Sympathetic Neuron Culture-All experiments with animals were approved by the Animal Care and Use Committee at Vanderbilt University. Superior cervical ganglia were dissected from postnatal day 5/6 Sprague-Dawley rats, C57BL/6J mice, or C57BL/6J p75 NTR(exonIII)Ϫ/Ϫ mice and dissociated with 0.08% trypsin (Worthington) and 0.3% collagenase (Sigma). Dissociated cells were then plated at a density of 5000 -7000 neurons/ 0.7 mm 2 on 8-well chamber slides (Thermo Scientific) or cell culture plates coated with poly-D-lysine (MP Biomedicals) and laminin (Invitrogen). All neurons were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), 40 ng/ml nerve growth factor (Harlan), 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 g/ml streptomycin (Invitrogen). To inhibit the proliferation of non-neuronal cells, the neurons were treated with 5-10 M cytosine arabinofuranoside (Sigma) 24 h after plating. Following 3 days of exposure, cytosine arabinofuranoside was removed for 24 h, and the neurons were then treated with the indicated concentrations of HNE or different pharmacological reagents. During pilot studies, we observed increased toxicity of HNE at lower cell densities, as has been reported for 6-hydroxydopamine (6-OHDA) (40), and, therefore, for all experiments, neuron plating densities and cytosine arabinofuranoside exposures were equivalent across all experimental conditions.
Cell Treatments-HNE was produced as described previously (41) as well as obtained from Calbiochem, and its concentration was determined by measuring the optical density at 224 nm and using a molar extinction coefficient of 13,750. HNE is a highly reactive lipid aldehyde, and some variability in toxicity was observed between different batches of the compound. Therefore, every effort was made to limit exposure of the HNE to oxygen, including its storage at Ϫ80°C under inert gas. For experiments with 6-OHDA (Sigma), the chemical was dissolved in cold phosphate-buffered saline with 0.02% ascorbate immediately prior to each experiment. For assessment of neurotrophin involvement in HNE-induced neurite degeneration and apoptosis, C57BL/6J sympathetic neurons were pretreated for 30 min with control serum or with 9650 immune serum containing ligand-blocking antibody specific for the p75 NTR extracellular domain (␣-p75 ECD, 1:500) (42). The neurons were then exposed to 12 M HNE for 20 h before fixation and quantification of neurite degeneration or cell survival.
Quantification of Neurite Degeneration-Analyses of neurite degeneration were performed as described previously (43)(44)(45), with slight modifications. Following the indicated treatments, sympathetic neurons in 8-well chamber slides were fixed with 4% paraformaldehyde and visualized via a ϫ20 optical lens on a Leica inverted fluorescence microscope. Phase-contrast images of five fields of view per well were captured with 16 -ms exposure by a Nikon DXM1200C digital camera. To ensure accurate measurement of neurites, images were captured from blindly selected regions with well separated axon tracts. Using an automated method of image analysis, the fragmentation of the neurites was then measured. Levels of neurite degeneration are reported as a degeneration index (DI), which is the ratio of the fragmented neurite area over the total neurite area. To process images for DI calculation, the auto-level function of the GNU image manipulation program software was first used to adjust image gray levels to objectively provide uniform background intensity to all of the images. ImageJ software was then utilized to binarize the image and to remove all cell bodies, rendering an image composed of black neurites on a white background. Although healthy neurites appear continuous, degenerating neurites have a disrupted, particulate structure because of blebbing and fragmentation. To measure the area of fragments from degenerating neurites, the Particle Analyzer algorithm of ImageJ was applied to identify regions of fragmentation on the basis of size (20 -10,000 pixels) and circularity (0.2-1.0). The total area of these detected neurite fragments was then divided by the total black neurite area to determine the DI. In agreement with other studies (43), a DI of 0.2 or greater accurately indicated neurite degeneration, whereas a DI of 1.0 would theoretically represent neurites that have completely degenerated into fragmented particles.
Quantification of Neuronal Death-Following the indicated treatments, sympathetic neurons in 8-well chamber slides were fixed with 4% paraformaldehyde and immunostained with neuron-specific anti-TUJ1 primary antibody (neuronal class III ␤-tubulin, 1:500, Covance, catalog no. MMS-435P) and Alexa Fluor 488 secondary antibody (1:1000, Invitrogen, catalog no. A11001). Slides were then mounted using Vectashield with 4,6diamidino-2-phenylindole (Vector Labs), and the neurons were scored blindly as apoptotic or non-apoptotic on the basis of the appearance of the nucleus, apoptotic nuclei being condensed or fragmented. At least 75 TUJ1-positive neurons were counted per condition in all experiments.
Measurement of Protein Carbonylation-Detection of protein carbonylation was performed using the Oxyblot kit (Millipore) following the instructions of the manufacturer. Briefly, cell lysates were treated with 2,4-dinitrophenylhydrazine to derivatize protein side chain carbonyl groups to 2,4-dinitrophenylhydrazone. Separately, as a negative control, aliquots of all cell lysates were treated with solution lacking 2,4-dinitrophenylhydrazine. Protein samples were then separated by polyacrylamide gel electrophoresis and analyzed by Western blotting using an anti-2,4-dinitrophenyl antibody.
In Vivo Assessment of 6-OHDA-induced Neurite Degeneration-Adult, age-matched p75 NTR(exonIII)Ϫ/Ϫ or ϩ/ϩ mice were administered 100 mg/kg 6-OHDA-hydrobromide (Sigma, freshly prepared in phosphate-buffered saline (pH 7.3) supplemented with 0.02% ascorbate) or vehicle solution by intraperitoneal injection once daily for 2 days. Animals were sacrificed 1 week later, and the spleens were collected for determination of norepinephrine concentrations or used for immunohistochemical localization of tyrosine hydroxylase (TH)-immunoreactive axons. For the latter studies, animals were perfused transcardially with heparized saline, followed by 4% paraformaldehyde. The spleens were then collected, post-fixed, cryosectioned at 12 m, and collected onto slides. Noradrenergic axons were detected by immunofluorescent localization of TH-immunoreactivity using a mouse anti-TH antibody (1:750, Abcam, Cambridge, MA). Splenic norepinephrine concentrations were determined by HPLC with electrochemical detection, following our method described previously (46).

P75 NTR Is Required for HNE-induced Neuronal Apoptosis-
Previous studies have demonstrated that sympathetic neurons exposed to the naturally produced oxidant HNE undergo caspase-dependent programmed cell death (47). To examine the effect of HNE on neuronal survival in our culture system, rat sympathetic neurons were treated with a range of concentrations of HNE and scored for apoptosis on the basis of nuclear morphology. HNE dose-dependently induced death of sympathetic neurons (Fig. 1A). We confirmed that the cell death induced by HNE was apoptotic, indicated by a marked increase in the levels of cleaved caspase 3 (Fig. 1B). HNE is known to promote apoptosis through its ability to form protein adducts and modify cell signaling (32). Additionally, HNE can propagate oxidative stress through mitochondrial impairment or depletion of antioxidants (36 -39). Because amino acid side chains are abundant targets of oxidation by reactive oxygen species and lipid-derived ␣,␤-unsaturated aldehydes, increased protein carbonylation is commonly used as a biomarker of oxidative stress (48). Treatment of sympathetic neurons with HNE caused a rapid increase in protein carbonylation, observed within 30 min of HNE treatment (Fig. 1C). These results indicate that exposure of sympathetic neurons to HNE models oxidative stress-induced apoptosis.
The p75 NTR has been implicated as a mediator of apoptosis in many pathological conditions involving oxidative stress (16, 20 -24). Therefore, we studied sympathetic neurons exposed to HNE to evaluate whether p75 NTR contributes to oxidative stress-induced neuronal apoptosis. Sympathetic neurons were cultured from p75 NTR knockout or wild-type mice and assessed for survival following exposure to various concentrations of HNE. Compared with neurons from wild-type mice, sympathetic neurons lacking p75 NTR were protected significantly from HNE-induced apoptosis (Fig. 2, A and B). These findings indicate that p75 NTR contributes to neuronal apoptosis induced by HNE.
HNE Stimulates p75 NTR -dependent Neurite Degeneration-During survival analysis of sympathetic neurons exposed to 12 M HNE, we observed extensive fragmentation of neuronal processes throughout the culture despite less than maximal cell death. Although the ability to induce neuronal apoptosis has been the most studied function of p75 NTR , recent investigations have also demonstrated a function for the receptor in promoting axonal degeneration (13,14,24). Because of our observations and because numerous pathological conditions related to oxidative stress have also been associated with axonal degeneration (49,50), we hypothesized that p75 NTR mediates the degeneration of axons caused by HNE. Therefore, sympathetic neurons were treated with 12 M HNE, and axonal degeneration was quantified from phase-contrast images. Using an automated method of image analysis, we measured the degeneration index, the ratio of the fragmented neurite area over the total neurite area (43)(44)(45)51). Remarkably, although HNEtreated neurons from wild-type animals had substantial neurite fragmentation, the processes from cells lacking p75 NTR were healthy and intact (Fig. 3A). Indeed, on the basis of the degeneration index, the p75 NTRϪ/Ϫ neurons were protected significantly (Fig. 3B). These results reveal that p75 NTR is necessary for HNE-induced neurite degeneration and suggest that oxidative stress invokes p75 NTR signaling to promote axon fragmentation.
Induction of p75 NTR -mediated Neurite Degeneration and Apoptosis by HNE Occurs through a Ligand-independent Mechanism-Because of the effects of p75 NTR on HNE-induced neurite degeneration and apoptosis, we speculated that oxidative stress promotes neurotrophin or proneurotrophin release, thereby leading to autocrine or paracrine activation of p75 NTR . We considered BDNF the most likely candidate because BDNF can be produced by sympathetic neurons (52,53) and can promote their apoptosis through activation of p75 NTR (5,6,11). Therefore, we collected lysates from neurons treated with 25 M HNE, the maximally effective dose, and measured BDNF by Western blotting. Surprisingly, however, no BDNF was detected, even after treatment with HNE (Fig. 4A). We next analyzed other neurotrophins. The precursor form of NGF, proNGF, is a known proapoptotic ligand for p75 NTR (17,19,54), whereas mature NGF is a well defined prosurvival factor for sympathetic neurons (55)(56)(57). We detected no proNGF in the neurons and found only low levels of mature NGF, likely because of its internalization from the media, which were unchanged in sympathetic neurons treated with vehicle or HNE (Fig. 4A). Similar analyses revealed that sympathetic neurons also do not produce NT-3 or NT-4 in response to HNE (Fig. 4A). Although substantial levels of proapoptotic neurotrophins would need to be present to induce neuronal death in the presence of NGF, which was in the media, it is theoretically possible that neurotrophins remaining in the neurons were below our detection limit. Therefore, we next used an antibody to the extracellular domain of p75 NTR that blocks neurotrophin-mediated activation of the receptor to further explore whether HNE-induced axon degeneration and apoptosis requires activation of p75 NTR by neurotrophins. As observed in previous studies (42), blockade of the extracellular domain with the p75 NTR antibody prevented BDNF-induced death of sym- pathetic neurons. However, the antibody failed to prevent HNE-induced neurite degeneration and apoptosis (Fig. 4, B and  C). Together, these data suggest that oxidative stress promotes p75 NTR -mediated axonal degeneration and apoptosis through a ligand-independent mechanism.
HNE Stimulates Proteolytic Cleavage of p75 NTR -Because our results indicated that the effects of HNE did not require ligand binding to p75 NTR , we hypothesized that oxidative stress triggers intracellular receptor signaling. We demonstrated previously that p75 NTR -mediated apoptosis in sympathetic neurons requires proteolytic cleavage of the receptor, first by the metalloprotease TACE/ADAM17 and then by ␥-secretase (5, 6). Therefore, we investigated whether HNE stimulates p75 NTR proteolysis. Sympathetic neurons were treated with various concentrations of HNE and subjected to Western blot analysis using an antibody that recognizes the intracellular domain of p75 NTR . Compared with neurons treated with vehicle, HNEtreated neurons had a robust and dose-dependent increase in the 25-and 20-kDa fragments of p75 NTR corresponding to the p75 NTR C-terminal fragment and p75 NTR ICD, respectively (Fig. 5A). Cleavage of p75 NTR in response to HNE was observed even after just 6 h of treatment (Fig. 5B), which was before apoptosis was visually apparent (data not shown), suggesting that proteolysis of the receptor precedes cell death. No change in the total expression level of p75 NTR was observed in response to HNE (Fig. 4D), indicating that cleavage of p75 NTR occurs through regulated activation of proteases rather than because of up-regulation of the full-length receptor. Because TACE and ␥-secretase have been shown to mediate cleavage of p75 NTR in response to neurotrophins, we hypothesized that similar enzymatic activities may be induced by oxidative stress. Treatment of sympathetic neurons with the TACE inhibitor TAPI-1 or with the ␥-secretase inhibitor DAPT blocked HNE-induced cleavage of p75 NTR (Fig. 5, C and D), thus indicating that HNE stimulates proteolytic cleavage of p75 NTR by TACE and ␥-secretase.
Cleavage of p75 NTR Is Required for HNE-induced Neurite Degeneration and Apoptosis-To determine whether proteolysis of p75 NTR is required for HNE-induced axon degeneration and apoptosis, we next blocked cleavage of p75 NTR by pretreating sympathetic neurons with the TACE inhibitor TAPI-1 and then assessed neurite integrity and neuronal death following exposure to HNE. Compared with neurons pretreated with vehicle, HNE-induced neurite fragmentation was reduced dramatically in sympathetic neurons pretreated with TAPI-1 (Fig.  6, A and B). Similarly, HNE-induced apoptosis was decreased significantly in neurons pretreated with TAPI-1 (Fig. 6C). Hence, receptor proteolysis is required for p75 NTR -mediated axon degeneration and apoptosis induced by HNE.
Oxidative Stress-associated Axonal Degeneration Requires p75 NTR in Vivo-We next sought to evaluate the effects of p75 NTR in axonal degeneration induced by oxidative stress in vivo. Because our aim was to promote oxidative stress specifically in neurons, we chose to use 6-OHDA, which is selectively taken up in cells expressing catecholinergic transporters (58), rather than HNE, which reacts with a wide variety of cell types (35). 6-OHDA is a neurotoxin that has long been used systemically to selectively induce degeneration of sympathetic axons  3). B, quantification of neuronal apoptosis induced by HNE after pretreatment with ligand-blocking ␣-p75 ECD antibody. Sympathetic neurons were pretreated with control serum or ␣-p75 ECD for 30 min and then exposed to 12 M HNE for 20 h. The neurons were then fixed, labeled with DAPI, and scored for apoptosis. To control for antibody efficacy, sympathetic neurons were pretreated similarly with ␣-p75 ECD before exposure to BDNF. After maintaining the neurons in 20 ng/ml NGF, the neurons were rinsed to remove the NGF and, to promote survival, refed with medium containing 12.5 mM KCl. The neurons were then pretreated with ␣-p75 ECD for 30 min, followed by 200 ng/ml BDNF for 24 h. Although ␣-p75-ECD significantly blocked BDNF-induced apoptosis, no significant effect of ␣-p75 ECD on HNE-induced cell death was observed. Data are mean Ϯ S.E. *, p Ͻ 0.05; NS, not significant; Student's t test). C, quantification of neurite degeneration after treatment of sympathetic neurons with 12 M HNE for 20 h following pretreatment with control serum or immune serum containing antibody specific for the ␣-p75 ECD for 30 min. Pretreatment with ␣-p75 ECD caused no significant change in neurite degeneration after exposure to 12 M HNE. Data are mean Ϯ S.E. (ANOVA with Bonferroni post hoc analysis). D, representative Western blot analysis of full-length p75 NTR from lysates of rat sympathetic neurons treated for 6 h with vehicle or 25 M HNE (n ϭ 3).
that express the norepinephrine transporter (59,60). It is thought to promote the degeneration of catecholaminergic neurons primarily by increasing intracellular levels of reactive oxygen species, partially because of its tendency to undergo auto-oxidation to generate the hydroxyl radical, quinones, and other reactive species (58,61,62). Before utilizing 6-OHDA to model oxidative stress in vivo, we first tested whether 6-OHDA activates p75 NTR signaling in cultured sympathetic neurons. As we observed following treatment with HNE, exposure of sympathetic neurons to 15 M 6-OHDA caused cleavage of p75 NTR , promoting a robust increase in the p75 NTR C-terminal and p75 NTR ICD fragments (Fig. 7A). We next administered 6-OHDA to adult wild-type or p75 NTR knockout mice to promote degeneration of sympathetic axons in vivo. One week after 6-OHDA treatment, a marked loss of TH-immunoreactive axons in the spleen was observed in wild-type mice (Fig.  7B). Interestingly in p75 NTRϪ/Ϫ mice treated with vehicle, the density of TH-immunoreactive axons in the spleen appeared lower than in vehicle-treated wild-type control animals. However, there also appeared to be partial protection from 6-OHDA-induced degeneration in the p75 NTR -null mice (Fig.  7B). To quantitatively assess the level of the sympathetic innervation, we determined the splenic norepinephrine content by HPLC. Administration of 6-OHDA caused a significantly greater loss of splenic norepinephrine in wild-type mice than in p75 NTR knockout mice (Fig. 7C), indicating that p75 NTR contributes to 6-OHDA-induced axonal degeneration in vivo.
Additionally, expression of the norepinephrine transporter was similar in sympathetic ganglia of wild-type and p75 NTRϪ/Ϫ mice (Fig. 7D), and, therefore, protection of the null animals from 6-OHDA-induced degeneration was likely not the result of altered 6-OHDA transport. These findings, together with the results obtained from cultured sympathetic neurons, suggest that oxidative stress promotes ligand-independent cleavage of p75 NTR by TACE and ␥-secretase, thereby leading to axonal degeneration and neuronal apoptosis.

DISCUSSION
The p75 NTR is up-regulated in response to a variety of conditions involving oxidative stress (16, 20 -23, 63), suggesting that the receptor may contribute to the associated cell death. However, a direct role for p75 NTR as an apoptotic mediator in response to oxidative stress had not been established. Here, we demonstrate that HNE, an endogenous product of oxidative stress, activates p75 NTR signaling by initiating proteolysis of the receptor, which results in axonal degeneration and neuronal apoptosis.
Oxidative stress can promote death through a variety of cell signaling mechanisms. Therefore, blocking an individual pathway may not be sufficient to confer significant protection. That neurons lacking p75 NTR were markedly protected from axonal degeneration and apoptosis induced by HNE or 6-OHDA is, therefore, quite remarkable because it indicates that p75 NTR is a critical regulator of neuronal responses to oxidative stress. Nev- ertheless, other signaling pathways also likely contribute to HNE-induced apoptosis because some apoptosis, although reduced, was still detected in cultures of p75 NTRϪ/Ϫ neurons exposed to the highest tested concentrations of HNE. Results from these studies were obtained from both cultured sympathetic neurons and from sympathetic target tissues in vivo. Sympathetic neurons are susceptible to a variety of neurodegenerative conditions. For example, they develop neurofibrillary tangles in association with tauopathies or myotonic dystrophy (64,65). Additionally, apart from the lower brainstem and olfactory bulb, peripheral autonomic nuclei are among the earliest cell populations affected by Parkinson disease, and sympathetic neurons of Parkinson disease patients are susceptible to Lewy pathology and progressive neurodegeneration (66 -68). The oxidant 6-OHDA has long been used to mimic Parkinson disease in the CNS and was initially characterized for its ability to selectively induce degeneration of sympathetic nerve terminals (58,68). Our findings suggest that the activation of p75 NTR by this oxidant plays a key role in promoting the breakdown of these axons. Interestingly, p75 NTR has also been detected in neurons of the substantia nigra (69), and the receptor has been reported to be up-regulated in a mouse model with Parkinsonian-like neuronal loss and motor deficits (63,70). Oxidative stress is widely regarded as a contributing factor to the pathogenesis of Parkinson disease (29), and, therefore, induction of p75 NTR signaling by reactive oxygen species may contribute to neurodegeneration caused by the disorder.
Interestingly, we did not observe an up-regulation of the p75 NTR in response to HNE. These findings suggest that the reported increases in p75 NTR expression observed under conditions associated with oxidative stress are likely not due to oxidants acting directly on neurons but, instead, are the result of neighboring glial cell activation, leading to the production of cytokines. Choi and Friedman (73,74) have shown that proinflammatory cytokines such as TNF␣ and IL-1␤, which can be released by microglia and astrocytes (71,72), up-regulate the expression of p75 NTR .
Although most investigations of p75 NTR -mediated cell death have focused on neurotrophin-or proneurotrophin-induced apoptosis, studies over expressing recombinant p75 NTR or its cleavage fragments have revealed the potential for ligand-independent apoptotic signaling by the receptor (75)(76)(77)(78)(79). Other non-apoptotic functions of p75 NTR have also been reported to occur independently of neurotrophin binding, such as inhibition of fibrinolysis through down-regulation of the serine protease tissue plasminogen activator (80). We did not observe any induction of NGF, BDNF, NT-3, or NT-4 expression in response to HNE, and use of a ligand-blocking antibody failed to prevent HNE-induced neurite degeneration and apoptosis, suggesting that initiation of these functions by p75 NTR occurs through a ligand-independent mechanism.
Numerous studies have demonstrated that ROS activate TACE. For example, H 2 O 2 has been found to activate TACE through a mechanism suggested to involve oxidative disruption of inhibitory interactions between the TACE prodomain and the Zn 2ϩ -containing catalytic site (81). More recently, a study by Walcheck and co-workers (82) revealed that the activity of purified TACE lacking its prodomain and intracellular region is enhanced by H 2 O 2 . Their results indicate that oxidation of conserved CXXC motifs within the extracellular domain of TACE promotes its activation (82). These and other studies (83,84) demonstrate that ROS can activate TACE through multiple mechanisms, and, therefore, we hypothesized that similar mechanisms could link oxidative stress to cleavage of p75 NTR . Fitting with this hypothesis, treatment of sympathetic neurons with HNE promoted the robust cleavage of p75 NTR , indicating that oxidative stress promotes the activation of the regulatory proteases of the receptor. Pretreatment with the matrix metalloprotease and TACE inhibitor TAPI-1 or the ␥-secretase inhibitor DAPT blocked HNE-induced p75 NTR cleavage. Although cleavage of the p75 NTR extracellular domain by metalloproteases other than TACE is also feasible, previous work has demonstrated that TACE is required for p75 NTR cleavage in sympathetic neurons (6). Induction of p75 NTR cleavage was observed not only after exposure of sympathetic neurons to HNE but also after treatment with 6-OHDA, indicating that different oxidants are capable of initiating p75 NTR signaling. Therefore, our results support a model in which oxidative stress promotes ligand-independent cleavage of p75 NTR by TACE and ␥-secretase, leading to axonal degeneration and programmed cell death. Although our findings demonstrate that oxidants can trigger activation of p75 NTR -mediated apoptotic signaling in neurons, a previous report using PC12 cells found the intracellular domain of the receptor to have antioxidant capability, thereby conferring resistance to ROS (85). Because p75 NTR has been shown to have cell-specific effects on survival, cleavage of the receptor in response to oxidative stress may confer death in specific populations of postmitotic neurons, but similar signaling mechanisms may lead to cell survival in other non-mitotic cell types. Fitting with this hypothesis is the fact that p75 NTR cleavage has been reported to promote cell survival by enhancing Trk receptor signaling in PC12 cells (8,86), but, in sympathetic neurons, cleavage of p75 NTR induces programmed cell death (5,6). Thus, p75 NTR may regulate cell survival in different cell populations through similar proteolytic signaling mechanisms that lead to cell-specific physiological responses.
Although previous studies have demonstrated that p75 NTR mediates axonal degeneration as part of developmental pruning (14), our findings indicate that this function of the receptor is also engaged in the response to oxidative stress. Blocking cleavage of p75 NTR with the metalloprotease inhibitor TAPI-1 significantly protected sympathetic neurons from HNE-induced neurite degeneration as well as apoptosis, indicating that proteolysis of the receptor is required for oxidative stress-induced neurodegeneration. These results provide the first evidence that p75 NTR -mediated axonal degeneration requires receptor proteolysis, similar to p75 NTR -mediated inhibition of axon out-growth and neuronal apoptosis. Although neuronal death and axonal degeneration were correlated in our in vitro studies, administration of 6-OHDA in vivo caused axonal loss without leading to apoptosis of sympathetic neurons (data not shown). These findings are in agreement with earlier studies of 6-OHDA administration in which axonal degeneration was detected without sympathetic neuron loss (59,60,87). Therefore, these functions of the receptor appear to have similar upstream components but, in particular situations, produce different functional outcomes. Further studies are needed to understand how the degenerative signaling of p75 NTR can be confined so that axonal regression occurs without neuronal apoptosis.