Transgenic Mice Expressing the Intracellular Domain of the p75 Neurotrophin Receptor Undergo Neuronal Apoptosis

MATERIALS AND METHODS

Creation of transgenic animals, genotyping, and breeding.A Tα1 minigene cloning cassette was constructed in which the 1.1 kb Tα1 α-tubulin promoter (Gloster et al., 1994) was separated from SV40 intron and polyadenylation sites by a short polylinker. For the Tα1:intracellular domain (ICD) construct, a cDNA encoding an initiator methionine, followed by amino acids 276–425 of the rat p75^NTR (Radeke et al., 1987), was subcloned into the polylinker. Completed minigenes were purified free of vector sequence and microinjected into pronuclei to produce founder transgenic animals at the Canadian NeuroScience Network Transgenic Core Facility (McGill University). Genotyping was performed on tail (postnatal animals) or hindlimb (embryos) DNA by Southern blotting with a fragment of the Tα1 α-tubulin promoter sequence or by PCR with primers derived from the Tα1 α-tubulin promoter and p75^NTR sequence. Seven lines of Tα1:ICD mice were identified; four of these were characterized to a moderate degree, and two were examined in detail (4163 Tα1:ICD and 4173 Tα1:ICD). For some studies Tα1:ICD lines were crossed to mice carrying a separate transgene in which the Tα1 α-tubulin promoter drives expression of nuclear LacZ (Gloster et al., 1994; Bamji and Miller, 1996). The resultant double transgenic lines were designated 4163 Tα1:ICD × Tα1:nlacZ and 4173 Tα1:ICD × Tα1:nlacZ.

Creation of stable PC12 sublines. pRC-CMV (Invitrogen, San Diego, CA) driving expression of the truncated intracellular domain of rat p75^NTR described above was transfected into PC12 cells, using Lipofectamine (Life Technologies, Gaithersburg, MD), and stable clones were selected in 400 μg/ml G418. Sixteen clones were analyzed for expression of the p75^NTR-ICD by immunoblot, as described below, and six lines showing expression were retained. The results shown in Figure 7 were repeated in two separate clonal lines.

Animals and surgical procedures. For biochemistry, animals were anesthetized with sodium pentobarbital (35 mg/kg), and tissues were removed immediately and processed for immunoblots. For morphometric analyses, animals were anesthetized with sodium pentobarbital (35 mg/kg) for 30 min, and ganglia were removed and immersion-fixed in 4% paraformaldehyde in phosphate buffer (PB) for 1 hr to overnight at 4°C (for morphometric analysis of peripheral ganglia) or in 1.6% glutaraldehyde in 0.1 m PB, pH 7.3 (for electron microscopy). For morphometric analysis of the cortex, animals were perfused transcardially with 4% paraformaldehyde in PB. Subsequent to transcardial perfusion, the brains were removed and post-fixed in 4% paraformaldehyde in PB. The brains subsequently were cryoprotected in graded sucrose solutions, sectioned on the cryostat, and stained with cresyl violet.

For facial nerve lesion studies, animals were anesthetized with Metophane, and the main branch of the facial nerve was resected as it exited the stylomastoid foramen, taking care not to injure the adjacent blood vessels. Seven days after axotomy, animals were anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. Brains were cryoprotected in graded sucrose solutions and sectioned on the cryostat. In total, three control and transgenic animals from each line were analyzed.

Histological and morphometric analyses. Embryos were prepared for β-galactosidase staining essentially as described (Gloster et al., 1994). In brief, embryos were fixed for 60 min at 4°C in freshly prepared 4% paraformaldehyde in 0.1 mNaH₂PO₄, pH 7.3, and then rinsed three times in 0.1 m NaH₂PO₄, pH 7.3, 2 mm MgCl₂, 0.01% sodium deoxycholate, and 0.02% Nonidet P40. The staining reaction was performed in rinse buffer supplemented with 1 mg/ml X-gal, 5 mm K₃Fe(CN)₆, and 5 mm K₄FE(CN)₆, typically for 3–5 hr at 37°C. Embryos were post-fixed up to 48 hr in 4% paraformaldehyde. For sectioning, tissues were cryopreserved and cryosectioned. For terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis, embryos first were stained in X-gal, post-fixed for 24 hr in 4% paraformaldehyde, cryosectioned, and then TUNEL-labeled by an Apoptag kit (Oncor, Gaithersburg, MD) as per the manufacturer’s instructions.

Morphometric analyses of motor and sensory neurons were performed with a computer-based image analysis system that prevents double measurement of profiles (Biocom, Paris, France). For counts of facial motor neurons, cryoprotected brains were frozen and mounted for cryosectioning, and 16 μm coronal sections were collected through the extent of the facial nuclei. Slides subsequently were stained with cresyl violet to visualize nuclei, and all of the neuronal profiles with a distinct nucleolus were counted on every fifth section. Quantitative comparisons were made, in all cases, between injured facial motor neurons on one side and the contralateral, uninjured motor neurons on the other side. Similar sections through the facial nucleus were analyzed for neuronal size. For determination of sensory neuron number, L3, L4, and L5 dorsal root ganglia (DRG) were serial-sectioned at 7 μm, and neurons containing a distinct nucleolus were counted in every fourth section, as per Coggeshall (Coggeshall, 1992; Coggeshall and Lekan, 1996). This approach does not correct for split nucleoli. For comparisons of cortical neuron number, cresyl violet-stained coronal sections at the appropriate level were photographed, and the total number of neuronal profiles was counted in 530-μm-wide strips that extended from pia to corpus collosum.

For electron microscopy of the dorsal cutaneous nerve (DCN), the DCN was dissected from adult animals, immersion-fixed overnight in 1.6% glutaraldehyde in 0.1 m PB, pH 7.3, post-fixed in 1% OsO₄ in sodium cacodylate, and dehydrated in a series of ascending ethanol gradients (70, 85, 95, and 100%). Samples were cleared in acetone, infiltrated, and embedded in spurr resin. Sections were collected on Formvar-coated 50 mesh copper grids, stained with lead citrate and uranyl acetate, and examined and photographed on a Hitachi H7100 transmission electron microscope. Myelinated axons were identified by their darkly stained sheaths, whereas unmyelinated axons were identified by their light appearance relative to the surrounding Schwann cell cytoplasm. These cross sections of the DCN were photographed and montaged, and the total number of myelinated and unmyelinated axons was counted. Statistical results were expressed as mean value ± SEM and were tested for significance by the one-tailed Student’s t test for paired differences.

Immunoblotting, immunoprecipitation, jnk assays, and NF-kB assays. For immunoblotting for the p75^NTR-ICD, whole brains from P20 animals were homogenized in 20 mmTris, pH 8.0, 154 mm NaCl, 10% glycerol, 1% Nonidet P-40, 1 μg/ml leupeptin, 100 μm phenylmethylsulfonyl fluoride, 5 mm phenanthroline, and 1 mmorthovanadate in a polytron, spun for 30 min at 12,000 rpm in a Beckman JA-14 rotor to remove insoluble material, and then assayed for protein content by the BCA assay (Pierce, Rockford, IL). Soluble brain extract (20 μg) from NP40 lysates was separated on 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with αP1, a rabbit polyclonal antibody directed against the p75^NTR intracellular domain (Barker et al., 1994). So that trk receptors in brain tissue could be characterized, trk receptors were immunoprecipitated from lysates prepared from neonatal or adult brains with the pan-trk antibody 203 (Hempstead et al., 1992), separated by 7.5% SDS-PAGE, and then immunoblotted either with 4G10, an anti-phosphotyrosine antibody, or with pan-trk 203, TrkB_out (Kaplan et al., 1993) or TrkC_in (Belliveau et al., 1997) to detect total trk, trkB, or trkC, respectively. For analysis of whole-cell lysate tyrosine phosphorylation, lysates of neural tissue were separated by 7.5% SDS-PAGE and immunoblotted with 4G10.

NF-kB and jun kinase activity assays were performed on nuclear and cytoplasmic fractions, respectively, of embryonic day 16 (E16), E17, and E18 transgenic and wild-type littermates. NF-kB levels were determined by electrophoretic mobility shift assays, using HIV-LTR as a probe, essentially as described (Singh and Aggarwal, 1995). Jun kinase assays were performed by using a GST-jun fusion protein encoding amino acids 1–91 of human c-jun, as described in Westwick and Brenner (1995).

RESULTS

Recent studies suggest a role for p75^NTR in the regulation of cellular apoptosis (Barrett and Bartlett, 1994;Casaccia-Bonnefil et al., 1996). To determine whether p75^NTR is capable of promoting neuronal apoptosis and to begin to examine molecular mechanisms underlying such effects, we expressed the cytoplasmic domain of the p75^NTR in neurons of transgenic mice, using a minigene composed of the 1.1 kb Tα1 α-tubulin promoter, an open reading frame encoding the intracellular domain of rat p75^NTR (amino acids 276–425), and an SV40 intron and polyadenylation signal (Fig.1A). The Tα1 α-tubulin promoter produces robust expression concomitant with or shortly after neuronal terminal mitosis and is maximally active during periods of process extension (Gloster et al., 1994). Tα1-driven expression decreases to lower levels in the mature nervous system (Bamji and Miller, 1996) and is reinduced after axonal injury (Gloster et al., 1994; Wu et al., 1997).

Genotyping revealed that Tα1:ICD animals were under-represented in litters, suggesting some embryonic lethality of the transgenic animals. Nonetheless, many animals survived to term and developed into fertile adults. Immunoblot analysis showed p75^NTR-ICD expression in neonatal brains of various Tα1:p75^NTR-ICD lines, with levels of expression varying considerably (Fig. 1B). Transgene-positive animals from these lines exhibited a phenotype indicative of nervous system deficits, including a general lack of coordination. By the third postnatal week Tα1:ICD mice exhibited ptosis, and several animals displayed self-mutilation, consistent with defects in sympathetic and sensory innervation, respectively. To examine the cellular basis for these defects, we chose the 4163 and 4173 Tα1:p75^NTR-ICD lines for detailed analysis.

DISCUSSION

Although p75^NTR was cloned over a decade ago, its physiological role remains unclear. Results presented in this paper indicate that the p75^NTR, like other members of this receptor family, is capable of autonomously signaling to mediate the death of selected populations of developing and injured neurons. Specifically, we have demonstrated that neuronal expression of the truncated intracellular domain (ICD) of p75^NTR is sufficient to cause developmental apoptosis of developing sensory and sympathetic neurons in the PNS but not of facial motor neurons. This deficit in neurons was not limited to TrkA- or p75^NTR-expressing neurons because there are decreased numbers of neurons in the neocortex, where TrkA is not expressed and where p75^NTR is expressed only transiently. Moreover, our data indicate that neurons are differentially vulnerable to the ICD at different points in their lifetime; inducible expression of the ICD in injured adult facial motor neurons led to increased neuronal death, whereas the ICD did not affect survival of these same neurons during development. This effect on neuronal apoptosis is not attributable to a direct effect on trk receptors, because trk protein levels and endogenous and induced activation levels all remained the same regardless of the presence or absence of the p75^NTR-ICD. On the basis of these data, we believe that the intracellular domain of the p75^NTR is a constitutively active autonomous signaling protein that, in the appropriate neuronal context, will mediate neuronal death.

The structural motif that defines the TNF receptor superfamily is a tandemly repeated extracellular cysteine-rich domain (Banner et al., 1993; Bazan, 1993); many of the receptors within this family play important roles in regulation of proliferation and apoptosis, particularly within the immune system (for review, see Baker and Reddy, 1996). The intracellular domains of these proteins contain no enzymatic activity, and, until recently, the signaling paths that they activated were poorly characterized. The only intracellular homology identified among members of the TNFR superfamily is a region of 80 amino acids that is found in TNF-R1, fas, DR3, and p75^NTR(Boldin et al., 1995b; Chapman, 1995; Chinnaiyan et al., 1996). In TNF-R1 and fas, this domain is required for ligand-mediated activation of apoptotic pathways, and it therefore has been termed the “death domain” (Tartaglia et al., 1993). A major advance in understanding signaling through this class of receptor has come with the identification of proteins, including TRADD, FADD, and RIP, that interact with TNF-R1 and fas intracellular domains (Chinnaiyan et al., 1995; Hsu et al., 1996). It is now clear that death domain interactions play a critical role in mediating the mobilization of these signaling molecules and the activation of a proteolytic apoptotic cascade (for review, see Nagata, 1997). The fact that a soluble cytoplasmic fragment of TNF-R1 can provoke cellular apoptosis indicates that overexpressed receptor fragments are capable of activating these signaling proteins (Boldin et al., 1995a).

The analysis of p75^NTR signaling in physiologically relevant settings is complicated by the fact that many p75^NTR-expressing cells also express trk receptors that activate distinct signaling cascades in response to the same ligand(s). p75^NTR functionally interacts with trk receptors (Ip et al., 1993; Barker and Shooter, 1994; Verdi et al., 1994), and trk receptor activation may inhibit signaling by p75^NTR (Dobrowsky et al., 1995), further complicating the analysis of autonomous p75^NTRsignaling cascades. We therefore took a transgenic expression approach to allow us to examine the effects of p75^NTR-dependent signaling in physiologically relevant settings irrespective of ligand binding. The neuronal apoptosis observed in the Tα1:ICD mice is consistent with the hypothesis that the truncated p75^NTR fragment does, indeed, act as a constitutive activator of signaling pathways within neurons and suggests that a p75^NTR-derived signal is capable of mediating apoptosis in a manner similar to other members of this receptor family. Like the intracellular domains of fas and TNF-R1, our evidence suggests that the p75^NTR-ICD constitutively may activate a cell death cascade. Mechanisms that may underlie this signaling path include activation of sphingomyelinase, JNK, NF-kB, or members of the TRAF/TRAD family. Our examination of NF-kB or JNK activity in embryonic brains from Tα1:ICD mice did not reveal differences from nontransgenic littermates, but this does not rule out the possibility that activation of these pathways contributes to the transgenic phenotype in subtle cell-specific manners not detected in our biochemical assays. It is possible that changes in neuronal jnk or NF-kB activation that may result from p75^NTR-ICD expression are obscured by high levels of activity from unaffected cell types or, alternatively, that high jnk or NF-kB activity occurs in a short developmental window that precedes cell loss. In this regard, the effects of the p75^NTR-ICD show an intriguing cellular specificity, with developmental loss of sympathetic and peripheral sensory neurons and of central neurons of the neocortex. One possible explanation for this is that only these susceptible populations express the signaling partners necessary for mediation of a p75^NTRapoptotic cascade. As p75^NTR signaling partners are identified and their expression patterns are determined, the basis of the cell specificity of the p75^NTR-ICD may become apparent.

Our data do not support the hypothesis that the intracellular p75^NTR fragment acts in a dominant-negative manner, either inhibiting p75^NTR-dependent survival signal or interfering with the normal action of trkA. If the p75^NTR intracellular domain had a dominant-negative effect on p75^NTR signaling, the transgenic phenotype should mimic that of the p75^NTR−/− mice. However, whereas p75^NTR-ICD transgenics show almost complete loss of sympathetic neurons of the SCG and increased motor neuron loss after injury, in p75^NTR−/− mice sympathetic neuron number is not reduced (Lee et al., 1992; Brennan et al., 1996), and reduced motor neuron death is observed after facial nerve lesion (K.-F. Lee, personal communication). Expression of the p75^NTR-ICD has no demonstrable effect on ligand-dependent activation of trkA (in stably transfected PC12 cell lines) or trkB (in transgenic brain). It is possible that negative selection has occurred within the transgenic mice such that only those neurons resistant to the effects of the p75^NTR-ICD are present at the time of our analysis. However, results from the PC12 cell sublines suggest that a direct effect of the p75^NTR-ICD on proximal events in trkA activation is unlikely. Rather, the observation that p75^NTR-ICD expression results in a highly significant loss of neurons from multiple cortical layers, which normally do not express trkA or p75^NTR (Allendoerfer et al., 1990), suggests that the receptor fragment can have a dominant effect, possibly by constitutively activating an apoptotic signaling cascade.

The findings reported here are consistent with several recent reports indicating that p75^NTR may, in some circumstances, play a role in cell death. Supernumerary basal forebrain cholinergic neurons are observed in the p75^NTR−/− mice, and their normal developmental loss in wild-type animals is reduced by blocking ligand binding to p75^NTR(Vanderzee et al., 1996). Similarly, cell loss that normally occurs in the developing retina is reduced by reagents that block NGF access to p75^NTR (Frade et al., 1996). So far, only NGF appears to be capable of mediating this effect; in the avian isthmo-optic nucleus (von Bartheld et al., 1994) and on cultured oligodendrocytes that express p75^NTR but not trkA (Casaccia-Bonnefil et al., 1996), only NGF treatment results in cell death. Together, these results suggest that a normal role of p75^NTR may be to aid in the process of developmental apoptosis that occurs within the maturing nervous system. The use ofin vivo models such as the Tα1:p75^NTR-ICD mice described here should, in the future, allow us to identify functionally relevant p75^NTR signaling pathways that contribute to neuronal apoptosis.