Previously, we documented loss of CRD-Nrg-1–expressing motor and sensory neurons and cranial nerve nuclei in CRD-Nrg-1−/−
mice (Wolpowitz et al., 2000
). Loss of CRD-Nrg-1–expressing neurons in spiral ganglia ( A) and hippocampus (not depicted) also was evident in E16 CRD-Nrg-1−/−
embryos. The fraction of the spiral ganglion volume and the CA3 region of the hippocampus occupied by neurons in wild-type or CRD-Nrg-1−/−
embryos were evaluated from 12-μM serial sections stained for GAP43 expression. The fraction of neurons in mutant hippocampus (CA3 region) and mutant spiral ganglia (SG) were decreased by 50 and 90%, respectively ( A and not depicted). In contrast, evaluation of the erbB4-expressing cells within the target cochlea revealed that neither the number nor the distribution of erbB4-expressing cells (Co) was altered in mutant embryos (compared with wild-type; A, top).
Figure 1. Interaction between CRD-Nrg-1 and erbB receptors enhances survival of CRD-Nrg-1–expressing neurons. (A) Spiral ganglion neurons are lost after genetic disruption of CRD-Nrg-1. Spiral ganglia from wild-type (left) or CRD-Nrg-1−/− (more ...)
The observations that disrupting the CRD-Nrg-1 gene resulted in the loss of neurons that would have expressed CRD-Nrg-1 protein, led us to ask whether interactions between erbB receptors and CRD-Nrg-1 promotes neuronal survival. To test this idea, neurons were isolated from E16 mouse embryos, dispersed, and maintained in vitro. Apoptosis was evaluated by the presence of nuclear shrinkage and chromatin condensation after staining with bisbenzimide ( B, arrows). The fraction of neurons that were undergoing apoptosis 48 h after plating was 0.19–0.22 (, untreated and Nrg-ECD–treated controls). When neurons were incubated overnight in media containing soluble forms of the ECDs of erbB2 and erbB4 (which, together, constitute a high affinity receptor for Nrg-1; Fitzpatrick et al., 1998
), the number of apoptotic neurons was reduced significantly ( C; *, P < 0.01). The protection against apoptosis was specific for erbB2:erbB4. Neither soluble erbB2 alone (which does not bind Nrg-1) nor the soluble erbB2:erbB4 preincubated with CRD-Nrg-1-ECD before treatment affected neuronal survival (fraction of apoptotic neurons: untreated controls, 0.21; CRD-Nrg-1-ECD, 0.20; erbB2:B4, 0.13; erbB2:B4 + CRD-Nrg-1-ECD, 0.17). Treatment with soluble erbBs also protected hippocampal neurons from staurosporine-induced apoptosis by ~50% (fraction apoptotic: untreated controls 0.2; staurosporine alone 0.62; staurosporine + erbB2:B4 0.38). In addition to the changes in the number of healthy versus dying neurons detected under these conditions, there were parallel changes in the percentage of cells with detectable immunoreactive Nrg-1-ICD, and the intensity of staining in general, and over nuclei in particular.
The apparent differences in localization and levels of Nrg-1 in erbB2:erbB4-treated cells were examined in greater detail (). Neuronal cultures were stained with antibodies recognizing the shared ECD of Nrg-1 or the COOH-terminal region of the longest ICD of Nrg-1 (Nrg-1-ICD; a-form). Both antibodies against Nrg-1-ECD and Nrg-1-ICD stained untreated cells in an overlapping distribution in all nonnuclear compartments. Immunoreactive Nrg-1 was more diffusely distributed in neuronal soma and along the processes of control neurons viewed at either the nuclear ( A, top left) or the neurite level ( A, bottom). Nonneuronal cells did not stain with Nrg-1 antibodies, and all immunostaining was completely blocked by preincubation of the antibodies with peptide antigen ( D). After treatment with soluble erbB2:B4, the Nrg-1 staining pattern changed in several ways. First, there was a decrease in diffuse staining along processes with both the extracellular and ICD antibodies. Second, diffuse staining was replaced by pronounced “patches” of immunoreactive Nrg-1 at various points along the processes. Third, there was an increase in somal staining, in particular of a Golgi-like area adjacent to nuclei. The patches and the Golgi-like staining were seen with both antibodies; and fourth, multiple discrete puncta were seen in nuclei (note that in A, all images are from 1-μM sections captured through the middle of the nucleus, except for the neurite pictures in the bottom). These puncta stained with ICD antibody, but not with ECD antibody. Thus, within 20 min after treatment with soluble erbB2:erbB4, the intracellular and the ECDs separated and the ICD entered the nucleus.
Figure 2. Interaction with erbB receptors, or depolarization, target Nrg-1-ICD to the nucleus in primary neurons. (A) Dispersed E16 spiral ganglion neurons were maintained in vitro for 3 d and stained with antibodies recognizing the ICD of the “a” (more ...)
The presence of strong patches of Nrg-1-ICD, but not Nrg-1-ECD, immunoreactivity in neuronal nuclei indicates that exposing neurons to soluble erbB2:erbB4 resulted in physical separation of Nrg-1 ECD from Nrg-1 ICD. We postulated that such separation and subsequent nuclear targeting of the Nrg-1-ICD might participate in Nrg-1:erbB-induced signaling, which is implicated in the survival of neurons in vivo (Wolpowitz et al., 2000
) and in vitro (). To verify, and to quantify the magnitude of this response, we measured the percentage of total neurons that had nuclear, immunoreactive Nrg-1-ICD under a variety of conditions. In untreated (control) cultures, 15–20% of neuronal nuclei were positive for Nrg-1-ICD staining. After 15 min of treatment with soluble erbB2:erbB4 or after 15 min of depolarization (see Materials and methods), >85% of nuclei stained positive for Nrg-1-ICD ( B). Treatment with soluble erbB2 alone did not affect nuclear staining.
To explore further the possibility that cell surface erbB–CRD-Nrg-1 interactions result in proteolytic release and nuclear translocation of the Nrg-1-ICD, we prepared nuclear fractions from dispersed neurons 15 min after treatment with erbB2, erbB2 + erbB4, or 50 mM KCl. Extract proteins were resolved electrophoretically and immunoblots were probed with the Nrg-1-ICD specific antibody ( C). A faint signal at ~50 kD was seen in nuclear extracts from untreated (control) or soluble erbB2-treated cells. Stimulation with either the soluble erbB2:erbB4 combination or with KCl elevated the amount of the ~50-kD band detected in nuclear extracts. Preincubating the antibody with immunizing peptide (Nrg-1 + pept) led to a loss of signal ( D). The size of this band is consistent with a peptide corresponding to the intracellular portion of Nrg-1a (see ; for review see Wang et al., 2001
Figure 4. Nuclear translocation of Nrg-1-ICD–GFP fusions can be visualized in living cells. (A) The schematic illustration at the top shows the NRG-1βa-GFP chimeric used in this work. ECD, extracellular domain; TM, transmembrane domain; (more ...)
These results are consistent with regulated cleavage and release of the Nrg-1-ICD from the membrane and its subsequent translocation to the nucleus. Regulated intramembranous proteolysis of Notch (Struhl and Adachi, 2000
; Struhl and Greenwald, 2001
), SRE-BP1 and 2 (Brown and Goldstein, 1997
; Brown et al., 2000
), and possibly βAPP (Cao and Sudhof, 2001
; Kimberly et al., 2001
) and erbB4 (Ni et al., 2001
; Lee et al., 2002
) result in ICD-dependent regulation of gene expression. To determine whether regulated nuclear targeting of the Nrg-1-ICD was associated with changes in gene expression, we isolated total RNA from neuronal cultures that were either untreated or treated for 2 h with soluble erbB2 + erbB4. Using these RNAs, we synthesized 32
P-labeled cDNAs and probed a mouse cDNA array. Clear differences in expression of Oct-3, p19INK4
, IL-11, Bcl-X, BAK, and RIP were seen and confirmed by RT-PCR (). Expression of Bcl-X, BAK, and RIP were repressed after treatment with the soluble erbBs and after depolarization with KCl. Expression of Oct-3, p19INK4
, and IL-11 increased after treatment of neurons with erbB2 + erbB4, but only Oct-3 expression increased in response to KCl. Thus, although both depolarization and erbB2:erbB4 treatment induced nuclear translocation of Nrg-1-ICD, the effects of these treatments on target gene expression differed.
Figure 3. Treatments that target Nrg-1-ICD to the nucleus alter gene expression. E13.5 spiral ganglion neurons maintained in culture overnight were untreated (control) or stimulated with soluble erbB2:B4 or 50 mM KCl for 2 h. Total RNA was isolated and the relative (more ...)
To confirm that the effects on gene expression seen after treatment of neuronal cultures with soluble erbB2 + erbB4 required the interaction of these proteins with the ECD of endogenously expressed Nrg-1, neuronal cultures were either untreated, treated with soluble erbB2 + erbB4, or treated with erbB2 + erbB4 that had been preincubated with the CRD-Nrg-1 ECD. Expression of BAK and p19INK4 were measured by RT-PCR analysis of total RNA. Preincubating erbB2 + erbB4 with CRD-Nrg-1 ECD blocked the effects on BAK and p19INK4 expression ().
To gain more insight into the dynamics of regulated nuclear targeting of the Nrg-1-ICD, we expressed a series of chimeric CRD-Nrg-1s in HEK 293T cells (). Subcellular targeting of Nrg-1 was followed in living cells transfected with a CRD-Nrg-1βa-GFP fusion protein by continuous monitoring of the distribution of GFP by collecting images through the z-axis of cells ( A). In control cells, the strongest CRD-Nrg-1-GFP signal was detected around the cell periphery and in a single intracellular region, consistent with previous reports of Nrg-1 localization in the plasma membrane, Golgi structure, and endoplasmic reticulum (Burgess et al., 1995
). This pattern remained essentially unchanged for up to 2 h of continuous observation. In contrast, within 2–4 min after treatment with soluble erbB2 + erbB4, the distribution of green fluorescence changed and distinct fluorescent aggregates were seen both in peripheral regions of the cells and near Golgi-like structures. By 16 min after erbB2:erbB4 treatment, these GFP aggregates moved along discrete paths and entered the nucleus (, arrows).
The induced targeting of Nrg-1-ICD to the nucleus indicated that the Nrg-1-ICD might contain an identifiable NLS. Inspection of the primary sequence of Nrg-1-ICDs identified two potential NLSs (http://psort.nibb.ac.jp
). The first, NLS-1, includes the first eight amino acids after the transmembrane domain (KTKKQRKK) and is found in all Nrg-1-ICDs. We expressed Nrg-1-ICD-GFP fusion proteins that contained or lacked these eight amino acids in 293T cells ( B; these fusion proteins included just the ICD of Nrg-1 fused to GFP and lacked the transmembrane domain). Strong nuclear and diffuse cytoplasmic staining was seen when Nrg-1βc-ICD-GFP was expressed. Nrg-1βc-ICDΔNLS1
-GFP, lacking the eight–amino acid NLS, was distributed diffusely throughout the cells and did not concentrate in nuclei, which is consistent with a requirement for this domain for accumulating Nrg-1-ICD in nuclei. The presence or absence of the second putative NLS (PRLREKK) had no effect on the cellular localization of GFP fusion proteins (unpublished data).
To test further that erbB2:erbB4-induced nuclear targeting of Nrg-1a-ICD was associated with proteolysis of the full-length transmembrane form of Nrg-1, we separated cytoplasmic and membrane fractions from nuclear extracts of HEK293T cells expressing a CRD-Nrg-1βa-HA fusion protein (full-length CRD-Nrg-1βa tagged at the COOH terminus with an 11–amino acid HA epitope). The Nrg-1 COOH terminus was detected by probing immunoblots with an anti-HA antibody. In cells incubated under control conditions (untreated or treated with soluble erbB2; C, erbB2), the ~110-kD full-length protein and several higher molecular mass bands were detected. These higher molecular mass bands likely correspond to highly glycosylated or possibly aggregated forms of Nrg-1(Wang et al., 2001
). Treatment of transfected cells with soluble erbB2 + erbB4 resulted in increased amounts of a mostly nuclear ~50-kD protein corresponding to the Nrg-1-ICD ( C, erbB2:B4).
As a further demonstration that the Nrg-1-ICD translocates into nuclei, we expressed fusion proteins comprised of CRD-Nrg-1βa and the chimeric transcription factor Gal4-VP16 (Nrg-1βa-Gal4-VP16), Nrg-1βa-ICD (lacking the ECD and the transmembrane domains) plus the DNA-binding domain from Gal4 (ICD-Gal4), but without the VP16 activation domain, or Nrg-1βa-ICDΔNLS plus the Gal4 DNA-binding domain (ICDΔNLS-Gal4). The distribution of these fusion proteins in HEK 293T cells was measured by assaying luciferase expression from a cotransfected Gal4-UAS–luciferase reporter plasmid (). Expression of the nonmembrane-tethered Nrg-1-ICD-Gal4DBD chimera increased luciferase activity ~10-fold compared with the full-length Nrg-1 fused to Gal4-VP16 or compared with Nrg-1-ICDΔNLS-Gal4DBD ( B; 33-fold vs. fourfold or 2.8-fold, respectively). This level of transactivation is roughly comparable to the levels seen in cells expressing a Gal4DBD-VP16AD chimera. As the Gal4DBD lacks a transactivation domain and a nuclear localization signal, luciferase activity indicates that the Nrg-1-ICD has an interaction domain that is able to recruit coactivators to the Gal4-UAS promoter.
Figure 5. Treatment of transfected cells with soluble erbB receptors stimulates Nrg-1-ICD cleavage and translocation to a transcriptionally active compartment. (A) Schematic illustrations showing organization of the Gal4-Nrg-1 fusion proteins used. Gal4, DNA-binding (more ...)
The appearance of a ~50-kD COOH-terminal fragment of Nrg-1 in nuclei after soluble erbB2:erbB4 ( C and C) or after depolarization ( C) is consistent with regulated cleavage of the transmembrane precursor form of CRD-Nrg-1. Constitutive and regulated extracellular cleavage of both type 1 and type III Nrg-1 has been characterized (Burgess et al., 1995
; Lu et al., 1995
; Liu et al., 1998b
; Loeb et al., 1998
; Han and Fischbach, 1999
; Montero et al., 2000
; Wang et al., 2001
), but events leading to the release of the Nrg-1-ICD from the membrane have not been studied. Because the first eight intracellular amino acids are required for nuclear translocation, the cleavage event that releases the ICD is expected to occur at the junction between this sequence and the transmembrane domain, or within the transmembrane domain. As such, we tested whether γ-secretases, enzymes known to catalyze intramembranous proteolysis (Struhl and Adachi, 1998
; Struhl and Greenwald, 1999
; Brown et al., 2000
; Ni et al., 2001
; Ebinu and Yankner, 2002
; Lee et al., 2002
), might be involved in Nrg-1-ICD processing. HEK 293T cells expressing CRD-Nrg-1βa-Gal4-VP16 were pretreated with γ-secretase inhibitors (; inhibitors 1, CM-265; 2, WPE(III)-36B; or 3, MW (III)-26A) for 8 h before treatment with soluble erbB2 + erbB4. In the absence of inhibitors, or in cells treated with an inactive analogue of these inhibitors (JT-326), exposure of cells to soluble erbB2:erbB4 significantly elevated luciferase levels ( C, columns 3 and 9 compared with columns 1, 2, or 5). The induction of luciferase expression was blocked by all three inhibitors, as well as by preincubating soluble erbB2:erbB4 with the CRD-Nrg-1 ECD. Therefore, the stimulated nuclear targeting of the CRD-Nrg-1-ICD, at least in part, is dependent on a γ-secretase–like activity. These results are consistent with a model in which CRD-Nrg-1–erbB interactions result in cleavage of Nrg-1 within the transmembrane domain and the subsequent release and nuclear targeting of the Nrg-1-ICD.