Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of α7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface α7 nAChRs, which results from a redistribution of preexisting intracellular pools of α7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting α7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function

Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation

Type III Nrg1, a member of the Nrg1 family of signaling proteins, is expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with the ErbB family of receptor tyrosine kinases (ErbB RTKs) [1]. Type III Nrg1 signaling as a receptor (Type III Nrg1 back signaling) can acutely activate phosphatidylinositol-3-kinase (PtdIns3K) signaling, as well as regulate levels of α7* nicotinic acetylcholine receptors, along sensory axons [2]. Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeable ion channel found in primary sensory neurons that is necessary for the detection of thermal pain and for the development of thermal hypersensitivity to pain under inflammatory conditions [3]. Cell surface expression of TRPV1 can be enhanced by activation of PtdIns3K [4], [5], [6], making it a potential target for regulation by Type III Nrg1. We now show that Type III Nrg1 signaling in sensory neurons affects functional axonal TRPV1 in a PtdIns3K-dependent manner. Furthermore, mice heterozygous for Type III Nrg1 have specific deficits in their ability to respond to noxious thermal stimuli and to develop capsaicin-induced thermal hypersensitivity to pain. Cumulatively, these results implicate Type III Nrg1 as a novel regulator of TRPV1 and a molecular mediator of nociceptive function

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Type III Neuregulin1 Signaling in Peripheral Sensory Neurons Affects Thermal Pain Sensation and Hyperalgesia

Type III Neuregulin1 (Nrg1), a member of the Nrg1 family of signaling proteins, is highly expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with members of the ErbB family of receptor tyrosine kinases (Bao, Wolpowitz et al. 2003; Falls 2003). Type III Nrg1 signaling as a receptor (Type III Nrg1 back signaling) can acutely activate phosphatidylinositol-3-kinase (PtdIns3K) signaling, as well as regulate levels of α7* nicotinic acetylcholine receptors (α7* nAChRs), along sensory axons (Hancock, Canetta et al. 2008). Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeable ion channel found in primary sensory neurons that is necessary for the detection of thermal pain and for the development of thermal hyperalgesia under inflammatory conditions (Caterina, Leffler et al. 2000). Levels of functional TRPV1 can be enhanced by activation of PtdIns3K (Bonnington and McNaughton 2003; Zhang, Huang et al. 2005; Stein, Ufret-Vincenty et al. 2006; Zhu and Oxford 2007), making it a potential target for regulation by Type III Nrg1. I now show that Type III Nrg1 signaling along sensory axons enhances levels of functional α7* nAChRs and TRPV1 in a PtdIns3K-dependent manner. Furthermore, mice heterozygous for Type III Nrg1 have specific deficits in their ability to respond to noxious thermal stimuli and to develop capsaicin-induced thermal hyperalgesia. Cumulatively, these results implicate Type III Nrg1 as a novel regulator of TRPV1 and a molecular mediator of nociceptive function

Dissociated sensory neurons from E11 chick embryos were cultured for 2 d in vitro and treated with either B2-ECD (control) or B4-ECD for 1 or 24 h

(A) Quantification of surface or total pools of α7* nAChR by I-αBgTx radiolabeling in sensory neurons treated with either B2-ECD (control) or B4-ECD for 24 h. In response to a 24-h B4-ECD treatment, we detected an ∼2.7-fold increase in surface I-αBgTx binding compared with control conditions (B2-ECD [control], 1,339.15 ± 329.77 cpm; and B4-ECD, 3,562.81 ± 1,111.19 cpm). B4-ECD treatment did not induce a change in total I-αBgTx binding as compared with the control (B2-ECD [control], 11,159.74 ± 1,059.79 cpm; and B4-ECD, 12,258.85 ± 580.11 cpm). The graph shows means ± SEM. Data were pooled from three independent experiments with greater than or equal to three wells per condition per experiment. Statistical significance was determined by ANOVA. *, P < 0.05 (Statview). (B) Immunoblot analysis of total α7 subunit protein in sensory neurons treated with B2-ECD (control) or B4-ECD treatment for 24 h. In response to B4-ECD treatment, we did not detect a difference in total α7 subunit protein. NF probing in bottom panel shows equivalent lysate loading. (C) Sensory neurons were treated with B2-ECD (control) or B4-ECD for 1 h. In parallel, neurons pretreated with CHX for 45 min were treated with B2-ECD or B4-ECD for 1 h. Neurons were labeled with αBgTx-488 (green), fixed, permeabilized, and colabeled for NF protein (blue). CHX treatment (c and d) did not affect either the basal number of αBgTx-488 clusters on control neurons (c) or the response to B4-ECD (d). Linescans of fluorescence intensity profiles of αBgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 μm. (D) Quantification of surface αBgTx-488 clusters along NF-labeled axons. B4-ECD treatment induced an ∼1.9-fold increase in surface αBgTx-488 clusters along axons, and B4-ECD treatment in the presence of CHX induced an ∼2.1-fold increase. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P = 0.01; **, P < 0.0001 (Statview). (E) Quantification of surface αBgTx-488 cluster area. B4-ECD treatment in the presence or absence of CHX induced an increase in αBgTx-488 cluster area. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance was determined by the Kolmogorov-Smirnov Test. *, P ≤ 0.0001 (Statview).<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

Dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1 h

In parallel, neurons were pretreated with WM or an Akt inh. for 45 min before treatment with B2-ECD or B4-ECD for an additional hour. Neurons were labeled for surface α7* nAChRs with αBgTx-488 (green), fixed, permeabilized, and costained for NF protein (blue). (A) Representative micrographs of αBgTx-488 staining along NF-positive axons. B4-ECD treatment increased surface αBgTx-488 clusters (b), which did not occur in the presence of WM (d). Linescans of fluorescence intensity profiles of αBgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 μm. (B) Quantification of surface αBgTx-488 clusters along sensory neuron axons represented in A. B4-ECD treatment induced an ∼1.9-fold increase of surface αBgTx-488 clusters but not in the presence of WM or Akt inh. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.0001 (Statview). (C) Quantification of surface αBgTx-488 cluster area. B4-ECD treatment induced an increase in αBgTx-488 cluster area but not in the presence of WM or Akt inh. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance determined by the Kolmogorov-Smirnov Test. *, P = 0.0001 (Statview).<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons-1

Re labeled for surface α7* nAChRs with αBgTx-488 (green), fixed, permeabilized, and labeled for NF protein (blue). (A) Representative micrographs of axons from WT (a and b) or Type III Nrg1 (c and d) sensory neurons under control (a and c) versus B4-ECD (b and d) conditions. B4-ECD treatment increased the number of surface αBgTx-488 clusters along NF-positive processes of WT neurons (b). Linescans of fluorescence intensity profile for αBgTx-488 staining along representative axons (see Materials and methods). Bar, 5 μm. (B) Quantification of surface αBgTx-488 clusters along NF-positive axons from WT versus Type III Nrg1 DRG explants treated with either B2-ECD (control) or B4-ECD for 24 h. In WT cultures, B4-ECD treatment induced an ∼1.6-fold increase in surface αBgTx clusters along NF-positive axons compared with the control. There was no detectable change in αBgTx clusters along axons of Type III Nrg1 neurons. The graph shows means ± SEM. Data were pooled from three independent experiments. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.03; **, P < 0.001 (Statview). (C) After 2 d in vitro, dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1, 2, 6, or 12 h and labeled as described in A. Axonal surface αBgTx-488 clusters were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Statview). (D) Axonal-bound B4-ECD and αBgTx-488 were detected in puncta along axons treated with B4-ECD (b, d, and e). Sensory neurons from E14.5 WT mouse embryos were cultured for 2 d in vitro and treated with B2-ECD (control) or B4-ECD for 1 h. Before fixation, surface α7* nAChRs and axonal-bound B2-ECD (control) or B4-ECD were labeled with αBgTx-488 (green) and an antibody against the human Fc domain (anti-Fc; red), respectively. c and d and e are magnifications of the areas shown in dotted squares in a and b, respectively. Bar: (a and b) 5 μm; (c–e) 1 μm. (E) Sensory neurons from E11 chick embryos were treated with B2-ECD (control), B4-ECD, or soluble Nrg1β peptide (Nrg1-ECD) for 1 h. In parallel, neurons pretreated with an ErbB tyrosine kinase inhibitor (ErbB inh.) for 45 min were treated with B2-ECD, B4-ECD, or Nrg1-ECD for 1 h. Neurons were labeled as described in A, and surface αBgTx-488 clusters along axons were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.005; **, P < 0.01 (Statview).<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

(A) Dissociated sensory neurons from E11 chick embryos were treated for 5 min with B2-ECD (control), B4-ECD, 50 ng/ml NGF, or 10 ng/ml of soluble Nrg1β peptide (Nrg1-ECD)

In parallel, neurons were treated with WM for 45 min before B4-ECD stimulation (WM + B4-ECD). Neurons were fixed, permeabilized, and costained for PIP (red) and tau protein (blue) to label axons. Both B4-ECD (g) and NGF (i) treatment induced puncta of PIP along tau-positive axons. Neither B4-ECD stimulation in the presence of WM (c and h) nor that of Nrg1-ECD (e and j) induced an increase in PIP. Confocal images were obtained with a 40× oil objective. Bar, 10 μm. (B) Immunoblot analysis of phospho-Akt (Ser 473) in WT or Type III Nrg1 sensory neurons treated with either B2-ECD (control) or B4-ECD for 10 min. In WT neurons, B4-ECD treatment induced an approximately threefold increase in phospho-Akt, whereas no response was detected in mutant neurons. Total Akt in the bottom panel shows equal lysate loading. The bar graph represents phospho-Akt normalized to total Akt immunoreactive bands. Data are representative of three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.002 (Statview). (C and D) E14.5 WT (a and b) or Type III Nrg1 (c and d) DRG explants were treated with B2-ECD (control) or B4-ECD for 10 min. Surface-bound B4-ECD or B2-ECD were labeled with an antibody against the human Fc domain (anti-Fc; green) before fixation. Neurons were fixed, permeabilized, and stained for phospho-Akt (red) and NF protein (blue). B4-ECD treatment increased phospho-Akt along Fc-positive axons of WT neurons (b and D) but did not do so along axons of mutant neurons (d). Note the close proximity of anti-FC and phospho-Akt puncta in the high-power micrograph shown in e. The asterisk denotes an axon negative for both anti-Fc and phospho-Akt immunolabeling (c). A 63× oil objective was used (a–d). Confocal imaging was obtained with a 100× oil objective (D). Bar: (a–d)10 μm; (D) 5 μm. (E) Quantification of the average fluorescence intensity (AFI) of phospho-Akt along axons of WT or Type III Nrg1 sensory neurons treated with B2-ECD (control) or B4-ECD for 10 min or 1, 2, or 6 h (see Materials and methods). Along WT axons, B4-ECD treatment induced increases in phospho-Akt. Along axons of mutant neurons, we did not detect an increase in phospho-Akt in response to B4-ECD treatment. The graph shows means ± SEM. Data are from three independent experiments. Statistical significance was determined by ANOVA. *, P < 0.02.<p><b>Copyright information:</b></p><p>Taken from "Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons"</p><p></p><p>The Journal of Cell Biology 2008;181(3):511-521.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364689.</p><p></p

Acute stimulation of Type III Nrg1 signaling in Type III Nrg1^+/− sensory axons does not rescue functional TRPV1 deficits.

<p>(A) Representative traces of intracellular calcium along Type III Nrg1^+/− sensory axons in response to repeated applications of 1 µM capsaicin followed by application of 56 mM KCl. The maximum percent change in intracellular calcium ([(F−F₀)/F₀]*100) in response to capsaicin decreased between the 4^th and 5^th capsaicin application in Type III Nrg1^+/− axons under control conditions (left) and did not increase when Type III Nrg1 signaling was stimulated by sErbB4-ECD application during that interval (right). (B) Quantification of percent change in maximum response to capsaicin between the 4^th and the 5^th capsaicin application ([(F₅−F₄)/F₄]*100) by treatment. Results from WT sensory axons are included for comparison (WT CON, n = 10; WT B4, n = 7; ***p<0.001). Type III Nrg1^+/− axons did not show a statistically significantly enhanced response to capsaicin when Type III Nrg1 signaling was stimulated (Type III Nrg1^+/− CON, n = 6 animals; Type III Nrg1^+/− B4, n = 8). All comparisons between genotypes and treatments were made using an ANOVA with a Holm-Sidak post-hoc test for multiple comparisons. Graph shows mean±SEM.</p