Progressive tauopathy disrupts breathing stability and chemoreflexes during presumptive sleep in mice

Rationale: Although sleep apnea occurs in over 50% of individuals with Alzheimer’s Disease (AD) or related tauopathies, little is known concerning the potential role of tauopathy in the pathogenesis of sleep apnea. Here, we tested the hypotheses that, during presumptive sleep, a murine model of tauopathy (rTg4510) exhibits: 1) increased breathing instability; 2) impaired chemoreflex function; and 3) exacerbation of these effects with tauopathy progression.Methods: rTg4510 mice initially develop robust tauopathy in the hippocampus and cortex, and eventually progresses to the brainstem. Type I and II post-sigh apnea, Type III (spontaneous) apnea, sigh, and hypopnea incidence were measured in young adult (5–6 months; n = 10–14/group) and aged (13–15 months; n = 22–24/group) non-transgenic (nTg), monogenic control tetracycline transactivator, and bigenic rTg4510 mice using whole-body plethysmography during presumptive sleep (i.e., eyes closed, curled/laying posture, stable breathing for >200 breaths) while breathing room air (21% O2). Peripheral and central chemoreceptor sensitivity were assessed with transient exposures (5 min) to hyperoxia (100% O2) or hypercapnia (3% and 5% CO2 in 21% O2), respectively.Results: We report significant increases in Type I, II, and III apneas (all p < 0.001), sighs (p = 0.002) and hypopneas (p < 0.001) in aged rTg4510 mice, but only Type III apneas in young adult rTg4510 mice (p < 0.001) versus age-matched nTg controls. Aged rTg4510 mice exhibited profound chemoreflex impairment versus age matched nTg and tTA mice. In rTg4510 mice, breathing frequency, tidal volume and minute ventilation were not affected by hyperoxic or hypercapnic challenges, in striking contrast to controls. Histological examination revealed hyperphosphorylated tau in brainstem regions involved in the control of breathing (e.g., pons, medullary respiratory column, retrotrapezoid nucleus) in aged rTg4510 mice. Neither breathing instability nor hyperphosphorylated tau in brainstem tissues were observed in young adult rTg4510 mice.Conclusion: Older rTg4510 mice exhibit profound impairment in the neural control of breathing, with greater breathing instability and near absence of oxygen and carbon-dioxide chemoreflexes. Breathing impairments paralleled tauopathy progression into brainstem regions that control breathing. These findings are consistent with the idea that tauopathy per se undermines chemoreflexes and promotes breathing instability during sleep

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B

Introduction: Club cells are precursors for mucus-producing goblet cells. Interleukin 1β (IL-1B) is an inflammatory mediator with pro-mucin activities that increases the number of mucus-producing goblet cells. IL-1B-mediated mucin production in alveolar adenocarcinoma cells requires activation of the cAMP response element-binding protein (CREB). Whether the pro-mucin activities of IL-1B require club cell CREB is unknown.Methods: We challenged male mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-1B or vehicle. Secondarily, we studied human “club cell-like” H322 cells.Results: IL-1B increased whole lung mRNA of secreted (Mucin 5ac, Mucin 5b) and tethered (Mucin 1, Mucin 4) mucins independent of genotype. However, loss of club cell Creb1 increased whole lung mRNA of member RAS oncogene family (Rab3D), decreased mRNA of the muscarinic receptor 3 (M3R) and prevented IL-1B mediated increases in purinergic receptor P2Y, (P2ry2) mRNA. IL-1B increased the density of goblet cells containing neutral mucins in wildtype mice but not in mice with loss of club cell Creb1. These findings suggested that club cell Creb1 regulated mucin secretion. Loss of club cell Creb1 also prevented IL-1B-mediated impairments in airway mechanics. Four days of pharmacologic CREB inhibition in H322 cells increased mRNA abundance of forkhead box A2 (FOXA2), a repressor of goblet cell expansion, and decreased mRNA expression of SAM pointed domain containing ETS transcription factor (SPDEF), a driver of goblet cell expansion. Chromatin immunoprecipitation demonstrated that CREB directly bound to the promoter region of FOXA2, but not to the promoter region of SPDEF. Treatment of H322 cells with IL-1B increased cAMP levels, providing a direct link between IL-1B and CREB signaling.Conclusion: Our findings suggest that club cell Creb1 regulates the pro-mucin properties of IL-1B through pathways likely involving FOXA2

Neuropeptides in asthma, chronic obstructive pulmonary disease and cystic fibrosis

Abstract The nervous system mediates key airway protective behaviors, including cough, mucus secretion, and airway smooth muscle contraction. Thus, its involvement and potential involvement in several airway diseases has become increasingly recognized. In the current review, we focus on the contribution of select neuropeptides in three distinct airway diseases: asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. We present data on some well-studied neuropeptides, as well as call attention to a few that have not received much consideration. Because mucus hypersecretion and mucus obstruction are common features of many airway diseases, we place special emphasis on the contribution of neuropeptides to mucus secretion. Finally, we highlight evidence implicating involvement of neuropeptides in mucus phenotypes in asthma, COPD and cystic fibrosis, as well as bring to light knowledge that is still lacking in the field

Expression and activity of acid-sensing ion channels in the mouse anterior pituitary.

Acid sensing ion channels (ASICs) are proton-gated cation channels that are expressed in the nervous system and play an important role in fear learning and memory. The function of ASICs in the pituitary, an endocrine gland that contributes to emotions, is unknown. We sought to investigate which ASIC subunits were present in the pituitary and found mRNA expression for all ASIC isoforms, including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4. We also observed acid-evoked ASIC-like currents in isolated anterior pituitary cells that were absent in mice lacking ASIC1a. The biophysical properties and the responses to PcTx1, amiloride, Ca2+ and Zn2+ suggested that ASIC currents were mediated predominantly by heteromultimeric channels that contained ASIC1a and ASIC2a or ASIC2b. ASIC currents were also sensitive to FMRFamide (Phe-Met-Arg-Phe amide), suggesting that FMRFamide-like compounds might endogenously regulate pituitary ASICs. To determine whether ASICs might regulate pituitary cell function, we applied low pH and found that it increased the intracellular Ca2+ concentration. These data suggest that ASIC channels are present and functionally active in anterior pituitary cells and may therefore influence their function

Acid-induced [Ca²⁺]_i increase in cultured anterior pituitary cells.

<p><b>A.</b> Fluorescence (F340/F380 ratio) was measured under indicated conditions. Top, representative image under indicated conditions. Bottom, average traces of F/F₀, (F340/F380)/(F340/F380 at pH 7.4). Applications of 2 µM ionomycin at the end of the recordings served as a positive control (n = 6). <b>B</b>. Average values of F/F₀. pH 5.0 increased the F340/F380 ratio to 118±5% compared to the ratio at pH 7.4. Amiloride inhibited the pH 5.0 induced ratio changes (104±2% as baseline), n = 6. * <i>P</i><0.05, one-way ANOVA with Tukey's post-hoc multiple comparison test.</p

The effects of extracellular Ca²⁺ on ASIC currents in freshly isolated anterior pituitary cells.

<p><b>A</b>. Representative ASIC currents elicited by pH 5.5 with various concentrations of extracellular Ca²⁺. Holding potential was −70 mV. Ca²⁺ concentrations from 0 µM to 10 mM at pH 7.4 were applied for 2 minutes and ASIC currents were activated by pH 5.5 for 5 sec with the same Ca²⁺ concentration. <b>B</b>. Summary data showing the dose-dependent block of ASIC currents by Ca²⁺. The IC₅₀ of Ca²⁺ block was 357±20 µM (n = 7).</p

Effects of FMRFamide on ASIC currents in freshly isolated anterior pituitary cells.

<p><b>A, C.</b> Representative traces of pH 5.0 activated ASIC currents, with and without 30 second pretreatment with 100 µM FMRFamide. FMRFamide potentiated the pH 5.0 induced peak (<b>A</b>) and sustained (<b>C</b>) currents. <b>B, D.</b> Average data showing potentiation of current by FMRFamide as shown in <b>A</b> and <b>C</b> (n = 7). * <i>P</i><0.05, Student's <i>t</i>-test.</p

The effects of ASIC channel blockers on ASIC currents in freshly isolated anterior pituitary cells.

<p><b>A</b>. Representative ASIC currents elicited by pH 5.5 with various concentrations of amiloride. Holding potential was −70 mV. The cells were held at pH 7.4 before and in between applications of pH 5.5. Amiloride was applied during acid application. <b>B</b>. Summary data showing the dose-dependent block of ASIC currents by amiloride in anterior pituitary cells (n = 7). Currents were normalized to the control condition (without amiloride). The IC₅₀ of amiloride block was 6.3±1.0 µM (n = 7). C. Representative ASIC currents in WT and ASIC2^-/- cells elicited by pH 5.5 with 100 nM PcTx1. Cells were pre-treated with PcTx1 in pH 7.4 for 1 min and then were perfused with pH 5.5 containing 100 nM PcTx1. Holding potential was −70 mV. D. Summary data showing the block of ASIC currents by 100 nM PcTx1 in anterior pituitary cells from WT (n = 7) and ASIC2^-/- mice (n = 6). * <i>P</i><0.05 compared to control, Student's <i>t</i>-test.</p

Table1_Sex-dependent regulation of mucin gene transcription and airway secretion and mechanics following intra-airway IL-13 in mice with conditional loss of club cell Creb1.docx

Introduction: Interleukin 13 (IL-13) is an important effector molecule in allergic asthma. IL-13-mediated mucin hypersecretion requires conversion of secretoglobin-positive club cells into goblet cells through suppression of forkhead box A2 (FOXA2) and induction of SAM pointed domain containing ETS transcription factor (SPDEF). IL-13-mediated mucin hypersecretion may also include modulation of purinergic and muscarinic receptors that control basal and stimulated mucin secretion. We recently found that the transcription factor cAMP response element-binding protein (Creb1) inhibits FOXA2 and modulates mucus secretion in mice.Methods: We tested the hypothesis that loss of club cell Creb1 mitigates the pro-mucin effects of IL-13. We challenged male and female mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-13 or vehicle. We also studied human “club cell-like” NCI-H322 cells.Results: Loss of club cell Creb1 augmented IL-13-mediated increases in mRNA for the gel-forming mucins Muc5ac and Muc5b and prevented IL-13-mediated decreases in muscarinic 3 receptor (M3R) mRNA in male airways. In female airways, loss of club cell Creb1 reduced M3R mRNA and significantly blunted IL-13-mediated increases in purinergic receptor P2Y2 (P2ry2) mRNA but did not impact Muc5ac and Muc5b mRNA. Despite changes in mucins and secretion machinery, goblet cell density following cholinergic stimulation was not impacted by loss of club cell Creb1 in either sex. IL-13 treatment decreased basal airway resistance across sexes in mice with loss of club cell Creb1, whereas loss of club cell Creb1 augmented IL-13-mediated increases in airway elastance in response to methacholine. NCI-H322 cells displayed IL-13 signaling components, including IL-13Rα1 and IL-4Rα. Pharmacologic inhibition of CREB reduced IL-13Rα1 mRNA, whereas recombinant CREB decreased IL-4Rα mRNA. Application of IL-13 to NCI-H322 cells increased concentrations of cAMP in a delayed manner, thus linking IL-13 signaling to CREB signaling.Conclusion: These data highlight sex-specific regulation of club cell Creb1 on IL-13-mediated mucin hypersecretion and airway mechanics.</p