Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

Dysregulation of miR-34a links neuronal development to genetic risk factors for bipolar disorder.

Bipolar disorder (BD) is a heritable neuropsychiatric disorder with largely unknown pathogenesis. Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD. Here we show that levels of miR-34a, which is predicted to target multiple genes implicated as genetic risk factors for BD, are increased in postmortem cerebellar tissue from BD patients, as well as in BD patient-derived neuronal cultures generated by reprogramming of human fibroblasts into induced neurons or into induced pluripotent stem cells (iPSCs) subsequently differentiated into neurons. Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets. Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration. Taken together, we propose that miR-34a serves as a critical link between multiple etiological factors for BD and its pathogenesis through the regulation of a molecular network essential for neuronal development and synaptogenesis.

Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons.

Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia and major depression, and a haplotype located in an intronic region of the L-type voltage-gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in iNs homozygous for the risk genotype, compared with non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human iNs, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease.

Cis-acting regulation of brain-specific ANK3 gene expression by a genetic variant associated with bipolar disorder.

Several genome-wide association studies for bipolar disorder (BD) have found a strong association of the Ankyrin 3 (ANK3) gene. This association spans numerous linked single-nucleotide polymorphisms (SNPs) in an ~250-kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of the six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as Ankyrin-G. Using RNA ligase-mediated rapid amplification of cDNA ends to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites and coupling of specific 5' ends with 3' mRNA splicing events in postmortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD-associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 that may be relevant to BD pathophysiology.

LY294002, but not wortmannin, increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells.

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.

Interleukin-4 rapidly inhibits calcium transients in response to carbachol in bovine airway smooth muscle cells.

To assess interleukin (IL)-4 effects on calcium signaling, bovine airway smooth-muscle (ASM) cells were loaded with fura-2 and cytosolic calcium ([Ca(2+)](i)) was measured in single cells by digital microscopy. Human recombinant IL-4 (50 ng/ml) caused small increases in [Ca(2+)](i). For single cells, carbachol-stimulated calcium transients were compared before (S1) and after (S2) exposure to IL-4 or IL-13. When cells were treated with IL-4 (50 ng/ml) for 20 min, the S2/S1 ratio was 0.17 +/- 0.04 (n = 7) even though IL-4 had been washed from the chamber for 10 min before the S2 response. In contrast, controls not treated with IL-4 had S2/S1 of 0.70 +/- 0.04 (n = 13, P < 0.01). Lower concentrations of IL-4 variably decreased transients and IL-13 had no effect. In other experiments, 5 min of IL-4 did not immediately decrease transients but did after a 25-min delay. Goat antihuman IL-4 antibody abolished the effect of IL-4. IL-4 (50 ng/ml) also inhibited responses to caffeine (S2/S1: 0.30 +/- 0.04 and 0.54 +/- 0.06 for IL-4-treated versus control). We conclude that IL-4 rapidly inhibited calcium transients. Because caffeine-stimulated transients were inhibited, IL-4 may act, at least in part, by depleting calcium stores. IL-4 inhibition of cholinergic signaling may be important for modulating ASM responses during inflammation.

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells.

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.

Symptom research on chronic cough: a historical perspective.

This review provides a perspective on how research on the management of cough has evolved, looks at key methodologic lessons that have been learned from this research and how they may relate to the management of other symptoms, identifies important methodologic challenges that remain to be solved, and lists important questions that still need to be answered. Three important methodologic lessons have been learned. First, cough must be evaluated systematically and according to a neuroanatomic framework. Second, the response to specific therapy must be noted to determine the cause or causes of cough and to characterize the strengths and limitations of diagnostic testing. Third, multiple conditions can simultaneously cause cough. Among the three methodologic challenges that still need to be solved are 1) definitively determining the diagnostic accuracy and reliability of 24-hour esophageal pH monitoring and how best to interpret pH test results, 2) definitively determining the role of nonacid reflux in cough due to gastroesophageal reflux disease, and 3) developing reliable and reproducible subjective and objective methods with which to assess the efficacy of cough therapy. Numerous important clinical questions are still unanswered: What role do empirical therapeutic trials play in diagnosing the cause of chronic cough? What is the most cost-effective approach to the diagnosis and treatment of chronic cough: empirical therapeutic trials or laboratory testing-directed therapeutic trials? How often is environmental air pollution, unrelated to allergies or smoking, responsible for chronic cough?

Loss of chemokine SDF-1alpha-mediated CXCR4 signalling and receptor internalization in human hepatoma cell line HepG2.

Expression of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is absent from many carcinomas, including hepatomas. We note an early signalling defect in the hepatocellular carcinoma (HCC) cell line HepG2 that expresses the CXCR4 receptor and binds biotin-labelled SDF, but fails to stimulate downstream signalling events after engagement with SDF. In HepG2, the SDF/CXCR4 interaction did not result in calcium influx, phosphorylation and internalization of CXCR4, nor in a rapid phosphorylation of p44/42 MAP kinase. There were no CXCR4 mutations in the second chemokine binding loop or C terminal phosphorylation and internalization domains. The downstream signalling machinery in HepG2 appears to be intact since transfection of wild-type CXCR4 restored functional responsiveness. We conclude that HepG2 is unresponsive to SDF stimulation because of a defect located after receptor binding but before the activation of the signalling cascade. A hypothetical blocking molecule could hinder receptor internalization or CXCR4 signalling.

Effects of salmeterol on muscarinic inhibition of adenylyl cyclase in bovine trachealis cells.

The goal was to assess whether salmeterol, a potent and long-acting beta-2-adrenergic agonist used in the treatment of asthma, also has non-beta-2-adrenergic effects on the stimulation or inhibition of adenylyl cyclase activity. Salmeterol (100 nM) maximally stimulated cAMP accumulation in enzyme dispersed bovine trachealis cells and this was entirely inhibited by propranolol, as expected for beta-adrenergic stimulation. However, the same concentration of salmeterol also antagonized carbachol inhibition of cAMP accumulation and altered binding of carbachol to muscarinic receptors. These effects of salmeterol were sensitive to washing of the cells and this was not consistent with a beta-2-adrenergic mechanism. The findings suggested that the maximal, beta-2-adrenergic stimulation of cAMP accumulation by salmeterol was accompanied by a non-beta-2-adrenergic interaction of salmeterol with muscarinic receptors that attenuated muscarinic inhibition of adenylyl cyclase.

The cough reflex and its relation to gastroesophageal reflux.

Each cough involves a complex reflex arc beginning with the stimulation of sensory nerves that function as cough receptors. There is evidence, primarily clinical, that the sensory limb of the reflex exists in and outside of the lower respiratory tract. Although myelinated, rapidly adapting pulmonary stretch receptors (RARs), also known as irritant receptors, are the most likely type of sensory nerve that stimulates the cough center in the brain, afferent C-fibers and slowly adapting pulmonary stretch receptors (SARs) also may modulate cough. RARS, C-fibers, and SARs have been identified in the distal esophageal mucosa; however, studies have not been performed to determine whether they can participate in the cough reflex. Although gastroesophageal reflux disease can potentially stimulate the afferent limb of the cough reflex by irritating the upper respiratory tract without aspiration and by irritating the lower respiratory tract by micro- or macroaspiration, there is evidence that strongly suggests that reflux commonly provokes cough by stimulating an esophageal-bronchial reflex. Theoretically, the pathways of this reflex may be modeled in a variety of ways, and these are speculated upon in this article. The predominant role of acid in triggering cough by means of this reflex is unclear because of conflicting results from provocative challenge studies. It is interesting to speculate that a distal esophageal-bronchial reflex evolved as an early warning defense so that coughing could be started, just in case the refluxate were to reach the inlet of the lower respiratory tract. That is, thinking teleologically, it is possible that an esophageal-bronchial reflex evolved as one of several mechanisms designed to protect the lungs from aspiration of gastric contents.

Anatomical diagnostic protocol in evaluating chronic cough with specific reference to gastroesophageal reflux disease.

Using the anatomic, diagnostic protocol, the cause of chronic cough can be determined 88% to 100% of the time, leading to specific therapy with success rates of 84% to 98%. Gastroesophageal reflux disease (GERD), along with postnasal drip syndrome (PNDS) and asthma, is one of the three most common causes of chronic cough in all age groups. When GERD is the cause of chronic cough, there may be no gastrointestinal (GI) symptoms up to 75% of the time, and, in these cases, the term "silent GERD" is used. The most sensitive and specific test for GERD is 24-hour esophageal pH monitoring. In interpreting this test, it is essential not only to evaluate the duration and frequency of the reflux episodes but also to determine the temporal relationship between reflux and cough events. Patients with normal standard reflux parameters still may have reflux diagnosed as the likely cause of cough if a temporal relationship exists. The definitive diagnosis of cough resulting from GERD can only be made if cough goes away with antireflux therapy. When 24-hour esophageal pH monitoring cannot be done, an empiric trial of antireflux medical therapy is appropriate when GERD is a likely cause of chronic cough. It is likely in the following settings: patients with prominent GI symptoms consistent with GERD and/or those with no GI complaints and normal chest x-rays, who are not taking angiotensin-converting enzyme inhibitors and who are not smoking, and in whom asthma and PNDS have been excluded. However, if empiric treatment fails, it cannot be assumed that GERD has been ruled out as a cause of chronic cough; rather, objective investigation for GERD is recommended, because the empiric therapy may not have been intensive enough or it may have failed. In treating patients with chronic cough resulting from GERD, cough has been reported to resolve with medical therapy 70% to 100% of the time. Mean time to recovery may take as long as 161 to 179 days, and patients may not start to get better for 2 to 3 months. In patients who fail to respond to maximal medical therapy, antireflux surgery can be successful.

Refilling of caffeine-sensitive intracellular calcium stores in bovine airway smooth muscle cells.

The goal of this study was to assess the mechanisms by which the caffeine-sensitive calcium stores of airway smooth muscle cells are refilled. Bovine trachealis cells were loaded with fura 2-AM (0.5 microM) for imaging of cytosolic calcium concentrations ([Ca2+]i) in the inner cytosol. After a first stimulation (S1) with caffeine, the response to a second stimulation (S2) depended on the presence of extracellular calcium during an intervening 80-s-long refilling phase. The S2-to-S1 ratio (S2/S1) was 0.11 +/- 0.05 (n = 13 cells) during calcium-free refilling but 0.72 +/- 0.04 (n = 36 cells) within 80 s of exposure to extracellular calcium. Maximum mean [Ca2+]i during the 80 s of refilling was not different for calcium-free (116 +/- 19 nM; n = 13 cells) versus extracellular calcium plus nickel (2 mM) (121 +/- 12 nM; n = 21 cells); despite this, significantly greater refilling (S2/S1 0.58 +/- 0.06; n = 24 cells) occurred in the presence of extracellular calcium plus nickel. The protein tyrosine kinase inhibitors genistein (100 microM) and ST-638 (50 microM) significantly decreased refilling over 80 s (S2/S1 0.35 +/- 0.06, n = 14 cells and 0.51 +/- 0.07, n = 14 cells, respectively). Daidzein (100 microM) had no effect on S2/S1. We concluded that [Ca2+]i of the inner cytosol during refilling correlated poorly with S2/S1 values and that, therefore, additional compartments not well detected by fura 2 contribute to refilling. The findings suggest that calcium influx for refilling is segregated from the inner cytosol of the cell, relatively insensitive to nickel, and regulated or modulated by protein tyrosine kinase activity.

Characterization of a human homologue of the Saccharomyces cerevisiae transcription factor spt3 (SUPT3H).

Spt3 is a Saccharomyces cerevisiae transcription factor that is required in vivo for the transcription of a number of RNA polymerase II-transcribed genes. We report the cloning of the gene encoding the human homologue of Spt3, SUPT3H, and its initial functional analysis. The human and yeast Spt3 homologues share an overall identity of 30% that defines three conserved regions, suggesting possible functional domains. To determine whether SUPT3H is a true functional Spt3 homologue, we tested for complementation of an spt3Delta mutation in yeast. While expression of the full-length SUPT3H is unable to complement an spt3Delta mutation, expression of a human-yeast chimeric gene that contains 42% human sequences can partially complement an spt3Delta mutation. These data suggest that the transcriptional control carried out by Spt3 has been conserved from yeast to human.

Chronic obstructive pulmonary disease.

Acute exacerbations of underlying COPD are a common cause of respiratory deterioration. Developments have been made in preventive measures, but admission to hospital for acute exacerbations can be expected to remain common. Several expert consensus guidelines have been published to define the appropriate management of COPD patients. These consensus guidelines generally agree, but all acknowledge a lack of large well-controlled clinical studies, especially studies focusing on the management of acute exacerbations. Consequently, many potential controversies exist about the details of managing patients with acute exacerbations. Although studies of many fundamental aspects of management are still needed, the results of controlled clinical trials are sufficient to emphasise the importance of a careful clinical assessment, supplemental oxygen, inhaled bronchodilators to partially improve airway obstruction, corticosteroids to decrease the likelihood of treatment failures and to speed recovery, antibiotics, especially in severe patients, and non-invasive positive-pressure ventilation for treatment of acute ventilatory failure in selected patients.

Identification and analysis of Mot3, a zinc finger protein that binds to the retrotransposon Ty long terminal repeat (delta) in Saccharomyces cerevisiae.

Spt3 and Mot1 are two transcription factors of Saccharomyces cerevisiae that are thought to act in a related fashion to control the function of TATA-binding protein (TBP). Current models suggest that while Spt3 and Mot1 do not directly interact, they do function in a related fashion to stabilize the TBP-TATA interaction at particular promoters. Consistent with this model, certain combinations of spt3 and mot1 mutations are inviable. To identify additional proteins related to Spt3 and Mot1 functions, we screened for high-copy-number suppressors of the mot1 spt3 inviability. This screen identified a previously unstudied gene, MOT3, that encodes a zinc finger protein. We show that Mot3 binds in vitro to three sites within the retrotransposon Ty long terminal repeat (delta) sequence. One of these sites is immediately 5' of the delta TATA region. Although a mot3 null mutation causes no strong phenotypes, it does cause some mild phenotypes, including a very modest increase in Ty mRNA levels, partial suppression of transcriptional defects caused by a mot1 mutation, and partial suppression of an spt3 mutation. These results, in conjunction with those of an independent study of Mot3 (A. Grishin, M. Rothenberg, M. A. Downs, and K. J. Blumer, Genetics, in press), suggest that this protein plays a varied role in gene expression that may be largely redundant with other factors.

Identification and analysis of homologues of Saccharomyces cerevisiae Spt3 suggest conserved functional domains.

Spt3 of Saccharomyces cerevisiae is a factor required for normal transcription from particular RNA polymerase II-dependent promoters. As a step towards analysing Spt3 structure-function relationships, we have identified and studied Spt3 homologues from three other yeasts: Kluyveromyces lactis, Clavispora opuntiae and Schizosaccharomyces pombe. Alignment of their predicted amino acid sequences shows an overall identity of 30% between all four homologues and suggests that three conserved domains are present in Spt3. When tested for function in S. cerevisiae, K. lactis SPT3 was shown to fully complement and S. pombe SPT3 to partially complement an spt3 delta mutation. These data demonstrate that Spt3 is functionally conserved among distantly related yeasts.