Genetic stress-reactivity, sex, and conditioning intensity affect stress-enhanced fear learning.

The Stress-Enhanced Fear Learning (SEFL) model of posttraumatic stress disorder (PTSD) reveals increased fear memory in animals exposed to stress prior to contextual fear conditioning (CFC), similar to the increased likelihood of developing PTSD in humans after prior stress. The present study utilized the SEFL model by exposing animals to restraint stress as the first stressor, followed by CFC using foot-shocks with 0.6 mA or 0.8 mA intensity. Adult males and females from the two nearly isogenic rat strains, the genetically more stress-reactive Wistar Kyoto (WKY) More Immobile (WMI), and the less stress-reactive WKY Less Immobile (WLI) were employed. Percent time spent freezing at acquisition and at recall differed between these strains in both prior stress and no stress conditions. The significant correlations between percent freezing at acquisition and at recall suggest that fear memory differences represent a true phenotype related to the stress-reactivity differences between the strains. This assumption is further substantiated by the lack of effect of either conditioning intensity on percent freezing in WLI males, while WMI males were affected by both intensities albeit with opposite directional changes after prior stress. Differences between the sexes in sensitivity to the two conditioning intensities became apparent by the opposite directional and inverse relationship between fear memory and the intensity of conditioning in WMI males and females. The present data also illustrate that although corticosterone (CORT) responses to prior stress are known to be necessary for SEFL, plasma CORT and percent freezing were positively correlated only in the stress less-reactive WLI strain. These differences in baseline fear acquisition, fear memory, and the percent freezing responses to the SEFL paradigm in the two genetically close inbred WMI and WLI strains provide a unique opportunity to study the genetic contribution to the variation in these phenotypes.

Pilot validation of blood-based biomarkers during pregnancy and postpartum in women with prior or current depression.

Major depressive disorder (MDD) is more common in women than in men, and evidence of gender-related subtypes of depression is emerging. Previously identified blood-based transcriptomic biomarkers distinguished male and female subjects with MDD from those without the disorder. In the present pilot study, we investigated the performance of these biomarkers in pregnant and postpartum women with prior major depressive episodes, some of whom had current symptomatology. The symptom scores of 13 pregnant and 15 postpartum women were identified by the Inventory of Depressive Symptoms (IDS-SR-30) at the time of blood sampling. Blood levels of the 20 transcriptomic biomarkers and that of estrogen receptor 2 (ESR2), membrane progesterone receptor alpha and beta (mPRα, mPRß) were measured. In pregnant women, transcript levels of ADCY3, ASAH1, ATP11C, CDR2, ESR2, FAM46A, mPRß, NAGA, RAPH1, TLR7, and ZNF291/SCAPER showed significant association with IDS-SR-30 scores, of which ADCY3, FAM46A, RAPH1, and TLR7 were identified in previous studies for their diagnostic potential for major depression. ASAH1 and ATP11C were previously also identified as potential markers of treatment efficacy. In postpartum women, transcript levels of CAT, CD59, and RAPH1 demonstrated a trend of association with IDS-SR-30 scores. Transcript levels of ADCY3, ATP11C, FAM46A, RAPH1, and ZNF291/SCAPER correlated with ESR2 and mPRß expressions in pregnant women, whereas these associations only existed for mPRß in postpartum women. These results suggest that a blood biomarker panel can identify depression symptomatology in pregnant women and that expression of these biomarker genes are affected by estrogen and/or progesterone binding differently during pregnancy and postpartum.

Heterogeneous stock rats: a model to study the genetics of despair-like behavior in adolescence.

Major depressive disorder (MDD) is a complex illness caused by both genetic and environmental factors. Antidepressant resistance also has a genetic component. To date, however, very few genes have been identified for major depression or antidepressant resistance. In this study, we investigated whether outbred heterogeneous stock (HS) rats would be a suitable model to uncover the genetics of depression and its connection to antidepressant resistance. The Wistar Kyoto (WKY) rat, one of the eight founders of the HS, is a recognized animal model of juvenile depression and is resistant to fluoxetine antidepressant treatment. We therefore hypothesized that adolescent HS rats would exhibit variation in both despair-like behavior and response to fluoxetine treatment. We assessed heritability of despair-like behavior and response to sub-acute fluoxetine using a modified forced swim test (FST) in 4-week-old HS rats. We also tested whether blood transcript levels previously identified as depression biomarkers in adolescent human subjects are differentially expressed in HS rats with high vs. low FST immobility. We demonstrate heritability of despair-like behavior in 4-week-old HS rats and show that many HS rats are resistant to fluoxetine treatment. In addition, blood transcript levels of Amfr, Cdr2 and Kiaa1539, genes previously identified in human adolescents with MDD, are differentially expressed between HS rats with high vs. low immobility. These data demonstrate that FST despair-like behavior will be amenable to genetic fine-mapping in adolescent HS rats. The overlap between human and HS blood biomarkers suggest that these studies may translate to depression in humans.

Hippocampus-dependent memory and allele-specific gene expression in adult offspring of alcohol-consuming dams after neonatal treatment with thyroxin or metformin.

Fetal alcohol spectrum disorder (FASD), the result of fetal alcohol exposure (FAE), affects 2-11% of children worldwide, with no effective treatments. Hippocampus-based learning and memory deficits are key symptoms of FASD. Our previous studies show hypothyroxinemia and hyperglycemia of the alcohol-consuming pregnant rat, which likely affects fetal neurodevelopment. We administered vehicle, thyroxine (T4) or metformin to neonatal rats post FAE and rats were tested in the hippocampus-dependent contextual fear-conditioning paradigm in adulthood. Both T4 and metformin alleviated contextual fear memory deficit induced by FAE, and reversed the hippocampal expression changes in the thyroid hormone-inactivating enzyme, deiodinase-III (Dio3) and insulin-like growth factor 2 (Igf2), genes that are known to modulate memory processes. Neonatal T4 restored maternal allelic expressions of the imprinted Dio3 and Igf2 in the adult male hippocampus, while metformin restored FAE-caused changes in Igf2 expression only. The decreased hippocampal expression of DNA methyltransferase 1 (Dnmt1) that maintains the imprinting of Dio3 and Igf2 during development was normalized by both treatments. Administering Dnmt1 inhibitor to control neonates resulted in FAE-like deficits in fear memory and hippocampal allele-specific expression of Igf2, which were reversed by metformin. We propose that neonatal administration of T4 and metformin post FAE affect memory via elevating Dnmt1 and consequently normalizing hippocampal Dio3 and Igf2 expressions in the adult offspring. The present results indicate that T4 and metformin, administered during the neonatal period that is equivalent to the third trimester of human pregnancy, are potential treatments for FASD and conceivably for other neurodevelopmental disorders with cognitive deficits.

Nature and nurture: environmental influences on a genetic rat model of depression.

In this study, we sought to learn whether adverse events such as chronic restraint stress (CRS), or 'nurture' in the form of environmental enrichment (EE), could modify depression-like behavior and blood biomarker transcript levels in a genetic rat model of depression. The Wistar Kyoto More Immobile (WMI) is a genetic model of depression that aided in the identification of blood transcriptomic markers, which successfully distinguished adolescent and adult subjects with major depressive disorders from their matched no-disorder controls. Here, we followed the effects of CRS and EE in adult male WMIs and their genetically similar control strain, the Wistar Kyoto Less Immobile (WLI), that does not show depression-like behavior, by measuring the levels of these transcripts in the blood and hippocampus. In WLIs, increased depression-like behavior and transcriptomic changes were present in response to CRS, but in WMIs no behavioral or additive transcriptomic changes occurred. Environmental enrichment decreased both the inherent depression-like behavior in the WMIs and the behavioral difference between WMIs and WLIs, but did not reverse basal transcript level differences between the strains. The inverse behavioral change induced by CRS and EE in the WLIs did not result in parallel inverse expression changes of the transcriptomic markers, suggesting that these behavioral responses to the environment work via separate molecular pathways. In contrast, 'trait' transcriptomic markers with expression differences inherent and unchanging between the strains regardless of the environment suggest that in our model, environmental and genetic etiologies of depression work through independent molecular mechanisms.

Stage-specific regulation of respiratory epithelial cell differentiation by Foxa1.

Foxa1 is a member of the winged helix family of transcription factors that is expressed in epithelial cells of the conducting airways and in alveolar type II cells of the lung. To determine the role of Foxa1 during lung morphogenesis, histology and gene expression were assessed in lungs from Foxa1-/- gene-targeted mice from embryonic day (E) 16.5 to postnatal day (PN) 13. Deletion of Foxa1 perturbed maturation of the respiratory epithelium at precise times during lung morphogenesis. While dilatation of peripheral lung saccules was delayed in Foxa1-/- mice at E16.5, sacculation was unperturbed later in development (E17.5-E18.5). At PN5, alveolarization was markedly delayed in Foxa1-/- mice; however, by PN13 lung histology was comparable to wild-type controls. Clara cell secretory protein (CCSP), prosurfactant protein (SP)-C, and SP-B protein content and immunostaining were decreased in Foxa1-/- mice between E16.5 and E18.5 but normalized after birth. Timing and sites of expression of thyroid transcription factor-1, Foxj1, and beta-tubulin were unaltered in lungs of Foxa1-/- mice. In vitro, Foxa1 regulated the activity of CCSP and SP-A, SP-B, SP-C, and SP-D promoters as assessed by luciferase reporter assays in HeLa, H441, and MLE15 cells. Although Foxa1 regulates respiratory epithelial differentiation and structural maturation of the lung at precise developmental periods, the delay in maturation is subsequently compensated at times to enable respiratory function and restore normal lung structure after birth.

Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation.

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5(-/-)) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5(+/+) mice, Aqp5(-/-) mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5(-/-) mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5(-/-) mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.

Immunolocalization of sonic hedgehog (Shh) in developing mouse lung.

Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), beta-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.

SP-D and GM-CSF regulate surfactant homeostasis via distinct mechanisms.

Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(-/-),GM(-/-)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(-/-),GM(-/-) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(-/-),GM(-/-) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(-/-) mice, degradation of [(3)H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(-/-) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(-/-) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.

Surfactant protein deficiency in familial interstitial lung disease.

OBJECTIVE: To determine the contribution of surfactant protein abnormalities to the development of chronic lung injury in a familial form of interstitial lung disease. STUDY DESIGN: An 11-year-old girl, her sister, and their mother who were diagnosed with chronic interstitial lung disease underwent laboratory investigation of surfactant protein expression in bronchoalveolar lavage fluid and lung biopsy specimens. Nineteen patients with idiopathic pulmonary fibrosis and 9 patients who were investigated for pulmonary malignancy but who did not have interstitial lung disease served as control subjects. RESULTS: The 3 family members were found to have absent surfactant protein C (SP-C) and decreased levels of SP-A and SP-B in bronchoalveolar lavage fluid (BALF). Immunostaining for pulmonary surfactant proteins in lung biopsy specimens obtained from both children demonstrated a marked decrease of pro-SP-C in the alveolar epithelial cells but strong staining for pro-SP-B, SP-B, SP-A, and SP-D. No deviations from published surfactant protein B or C coding sequences were identified by DNA sequence analysis. All control subjects had a detectable level of SP-C in the BALF. CONCLUSION: The apparent absence of SP-C and a decrease in the levels of SP-A and SP-B are associated with familial interstitial lung disease.

Expression of thyroid transcription factor-1, surfactant proteins, type I cell-associated antigen, and Clara cell secretory protein in pulmonary hypoplasia.

Pulmonary hypoplasia (PH) is a developmental abnormality characterized by diminished distal lung parenchyma. Recent studies have demonstrated that thyroid transcription factor 1 (TTF-1), a member of NKx2 family of homeodomain transcription factors, plays an important role in lung organogenesis and lung epithelial gene expression. In order to evaluate whether abnormal expression of TTF-1 contributes to the pathophysiology of PH, we studied the expression of TTF-1, as well as that of the surfactant proteins (SPs), Clara cell secretory protein (CCSP), and type I cell-associated antigen (T1 cell-Ag), in PH. Immunolocalization patterns of these proteins were evaluated in 15 cases of PH with different associated diseases and compared with those of 14 matched controls. Our study demonstrated that the concentration gradient of TTF-1 along the proximal-distal axis in normal fetal lung is disrupted in PH after 24 weeks gestational age, while the expression of the SPs, CCSP, and T1 cell-Ag seemed to be preserved. We conclude that a normal TTF-1 expression pattern might be crucial in the control of distal lung development. Failure to switch off expression of TTF-1 in PH of more than 24 weeks gestational age may be a final common pathway leading to PH associated with the disease processes investigated in this study.

Surfactant proteins and cell markers in the respiratory epithelium of the amphibian, Ambystoma mexicanum.

The respiratory tract is lined by diverse epithelial cell types whose morphology, gene expression and functions are highly specialized along the cephalo-caudal axis of the lung. Pulmonary gas exchange, surface tension reduction, host defense, fluid and electrolyte transport are functions shared by various vertebrate species, each organism facing similar requirements for adaptation to air breathing. Consistent with this concept, we have identified distinct respiratory epithelial cell populations in the amphibian, Ambystoma mexicanum, using morphologic, histochemical and immunochemical techniques. Thyroid transcription factor-1 (TTF-1), a homeodomain nuclear transcription factor critical to lung formation, and surfactant protein B (SP-B), an amphipathic polypeptide required for surfactant function, were detected in the peripheral respiratory epithelial cells of the axolotl lung, in cells with characteristics of Type II alveolar epithelial cells in mammals. beta-Tubulin and carbohydrate staining identified distinct subsets of ciliated and goblet cells. SP-D, a member of the collectin family of innate host defense proteins, was also detected in peripheral epithelial cells of the axolotl lung. Pulmonary surfactant and host defense proteins are shared across diverse phyla supporting the concept that pulmonary structure and function have evolved from common ancestors.

FGF-10 disrupts lung morphogenesis and causes pulmonary adenomas in vivo.

Transgenic mice in which fibroblast growth factor (FGF)-10 was expressed in the lungs of fetal and postnatal mice were generated with a doxycycline-inducible system controlled by surfactant protein (SP) C or Clara cell secretory protein (CCSP) promoter elements. Expression of FGF-10 mRNA in the fetal lung caused adenomatous malformations, perturbed branching morphogenesis, and caused respiratory failure at birth. When expressed after birth, FGF-10 caused multifocal pulmonary tumors. FGF-10-induced tumors were highly differentiated papillary and lepidic pulmonary adenomas. Epithelial cells lining the tumors stained intensely for thyroid transcription factor (TTF)-1 and SP-C but not CCSP, indicating that FGF-10 enhanced differentiation of cells to a peripheral alveolar type II cell phenotype. Withdrawal from doxycycline caused rapid regression of the tumors associated with rapid loss of the differentiation markers TTF-1, SP-B, and proSP-C. FGF-10 disrupted lung morphogenesis and induced multifocal pulmonary tumors in vivo and caused reversible type II cell differentiation of the respiratory epithelium.

Temporal/spatial expression of nuclear receptor coactivators in the mouse lung.

Our laboratory has previously demonstrated that retinoic acid nuclear receptor, thyroid transcription factor-1 (TTF-1), and nuclear receptor coactivators such as cAMP response element binding protein (CREB) binding protein (CBP)/p300 and steroid receptor coactivator-1 (SRC-1) form an enhanceosome on the 5'-enhancer region of the human surfactant protein B gene. Immunohistochemistry was used to identify cells that coexpressed CBP/p300, SRC-1, retinoid X receptor, and TTF-1 in the developing and mature lung. CBP/p300 and SRC-1 were expressed in the adult mouse lung, CBP and p300 being present in both alveolar type I and type II epithelial cells and SRC-1 and TTF-1 being restricted to type II epithelial cells. CBP/p300, SRC-1, and TTF-1 were readily detected in the nuclei of developing respiratory epithelial tubules in fetal mice from embryonic days 10 to 18. CBP/p300 and SRC-1 were also detected in developing mesenchymal cells. These coactivators were coexpressed with TTF-1 and SP-B in human pulmonary adenocarcinoma cells (H441 cells) in vitro. Interaction assays with a two-hybrid reporter analysis demonstrated direct interactions among TTF-1, SRC-1, and CBP/p300 in H441 cells. These findings support a role for retinoic acid receptor and nuclear receptor coactivators in the regulation of SP-B gene expression in the respiratory epithelium.

Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo.

Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA). To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR. A subacute intratracheal dose of 3 x 10(6) bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/-). In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 x 10(7) PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/-, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR-/-, the latter lacking expression of mCFTR in the lung. However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice. Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments. Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression. The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.

Plasminogen did not affect lung function in surfactant-treated preterm lambs.

Preterm infants with respiratory distress syndrome develop fibrin-rich hyaline membranes within the alveoli and have depressed fibrinolytic activity, which is thought to be due to a relative deficiency of plasminogen. Local fibrin deposition inhibits surfactant function and amplifies inflammation. We hypothesized that plasminogen administration to surfactant-treated preterm lambs would prevent fibrin-rich hyaline membrane formation, resulting in the amelioration of lung pathology and improved lung function. We randomly treated preterm lambs (gestational age 127-129 days) with either 16 mg of lysine-plasminogen (n = 10) or saline (n = 10), and ventilated them for 5 h. There were no significant differences in physiologic measurements of lung function (ventilation efficiency index, oxygenation index, dynamic compliance, quasi-static pressure volume curve), measures of lung injury (alveolar wash protein content and (125)I-albumin recovery) or surfactant pool size. The degree and extent of bronchiolar erosion and hyaline membrane formation were similar in the two groups. Plasminogen administration did not improve lung function or prevent hyaline membrane formation in surfactant-treated lambs.

Recurrent familial neonatal deaths: hereditary surfactant protein B deficiency.

Hereditary surfactant protein B (SP-B) deficiency is an uncommon autosomal recessive lung disorder that causes hypoxemic respiratory failure in mature, morphologically normal infants. Recognition and diagnosis of this condition is of paramount importance, as it has significant implications for future pregnancies with a recurrence risk of 25%. In a family with three neonatal deaths over 20 years, SP-B deficiency was diagnosed following the death of the fourth affected infant. Previous deaths were mistakenly attributed to hyaline membrane disease (HMD), congenital Mycoplasma hominis infection, and pulmonary hypertension, however, following the diagnosis in the proposita, SP-B deficiency was also confirmed in her deceased siblings by immunohistochemical staining of autopsy specimens. This case highlights the presentation, postnatal course, diagnosis, and therapeutic options of SP-B deficiency in addition to the mode of inheritance and the possibility of antenatal diagnosis. Genetic consultation is imperative in the investigations of recurrent neonatal deaths, especially in cases of remote events. The recent enormous advances in human genetics have shown that many conditions previously ascribed to environmental agents have a genetic basis.

Prolonged survival in hereditary surfactant protein B (SP-B) deficiency associated with a novel splicing mutation.

Hereditary surfactant protein B (SP-B) deficiency has been lethal in the first year of life without lung transplantation. We tested the hypothesis that SP-B gene mutations may result in milder phenotypes by investigating the mechanisms for lung disease in two children with less severe symptoms than have been previously observed in SP-B deficiency. Immunostaining patterns for pulmonary surfactant proteins were consistent with SP-B deficiency in both children. DNA sequence analysis indicated that both children were homozygous for a mutation in exon 5 that created an alternative splice site. Reverse transcriptase PCR and sequence analysis confirmed use of this splice site, which resulted in a frameshift and a premature termination codon in exon 7. The predominant reverse transcriptase PCR product, however, lacked exon 7, which restored the reading frame but would not allow translation of the exons that encode mature SP-B. Western blot analysis detected reduced amounts of mature SP-B as well as an aberrant SP-B proprotein that corresponded to the size expected from translation of the abnormal transcript. We conclude that a novel splicing mutation was the cause of lung disease in these children and that hereditary SP-B deficiency can be the cause of lung disease in older children.