Ciliopathy is differentially distributed in the brain of a Bardet-Biedl syndrome mouse model

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous inherited human disorder displaying a pleotropic phenotype. Many of the symptoms characterized in the human disease have been reproduced in animal models carrying deletions or knock-in mutations of genes causal for the disorder. Thinning of the cerebral cortex, enlargement of the lateral and third ventricles, and structural changes in cilia are among the pathologies documented in these animal models. Ciliopathy is of particular interest in light of recent studies that have implicated primary neuronal cilia (PNC) in neuronal signal transduction. In the present investigation, we tested the hypothesis that areas of the brain responsible for learning and memory formation would differentially exhibit PNC abnormalities in animals carrying a deletion of the Bbs4 gene (Bbs4-/-). Immunohistochemical localization of adenylyl cyclase-III (ACIII), a marker restricted to PNC, revealed dramatic alterations in PNC morphology and a statistically significant reduction in number of immunopositive cilia in the hippocampus and amygdala of Bbs4-/- mice compared to wild type (WT) littermates. Western blot analysis confirmed the decrease of ACIII levels in the hippocampus and amygdala of Bbs4-/- mice, and electron microscopy demonstrated pathological alterations of PNC in the hippocampus and amygdala. Importantly, no neuronal loss was found within the subregions of amygdala and hippocampus sampled in Bbs4-/- mice and there were no statistically significant alterations of ACIII immunopositive cilia in other areas of the brain not known to contribute to the BBS phenotype. Considered with data documenting a role of cilia in signal transduction these findings support the conclusion that alterations in cilia structure or neurochemical phenotypes may contribute to the cognitive deficits observed in the Bbs4-/- mouse mode. © 2014 Agassandian et al

Early over expression of messenger RNA for multiple genes, including insulin, in the Pancreatic Lymph Nodes of NOD mice is associated with Islet Autoimmunity

<p>Abstract</p> <p>Background</p> <p>Autoimmune diabetes (T1D) onset is preceded by a long inflammatory process directed against the insulin-secreting β cells of the pancreas. Deciphering the early autoimmune mechanisms represents a challenge due to the absence of clinical signs at early disease stages. The aim of this study was to identify genes implicated in the early steps of the autoimmune process, prior to inflammation, in T1D. We have previously established that insulin autoantibodies (E-IAA) predict early diabetes onset delineating an early phenotypic check point (window 1) in disease pathogenesis. We used this sub-phenotype and applied differential gene expression analysis in the pancreatic lymph nodes (PLN) of 5 weeks old Non Obese Diabetic (NOD) mice differing solely upon the presence or absence of E-IAA. Analysis of gene expression profiles has the potential to provide a global understanding of the disease and to generate novel hypothesis concerning the initiation of the autoimmune process.</p> <p>Methods</p> <p>Animals have been screened weekly for the presence of E-IAA between 3 and 5 weeks of age. E-IAA positive or negative NOD mice at least twice were selected and RNAs isolated from the PLN were used for microarray analysis. Comparison of transcriptional profiles between positive and negative animals and functional annotations of the resulting differentially expressed genes, using software together with manual literature data mining, have been performed.</p> <p>Results</p> <p>The expression of 165 genes was modulated between E-IAA positive and negative PLN. In particular, genes coding for insulin and for proteins known to be implicated in tissue remodelling and Th1 immunity have been found to be highly differentially expressed. Forty one genes showed over 5 fold differences between the two sets of samples and 30 code for extracellular proteins. This class of proteins represents potential diagnostic markers and drug targets for T1D.</p> <p>Conclusion</p> <p>Our data strongly suggest that the immune related mechanisms taking place at this early age in the PLN, correlate with homeostatic changes influencing tissue integrity of the adjacent pancreatic tissue. Functional analysis of the identified genes suggested that similar mechanisms might be operating during pre-inflammatory processes deployed in tissues i) hosting parasitic microorganisms and ii) experiencing unrestricted invasion by tumour cells.</p

ATP-binding cassette (ABC) transporters in normal and pathological lung

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters

Glutamatergic phenotype of glucagon-like peptide 1 neurons in the caudal nucleus of the solitary tract in rats

The expression of a vesicular glutamate transporter (VGLUT) suffices to assign a glutamatergic phenotype to neurons and other secretory cells. For example, intestinal L cells express VGLUT2 and secrete glutamate along with glucagon-like peptide 1 (GLP1). We hypothesized that GLP1-positive neurons within the caudal (visceral) nucleus of the solitary tract (cNST) also are glutamatergic. To test this, the axonal projections of GLP1 and other neurons within the cNST were labeled in rats via iontophoretic delivery of anterograde tracer. Dual immunofluorescence and confocal microscopy was used to visualize tracer-, GLP1-, and VGLUT2-positive fibers within brainstem, hypothalamic, and limbic forebrain nuclei that receive input from the cNST. Electron microscopy was used to confirm GLP1 and VGLUT2 immunolabeling within the same axon varicosities, and fluorescent in situ hybridization was used to examine VGLUT2 mRNA expression by GLP1-positive neurons. Most anterograde tracer-labeled fibers displayed VGLUT2-positive varicosities, providing new evidence that ascending axonal projections from the cNST are primarily glutamatergic. Virtually all GLP1-positive varicosities also were VGLUT2-positive. Electron microscopy confirmed the colocalization of GLP1 and VGLUT2 immunolabeling in axon terminals that formed asymmetric (excitatory-type) synapses with unlabeled dendrites in the hypothalamus. Finally, in situ hybridization confirmed that GLP1-positive cNST neurons express VGLUT2 mRNA. Thus, hindbrain GLP1 neurons in rats are equipped to store glutamate in synaptic vesicles, and likely co-release both glutamate and GLP1 from axon varicosities and terminals in the hypothalamus and other brain regions

Altered ADAR 2 equilibrium and 5HT2CR editing in the prefrontal cortex of ADAR 2 transgenic mice

Modulation of serotonin signaling by RNA editing of the serotonin 2C receptor (5HT2CR) may be relevant to affective disorder as serotonin functions regulate mood and behavior. Previously, we observed enhanced endogenous behavioral despair in ADAR2 transgenic mice. As the transcript of the 5HT2CR is a substrate of ADAR2, we hypothesized that perturbed ADAR2 equilibrium in the prefrontal cortex of ADAR2 transgenic mice alters the normal distribution of edited amino acid isoforms of the 5HT2CR and modifies the receptor function in downstream basal extracellular signal-regulated kinase (ERK) signaling. We examined groups of naive control and ADAR2 transgenic mice and found significantly increased ADAR2 expression, increased RNA editing at A, C, D and E sites and significantly altered normal distribution of edited amino acid isoforms of the 5HT2CR with increased proportions of valine asparagine valine, valine serine valine, valine asparagine isoleucine, isoleucine asparagine valine and decreased isoleucine asparagine isoleucine amino acid isoforms of the 5HT2CR in ADAR2 transgenic mice. Localized serotonin levels (5-HT) were unchanged and perturbed ADAR2 equilibrium coincides with dysregulated edited amino acid isoforms of the 5HT2CR and reduced basal ERK signaling. These results altogether suggest that altered 5HT2CR function could be contributing to enhanced depression-like behavior of ADAR2 transgenic mice and further implicate ADAR2 as a contributing factor in cases of affective disorder

Fbxl12 triggers G1 arrest by mediating degradation of calmodulin kinase I

AbstractCell cycle progression through its regulatory control by changes in intracellular Ca2+ levels at the G1/S transition mediates cellular proliferation and viability. Ca2+/CaM-dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex essential for G1 progression, but how this occurs is unknown. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27. Here, we show that a ubiquitin E3 ligase component, F-box protein Fbxl12, mediates CaMKI degradation via a proteasome-directed pathway leading to disruption of cyclin D1/cdk4 complex assembly and resultant G1 arrest in lung epithelia. We also demonstrate that i) CaMKI phosphorylates p27 at Thr157 and Thr198 in human cells and at Thr170 and Thr197 in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression. Lastly, known inducers of G1 arrest significantly increase Fbxl12 levels in cells. Thus, Fbxl12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism-based therapy

Ablation of the Leptin Receptor in the Hypothalamic Arcuate Nucleus Abrogates Leptin-Induced Sympathetic Activation

Job file for the creation/design of stained glass from either the Charles J. Connick Studio (1912-1945) or the Charles J. Connick Associates studio (1945-1986). The job file contains a job number, location information, date of completion, size, contact information, price, and a description of the project. This particular job file contains information on a job located at: Riverside, California. First Methodist Church

VCAM-1 is a TGF-β1 inducible gene upregulated in idiopathic pulmonary fibrosis

AbstractIdiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease of unknown etiology. We previously reported that high plasma levels of vascular cell adhesion molecule 1 (VCAM-1) predict mortality in IPF subjects. Here we investigated the cellular origin and potential role of VCAM-1 in regulating primary lung fibroblast behavior. VCAM-1 mRNA was significantly increased in lungs of subjects with IPF compared to lungs from control subjects (p=0.001), and it negatively correlated with two markers of lung function, forced vital capacity (FVC) and pulmonary diffusion capacity for carbon monoxide (DLCO). VCAM-1 protein levels were highly expressed in IPF subjects where it was detected in fibrotic foci and blood vessels of IPF lung. Treatment of human lung fibroblasts with TGF-β1 significantly increased steady-state VCAM1 mRNA and protein levels without affecting VCAM1 mRNA stability. Further, cellular depletion of VCAM-1 inhibited fibroblast cell proliferation and reduced G2/M and S phases of the cell cycle suggestive of cell cycle arrest. These effects on cell cycle progression triggered by VCAM1 depletion were associated with reductions in levels of phosphorylated extracellular regulated kinase 1/2 and cyclin D1. Thus, these observations suggest that VCAM-1 is a TGF-β1 responsive mediator that partakes in fibroblast proliferation in subjects with IPF