Growth State-Dependent Expression of Arachidonate Lipoxygenases in the Human Endothelial Cell Line EA.hy926.

Endothelial cells regulate vascular homeostasis through the secretion of various paracrine molecules, including bioactive lipids, but little is known regarding the enzymes responsible for generating these lipids under either physiological or pathophysiological conditions. Arachidonate lipoxygenase (ALOX) expression was therefore investigated in confluent and nonconfluent EA.h926 endothelial cells, which represent the normal quiescent and proliferative states, respectively. mRNAs for ALOX15, ALOX15B, and ALOXE3 were detected in EA.hy926 cells, with the highest levels present in confluent cells compared to nonconfluent cells. In contrast, ALOX5, ALOX12, and ALOX12B mRNAs were not detected. At the protein level, only ALOX15B and ALOXE3 were detected but only in confluent cells. ALOXE3 was also observed in confluent human umbilical artery endothelial cells (HUAEC), indicating that its expression, although previously unreported, may be a general feature of endothelial cells. Exposure to laminar flow further increased ALOXE3 levels in EA.hy926 cells and HUAECs. The evidence obtained in this study indicates that proliferative status and shear stress are both important factors that mediate endothelial ALOX gene expression. The presence of ALOX15B and ALOXE3 exclusively in quiescent human endothelial cells suggests their activity likely contributes to the maintenance of a healthy endothelium.

Muscarinic Acetylcholine Type 1 Receptor Activity Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons.

The muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein-coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking. We overexpressed M1R in primary cultures of adult rat sensory neurons and cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. In adult primary neurons, overexpression of M1R caused disruption of the tubulin, but not actin, cytoskeleton and significantly reduced neurite outgrowth. Over-expression of a M1R-DREADD mutant comparatively increased neurite outgrowth suggesting that acetylcholine released from cultured neurons interacts with M1R to suppress neurite outgrowth. M1R-dependent constraint on neurite outgrowth was removed by selective (pirenzepine) or specific (muscarinic toxin 7) M1R antagonists. M1R-dependent disruption of the cytoskeleton also diminished mitochondrial abundance and trafficking in distal neurites, a disorder that was also rescued by pirenzepine or muscarinic toxin 7. M1R activation modulated cytoskeletal dynamics through activation of the G protein (Gα13) that inhibited tubulin polymerization and thus reduced neurite outgrowth. Our study provides a novel mechanism of M1R control of Gα13 protein-dependent modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. This novel pathway could be harnessed to treat dying-back neuropathies since anti-muscarinic drugs are currently utilized for other clinical conditions.

Ammonia as a Potential Neurotoxic Factor in Alzheimer's Disease.

Ammonia is known to be a potent neurotoxin that causes severe negative effects on the central nervous system. Excessive ammonia levels have been detected in the brain of patients with neurological disorders such as Alzheimer disease (AD). Therefore, ammonia could be a factor contributing to the progression of AD. In this review, we provide an introduction to the toxicity of ammonia and putative ammonia transport proteins. We also hypothesize how ammonia may be linked to AD. Additionally, we discuss the evidence that support the hypothesis that ammonia is a key factor contributing to AD progression. Lastly, we summarize the old and new experimental evidence that focuses on energy metabolism, mitochondrial function, inflammatory responses, excitatory glutamatergic, and GABAergic neurotransmission, and memory in support of our ammonia-related hypotheses of AD.

Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation.

The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype.

Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform.

BACKGROUND: The Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied. RESULTS: In addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay. CONCLUSIONS: The mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.