Myogenic vasoconstriction requires canonical Gq/11 signaling of the angiotensin II type 1a receptor in the murine vasculature

BACKGROUND: The myogenic response is an inherent vasoconstrictive property of resistance arteries to keep blood flow constant in response to increases in intravascular pressure. Angiotensin II (Ang II) type 1 receptors (AT1R) are broadly distributed, mechanoactivated receptors, which have been proposed to transduce myogenic vasoconstriction. However, the AT1R subtype(s) involved and their downstream G protein- and β-arrestin-mediated signaling pathways are still elusive. OBJECTIVE: To characterize the function of AT1aR and AT1bR in the regulation of the myogenic response of resistance size arteries and possible downstream signaling cascades mediated by G(q/11) and/or β-arrestins. METHODS: We used Agtr1a(-/-), Agtr1b(-/-) and tamoxifen-inducible smooth muscle-specific AT1aR knockout mice (SM-Agtr1a mice). FR900359, [Sar1, Ile4, Ile8] Ang II (SII) and TRV120055 were used as selective G(q/11) protein inhibitor and biased agonists to activate non-canonical β-arrestin and canonical G(q/11) signaling of the AT1R, respectively. RESULTS: Myogenic and Ang II-induced vasoconstrictions were diminished in the perfused renal vasculature of Agtr1a(-/-) and SM-Agtr1a mice. Similar results were observed in isolated pressurized mesenteric and cerebral arteries. Myogenic tone and Ang II-induced vasoconstrictions were normal in arteries from Agtr1b(-/-) mice. The G(q/11) blocker FR900359 decreased myogenic tone and Ang II vasoconstrictions while selective biased targeting of AT1R β-arrestin signaling pathways had no effects. CONCLUSION: The present study demonstrates that myogenic arterial constriction requires G(q/11)-dependent signaling pathways of mechanoactivated AT1aR but not G protein-independent, noncanonical alternative signaling pathways in the murine mesenteric, cerebral and renal circulation

Identification of novel SNPs of ABCD1, ABCD2, ABCD3, and ABCD4 genes in patients with X-linked adrenoleukodystrophy (ALD) based on comprehensive resequencing and association studies with ALD phenotypes

Adrenoleukodystrophy (ALD) is an X-linked disorder affecting primarily the white matter of the central nervous system occasionally accompanied by adrenal insufficiency. Despite the discovery of the causative gene, ABCD1, no clear genotype–phenotype correlations have been established. Association studies based on single nucleotide polymorphisms (SNPs) identified by comprehensive resequencing of genes related to ABCD1 may reveal genes modifying ALD phenotypes. We analyzed 40 Japanese patients with ALD. ABCD1 and ABCD2 were analyzed using a newly developed microarray-based resequencing system. ABCD3 and ABCD4 were analyzed by direct nucleotide sequence analysis. Replication studies were conducted on an independent French ALD cohort with extreme phenotypes. All the mutations of ABCD1 were identified, and there was no correlation between the genotypes and phenotypes of ALD. SNPs identified by the comprehensive resequencing of ABCD2, ABCD3, and ABCD4 were used for association studies. There were no significant associations between these SNPs and ALD phenotypes, except for the five SNPs of ABCD4, which are in complete disequilibrium in the Japanese population. These five SNPs were significantly less frequently represented in patients with adrenomyeloneuropathy (AMN) than in controls in the Japanese population (p = 0.0468), whereas there were no significant differences in patients with childhood cerebral ALD (CCALD). The replication study employing these five SNPs on an independent French ALD cohort, however, showed no significant associations with CCALD or pure AMN. This study showed that ABCD2, ABCD3, and ABCD4 are less likely the disease-modifying genes, necessitating further studies to identify genes modifying ALD phenotypes

The PPAR-gamma agonist pioglitazone protects cortical neurons from inflammatory mediators via improvement in peroxisomal function

<p>Abstract</p> <p>Background</p> <p>Inflammation is known to play a pivotal role in mediating neuronal damage and axonal injury in a variety of neurodegenerative disorders. Among the range of inflammatory mediators, nitric oxide and hydrogen peroxide are potent neurotoxic agents. Recent evidence has suggested that oligodendrocyte peroxisomes may play an important role in protecting neurons from inflammatory damage.</p> <p>Methods</p> <p>To assess the influence of peroxisomal activation on nitric oxide mediated neurotoxicity, we investigated the effects of the peroxisomal proliferator activated receptor (PPAR) gamma agonist, pioglitazone in primary cortical neurons that were either exposed to a nitric oxide donor or co-cultured with activated microglia.</p> <p>Results</p> <p>Pioglitazone protected neurons and axons against both nitric-oxide donor-induced and microglia-derived nitric oxide-induced toxicity. Moreover, cortical neurons treated with this compound showed a significant increase in the protein and gene expression of PPAR-gamma, which was associated with a concomitant increase in the enzymatic activity of catalase. In addition, the protection of neurons and axons against hydrogen peroxide-induced toxicity afforded by pioglitazone appeared to be dependent on catalase.</p> <p>Conclusions</p> <p>Collectively, these observations provide evidence that modulation of PPAR-gamma activity and peroxisomal function by pioglitazone attenuates both NO and hydrogen peroxide-mediated neuronal and axonal damage suggesting a new therapeutic approach to protect against neurodegenerative changes associated with neuroinflammation.</p

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies

Oligodendrocytes: biology and pathology

Oligodendrocytes are the myelinating cells of the central nervous system (CNS). They are the end product of a cell lineage which has to undergo a complex and precisely timed program of proliferation, migration, differentiation, and myelination to finally produce the insulating sheath of axons. Due to this complex differentiation program, and due to their unique metabolism/physiology, oligodendrocytes count among the most vulnerable cells of the CNS. In this review, we first describe the different steps eventually culminating in the formation of mature oligodendrocytes and myelin sheaths, as they were revealed by studies in rodents. We will then show differences and similarities of human oligodendrocyte development. Finally, we will lay out the different pathways leading to oligodendrocyte and myelin loss in human CNS diseases, and we will reveal the different principles leading to the restoration of myelin sheaths or to a failure to do so

The peroxisome: still a mysterious organelle

More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed

In vivo labeling of peroxisomes by photoconvertible mEos2 in myelinating glia of mice

AbstractMutations of several genes encoding peroxisomal proteins have been associated with human diseases. Some of these display specific white matter abnormalities in the brain, although the affected proteins are ubiquitously expressed. To better understand the etiology of peroxisomal myelin diseases, we aimed to label these organelles in vivo and in a cell type specific fashion. We had previously shown that in oligodendrocytes and Schwann cells numerous peroxisomes reside in the cytoplasmic channels of “non-compacted” myelin. These organelles are smaller and biochemically distinct from non-myelin peroxisomes. Targeting peroxisomal functions in various cell types of the brain has demonstrated that oligodendroglial peroxisomes are specifically important for long-term integrity of the CNS. To visualize myelin peroxisomes in intact cells and tissues by live imaging, we have generated a novel line of transgenic mice for the expression of fluorescently tagged peroxisomes specifically in myelinating glia. This was achieved by modifying the gene for a photoconvertible mEos2 with a peroxisomal targeting signal type 1 (PTS1) and generating a fusion gene with the myelin-specific Cnp1 promoter. In the brain of resulting transgenic mice, peroxisomes are selectively labeled in oligodendrocytes. In this novel genetic tool, photoconversion of single peroxisomes from green to red fluorescence can be used to monitor the fate of single organelles and to determine the dynamics of PTS1-mediated protein import in the context of myelin diseases that affect peroxisomal functions

A role for myelin-associated peroxisomes in maintaining paranodal loops and axonal integrity

Demyelinating diseases of the nervous system cause axon loss but the underlying mechanisms are not well understood. Here we show by confocal and electron microscopy that in myelin-forming glia peroxisomes are associated with myelin membranes. When peroxisome biogenesis is experimentally perturbed in Pex5 conditional mouse mutants, myelination by Schwann cells appears initially normal. However, in nerves of older mice paranodal loops become physically unstable and develop swellings filled with vesicles and electron-dense material. This novel model of a demyelinating neuropathy demonstrates that peroxisomes serve an important function in the peripheral myelin compartment, required for long-term axonal integrity. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved