James Hewinson

Essential role of Phox2b-expressing ventrolateral brainstem neurons in the chemosensory control of inspiration and expiration.

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO2/pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Using in vivo (conscious and anesthetized rats) and in situ (arterially perfused rat brainstem–spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO2-evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupled Drosophila allatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and the in situ rat preparations. Postinspiratory activity was also reduced in situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO2-evoked abdominal expiratory activity in anesthetized rats and in situ preparations. Inspiratory responses evoked by rising levels of CO2 in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO2-evoked increases of inspiratory activity.

A Key Role for Redox Signaling in Rapid P2X7 Receptor-Induced IL-1β Processing in Human Monocytes

P2X7 receptors (P2X7Rs) are ATP-gated ion channels that trigger caspase-1 activation in the presence of TLR ligands. Inflammatory caspase-1 is responsible for the proteolytic activation of IL-1β. However, the signaling events that couple P2X7Rs to caspase-1 activation remain undefined. In this study we demonstrate that ATP-induced cellular oxidation is critical for caspase-1 activation and subsequent IL-1β processing. Purinergic receptor stimulation, including P2X7Rs, of endotoxin-primed human monocytes augments NADPH oxidase activity whereas concurrent purinergic receptor stimulation triggers protein denitroyslation, leading to the formation of peroxynitrite. IL-1β cleavage is blocked under conditions where superoxide anion formation is blocked or monocytes are treated with antioxidants or a peroxynitrite scavenger. Nigericin, a K+/H+ antiporter, also increases NADPH oxidase activity, leading to IL-1β and caspase-1 processing that is blocked by a peroxynitrite scavenger or inhibition of NADPH oxidase. These data demonstrate that signaling via NADPH oxidase activity is fundamental for the processing of mature IL-1β induced by P2X7R stimulation.

Optogenetic experimentation on astrocytes

We briefly review the current literature where optogenetics has been used to study various aspects of astrocyte physiology in vitro and in vivo. This includes both genetically engineered Ca2+ sensors and effector proteins, such as channelrhodopsin. We demonstrate how the ability to target astrocytes with cell‐specific viral vectors to express optogenetic constructs helped to unravel some previously unsuspected roles of these inconspicuous cells.

Optoactivation of Locus Ceruleus Neurons Evokes Bidirectional Changes in Thermal Nociception in Rats

Pontospinal noradrenergic neurons are thought to form part of a descending endogenous analgesic system that exerts inhibitory influences on spinal nociception. Using optogenetic targeting, we tested the hypothesis that excitation of the locus ceruleus (LC) is antinociceptive. We transduced rat LC neurons by direct injection of a lentiviral vector expressing channelrhodopsin2 under the control of the PRS promoter. Subsequent optoactivation of the LC evoked repeatable, robust, antinociceptive (+4.7°C ± 1.0, p < 0.0001) or pronociceptive (−4.4°C ± 0.7, p < 0.0001) changes in hindpaw thermal withdrawal thresholds. Post hoc anatomical characterization of the distribution of transduced somata referenced against the position of the optical fiber and subsequent further functional analysis showed that antinociceptive actions were evoked from a distinct, ventral subpopulation of LC neurons. Therefore, the LC is capable of exerting potent, discrete, bidirectional influences on thermal nociception that are produced by specific subpopulations of noradrenergic neurons. This reflects an underlying functional heterogeneity of the influence of the LC on the processing of nociceptive information.

Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers

Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies.

A novel role for P2X7 receptor signalling in the survival of mouse embryonic stem cells.

The growth of a pluripotent embryonic stem (ES) cell population is dependent on cell survival, proliferation and self-renewal. The nucleotide ATP represents an important extracellular signalling molecule that regulates the survival of differentiated cells, however, its role is largely undefined in embryonic stem cells. Here we report a role for ATP-gated P2X7 receptors in ES cell survival. The functional expression of P2X7 receptors in undifferentiated mouse ES cells is demonstrated using a selective P2X7 antagonist and small interfering RNA knockdown of these receptors. Our data illustrate a key role for the P2X7 receptor as an essential pro-survival signal required for optimal ES cell colony growth in the presence of leukemia inhibitor factor (LIF). However, chronic exposure to exogenous ATP leads to rapid P2X7-dependent cell death via necrosis. Together, these data demonstrate a novel role for P2X7 receptors in regulation of ES cell behaviour where they can mediate either a pro-survival or pro-death signal depending on the mode of activation.

Upregulation of junctional adhesion molecule-A is a putative prognostic marker of hypertension

Aims Establishing biochemical markers of pre-hypertension and early hypertension could help earlier diagnostics and therapeutic intervention. We assess dynamics of junctional adhesion molecule-A (JAM-A) expression in rat models of hypertension and test whether JAM-A expression could be driven by angiotensin (ANG) II and whether JAM-A contributes to the progression of hypertension. We also compare JAM-A expression in normo- and hypertensive humans. Methods and results In pre-hypertensive and spontaneously hypertensive rats (SHRs), JAM-A protein was overexpressed in the brainstem microvasculature, lung, liver, kidney, spleen, and heart. JAM-A upregulation at early and late stages was even greater in the stroke-prone SHR. However, JAM-A was not upregulated in leucocytes and platelets of SHRs. In Goldblatt 2K-1C hypertensive rats, JAM-A expression was augmented before any increase in blood pressure, and similarly JAM-A upregulation preceded hypertension caused by peripheral and central ANG II infusions. In SHRs, ANG II type 1 (AT1) receptor antagonism reduced JAM-A expression, but the vasodilator hydralazine did not. Body-wide downregulation of JAM-A with Vivo-morpholinos in juvenile SHRs delayed the progression of hypertension. In the human saphenous vein, JAM-A mRNA was elevated in hypertensive patients with untreated hypertension compared with normotensive patients but reduced in patients treated with renin–angiotensin system antagonists. Conclusion Body-wide upregulation of JAM-A in genetic and induced models of hypertension in the rat precedes the stable elevation of arterial pressure. JAM-A upregulation may be triggered by AT1 receptor-mediated signalling. An association of JAM-A with hypertension and sensitivity to blockers of ANG II signalling were also evident in humans. We suggest a prognostic and possibly a pathogenic role of JAM-A in arterial hypertension.

Viral gene delivery: optimized protocol for production of high titer lentiviral vectors.

HIV-derived lentiviral vectors (LVV) are among the most commonly used gene delivery vehicles. Their production in high quantities, which enables concentration of viral particles to high titers, is important for their successful application in both biomedical research and gene therapy. LVV are produced by co-transfection of three or more plasmids into a packaging cell line followed by several purification and concentration steps. Protocols currently in circulation differ from each other but the direct comparison of their efficacy based on the published information is extremely difficult because more than one variable may be changed and essential information may be omitted. We systematically evaluated three protocols and found that one single modification described here, using FuGene(®) 6 in the co-transfection step, increase LVV output almost 20 times as compared to the most commonly used calcium phosphate (CaPO4) transfection technique. Unexpectedly FuGene(®) 6 was also much more efficient than another widely used reagent, Superfect. Dependent on requirements, this permits a dramatic downscaling of the packaging stage of viral production, and/or super-concentration of LVV to achieve stronger expression. For example we were able to prepare ∼25 μL of high titer LVV suitable for injections into rodent brain using a single T75 cm(2) cell culture flask of packaging cells. The same output would require up to 20 times more packaging cells and reagents following conventional protocols. We illustrate the potential of our approach using transfection of primary neuronal cultures with LVV expressing an optogenetic actuator channelrhodopsin-2. Our observations should help to achieve reproducible production of high titer LVV for experimental and potential therapeutic applications.

A population-based analysis of germline BAP1 mutations in melanoma

Abstract Germline mutation of the BRCA1 associated protein-1 (BAP1) gene has been linked to uveal melanoma, mesothelioma, meningioma, renal cell carcinoma and basal cell carcinoma. Germline variants have also been found in familial cutaneous melanoma pedigrees, but their contribution to sporadic melanoma has not been fully assessed. We sequenced BAP1 in 1,977 melanoma cases and 754 controls and used deubiquitinase assays, a pedigree analysis, and a histopathological review to assess the consequences of the mutations found. Sequencing revealed 30 BAP1 variants in total, of which 27 were rare (ExAc allele frequency <0.002). Of the 27 rare variants, 22 were present in cases (18 missense, one splice acceptor, one frameshift and two near splice regions) and five in controls (all missense). A missense change (S98R) in a case that completely abolished BAP1 deubiquitinase activity was identified. Analysis of cancers in the pedigree of the proband carrying the S98R variant and in two other pedigrees carrying clear loss-of-function alleles showed the presence of BAP1-associated cancers such as renal cell carcinoma, mesothelioma and meningioma, but not uveal melanoma. Two of these three probands carrying BAP1 loss-of-function variants also had melanomas with histopathological features suggestive of a germline BAP1 mutation. The remaining cases with germline mutations, which were predominantly missense mutations, were associated with less typical pedigrees and tumours lacking a characteristic BAP1-associated histopathological appearances, but may still represent less penetrant variants. Germline BAP1 alleles defined as loss-of-function or predicted to be deleterious/damaging are rare in cutaneous melanoma.

Vascular physiology and pathology of circulating xanthine oxidoreductase: from nucleotide sequence to functional enzyme

Abstract The evolutionarily conserved, cofactor-dependent, enzyme xanthine oxidoreductase exists in both cell-associated and circulatory forms. The exact role of the circulating form is not known; however, several putative physiological and pathological functions have been suggested that range from purine catabolism to a mediator of acute respiratory distress syndrome. Regulation of gene expression, cofactor synthesis and insertion, post-translational conversion, entry into the circulation, and putative physiological and pathological roles for human circulating xanthine oxidoreductase are discussed.