Immune activation in Retinal Aging: A Gene Expression Study.

PURPOSE
To investigate changes in gene expression during aging of the retina in the mouse.

METHODS
Total RNA was extracted from the neuroretina of young (3-month-old) and old (20-month-old) mice and processed for microarray analysis. Age-related, differentially expressed genes were assessed by the empiric Bayes shrinkagemoderated t-statistics method. Statistical significance was based on dual criteria of a ratio of change in gene expression >2 and a P < 0.01. Differential expression in 11 selected genes was further verified by real-time PCR. Functional pathways involved in retinal ageing were analyzed by an online software package (DAVID-2008) in differentially expressed gene lists. Age-related changes in differential expression in the identified retinal molecular pathways were further confirmed by immunohistochemical staining of retinal flat mounts and retinal cryosections.

RESULTS
With ageing of the retina, 298 genes were upregulated and 137 genes were downregulated. Functional annotation showed that genes linked to immune responses (Ir genes) and to tissue stress/injury responses (TS/I genes) were most likely to be modified by ageing. The Ir genes affected included those regulating leukocyte activation, chemotaxis, endocytosis, complement activation, phagocytosis, and myeloid cell differentiation, most of which were upregulated, with only a few downregulated. Increased microglial and complement activation in the aging retina was further confirmed by confocal microscopy of retinal tissues. The most strongly upregulated gene was the calcitonin receptor (Calcr; >40-fold in old versus young mice).

CONCLUSIONS
The results suggest that retinal ageing is accompanied by activation of gene sets, which are involved in local inflammatory responses. A modified form of low-grade chronic inflammation (para-inflammation) characterizes these aging changes and involves mainly the innate immune system. The marked upregulation of Calcr in ageing mice most likely reflects this chronic inflammatory/stress response, since calcitonin is a known systemic biomarker of inflammation/sepsis. © Association for Research in Vision and Ophthalmology.

Persistent measles virus infection of mouse neural cells lacking known human entry receptors

Aims: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry.

Methods: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real-time reverse transcriptase polymerase chain reaction.

Results: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections.

Conclusions: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.

Proteomic profiling of the retinal dysplasia and degeneration chick retina

Purpose: In our previous paper we undertook proteomic analysis of the normal developing chick retina to identify proteins that were differentially expressed during retinal development. In the present paper we use the same proteomic approach to analyze the development and onset of degeneration in the retinal dysplasia and degeneration (rdd) chick. The pathology displayed by the rdd chick resembles that observed in some of the more severe forms of human retinitis pigmentosa.

Isolation and culture of mouse intestinal cells.

Complex cell signal transduction mechanisms regulate intestinal epithelial shape, polarity, motility, organelles, cell membrane components as well as physical and mechanical properties to influence alimentary digestion, absorption, secretion, detoxification and fluid balance. Interactions between the epithelial cells and adjacent mesenchyme are central to intestinal homeostasis although the key regulatory molecules of specific differentiation steps remain unclear. Isolation and primary culture of heterotypic murine intestinal cells provides a model system for elucidation of essential molecular cross-talk between epithelium and mesenchyme that may provide several biological and practical advantages over transformed cell lines. An in vitro primary culture system for neonatal rat or mouse intestinal cells has been established that forms monolayers, expresses intestine-specific epithelial features including intestinal brush borders and appropriate hydrolase enzymes. Our studies confirm the promise of this method which may advance our understanding of heterotypic cellular interactions implicated in intestinal function and may provide important insights into the pathobiology of disease.

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses but the role of different ROS sources remains unclear. Here, we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level which increased in cardiomyocytes during stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte Hif1 and the release of VEGF, resulting in an increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a novel inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of non-specific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

Cannabinoid Receptor influences Chromatin Remodelling in Mouse Spermatids by affecting Content of Transition Protein 2 mRNA and Histone Replacement

Marijuana smokers and animals treated with ?9-tetrahydrocannabinol, THC, the principal component of marijuana, show alterations of sperm morphology suggesting a role for cannabinoids in sperm differentiation and/or maturation. Since the cannabinoid receptor 1 (CNR1) activation appears to play a pivotal role in spermiogenesis, the developmental stage where DNA is remodeled, we hypothesized that CNR1 receptors might also influence chromatin quality in sperm. We used Cnr1 null mutant (Cnr1-/-) mice to study the possible role of endocannabinoids on sperm chromatin during spermiogenesis. We demonstrated that CNR1 activation regulated chromatin remodeling of spermatids by either increasing Tnp2 levels or enhancing histone displacement. Comparative analysis of WT, Cnr1+/- and Cnr1-/- animals suggested the possible occurrence of haploinsufficiency for Tnp2 turnover control by CNR1, while histone displacement was disrupted to a lesser extent. Further, flow cytometry analysis demonstrated that the genetic loss of Cnr1 decreased sperm chromatin quality and was associated with sperm DNA fragmentation. This damage increased during epididymal transit, from caput to cauda. Collectively, our results show that the expression/activity of CNR1 controls the physiological alterations of DNA structure during spermiogenic maturation and epididymal transit. Given the deleterious effects of sperm DNA damage on male fertility, we suggest that the reproductive function of marijuana users may also be impaired by deregulation of the endogenous endocannabinoid system.