Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones.

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A2 (PLA2) activities. The PLA2 activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal-glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases.

Monoamine oxidases in development.

Monoamine oxidases (MAOs) are flavoproteins of the outer mitochondrial membrane that catalyze the oxidative deamination of biogenic and xenobiotic amines. In mammals there are two isoforms (MAO-A and MAO-B) that can be distinguished on the basis of their substrate specificity and their sensitivity towards specific inhibitors. Both isoforms are expressed in most tissues, but their expression in the central nervous system and their ability to metabolize monoaminergic neurotransmitters have focused MAO research on the functionality of the mature brain. MAO activities have been related to neurodegenerative diseases as well as to neurological and psychiatric disorders. More recently evidence has been accumulating indicating that MAO isoforms are expressed not only in adult mammals, but also before birth, and that defective MAO expression induces developmental abnormalities in particular of the brain. This review is aimed at summarizing and critically evaluating the new findings on the developmental functions of MAO isoforms during embryogenesis.

Monoamine oxidase a expression is vital for embryonic brain development by modulating developmental apoptosis.

Monoamine oxidases (MAO-A, MAO-B) metabolize biogenic amines and have been implicated in neuronal apoptosis. Although apoptosis is an important process in embryo development, the role of MAO isoenzymes has not been investigated in detail. We found that expression of MAO-A and MAO-B can be detected early on during embryo development. Expression levels remained constant until around midgestation but then dropped to almost undetectable levels toward birth. Similar expression kinetics were observed in the brain. Isoform-specific expression silencing of MAO-A mediated by siRNA during in vitro embryogenesis induced developmental defects, as indicated by a reduction of the crown rump length and impaired cerebral development. These alterations were paralleled by elevated serotonin levels. Similar abnormalities were observed when embryos were cultured in the presence of the MAO-A inhibitor clorgyline or when the transcriptional inhibitor of MAO-A expression R1 was overexpressed. In contrast, no such alterations were detected when expression of MAO-B was knocked down. To explore the underlying mechanisms for the developmental abnormalities in MAO-A knockdown embryos, we quantified the degree of developmental apoptosis in the developing brain. MAO-A knockdown reduced the number of apoptotic cells in the neuroepithelium, which coincided with impaired activation of caspases 3 and 9. Moreover, we observed reduced cyclin D1 levels as an indicator of impaired cell proliferation in MAO-A knockdown embryos. This data highlights MAO-A as a vital regulator of embryonic brain development.

Heparan sulfate proteoglycans are receptors for the cell-surface trafficking and biological activity of transglutaminase-2.

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.

Alzheimer's disease modifies progenitor cell expression of monoamine oxidase B in the subventricular zone.

In the adult brain, progenitor cells remaining in the subventricular zone (SVZ) are frequently identified as glial fibrillary acidic protein (GFAP)-positive cells that retain attributes reminiscent of radial glia. Because the very high expression of monoamine oxidase B (MAO-B) in the subventricular area has been related to epithelial and astroglial expression, we sought to ascertain whether it was also expressed by progenitor cells of human control and Alzheimer's disease (AD) patients. In the SVZ, epithelial cells and astrocyte-like cells presented rich MAO-B activity and immunolabeling. Nestin-positive cells were found in the same area, showing a radial glia-like morphology. When coimmunostaining and confocal microscopy were performed, most nestin-positive cells showed MAO-B activity and labeling. The increased progenitor activity in SVZ proposed for AD patients was confirmed by the positive correlation between the SVZ nestin/MAO-B ratio and the progression of the disease. Nestin/GFAP-positive cells, devoid of MAO-B, can represent a distinct subpopulation of an earlier phase of maturation. This would indicate that MAO-B expression takes place in a further step of nestin/GFAP-positive cell differentiation. In the early AD stages, the discrete MAO-B reduction, different from the severe GFAP decrease, would reflect the capacity of this population of MAO-B-positive progenitor cells to adapt to the neurodegenerative process.

An assessment of the aversive nature of an animal management procedure (clipping) using behavioral and physiological measures.

Animal management often involves procedures that, while unlikely to cause physical pain, still cause aversive responses. The domestic horse (Equus caballus) regularly has excessive hair clipped off to facilitate its use as a riding/driving animal and this procedure causes adverse behavioral responses in some animals. The aim of this study was to compare behavioral and physiological measures to assess the aversive effect of this procedure. Ten horses were selected on the basis of being either compliant (C: n=5) or non-compliant (NC: n=5) during this procedure. The horses were subjected to a sham clipping procedure (SC: where the blades had been removed from the clippers) for a period of ten minutes. Measures were taken pre, during and post SC (-10min to +30min) and mean values calculated for ALL horses and for C and NC separately. Behavioral activity was scored (scale 1-5) by twenty students from video footage in (phase/group-blind scoring). Heart rate (HR), salivary cortisol and eye temperature were monitored throughout the procedure. The NC horses were found to be significantly more behaviorally active/less relaxed throughout the trial than C horses (p<0.05) with the greatest difference occurring during the SC procedure (p<0.01). NC horses were more active/less relaxed during, compared with pre or post SC (p<0.05), but showed no behavioral difference pre and post SC. HR of the NC horses was higher than that of the C horses throughout the trial but only significantly so after 10min of SC (p<0.01). ALL horses showed a significant increase in HR between +5 and +10min into the procedure (p<0.05). There was a significant increase in salivary cortisol concentration in ALL horses post procedure (p<0.01) with levels peaking at 20minute post SC. No significant differences in salivary cortisol concentration between C and NC were found at any stage of the trial. Eye temperature increased significantly in ALL horses during SC, peaking at +10min into the procedure (p<0.05) and then decreased substantially when SC had ceased (p<0.01). Although no significant differences were found between C and NC per se, there was a significant interaction between group and phase of trial (p<0.05) with the NC group showing a greater decrease in eye temperature post SC. There was a significant positive correlation between changes in salivary cortisol concentration and eye temperature (p<0.01) but no correlation between any of the other measures. Although the behavioral response of C and NC to this procedure was significantly different the physiological responses indicated that ALL horses found the procedure aversive. Eye temperature could be used as an objective and immediate measure of how an animal is responding to a specific situation in order to evaluate management procedures and adapt them where appropriate to reduce the negative impact on animal health and welfare.

An investigation of the effects of MitoQ on human peripheral mononuclear cells.

MitoQ is a ubiquinone derivative targeted to mitochondria which is known to have both antioxidant and anti-apoptotic properties within mammalian cells. Previous research has suggested that the age-related increase in oxidative DNA damage in T lymphocytes might contribute to their functional decline with age. This paper describes the impact of mitoQ on unchallenged or oxidatively challenged ex vivo human peripheral blood mononuclear cells from healthy 25-30 or 55-60 year old volunteers. When cells were challenged with hydrogen peroxide (H(2)O(2)), following mitoQ treatment (0.1-1.0 µM), the ratio of reduced to oxidized forms of glutathione increased, the levels of oxidative DNA damage decreased and there was an increase in the mitochondrial membrane potential. Low levels of mitoQ (0.1 or 0.25 µM) had no impact on endogenous DNA damage, whilst higher levels (0.5 and 1.0 µM) of mitoQ significantly reduced endogenous levels of DNA damage. The results of this investigation suggest that mitoQ may have anti-immunosenescent potential.

Reduced placental vascular reactivity to 5-hydroxytryptamine in pre-eclampsia and the status of 5HT(2A) receptors.

This study investigates the contractile response to 5 hydroxytryptamine (5HT) of chorionic artery and vein segments from normotensive (NT) and pre-eclamptic (PE) placentae. It also looked at the effectiveness of ketanserin (KET), a 5HT(2A) receptor antagonist, in reducing 5HT-mediated vasoconstriction. 5HT induced vasoconstriction in all of the vessels was studied. Compared to NT vessels, Emax (%KCl) was significantly reduced in PE arteries (p<0.05) and veins (p<0.0005). The mean Emax for NT arteries was 104.1 (±10.71) whilst PE arteries showed a mean Emax of 57.02 (±12.13). KET produced a statistically significant reduction of Emax in both vessels in NT and the arteries in PE. However the antagonistic effect of KET was not pronounced in PE veins. The EC50 values for NT and PE arteries and veins did not change significantly. There were no noticeable changes in the expression profiles of 5HT(2A) receptor mRNA and protein expressions. The data from this study suggest that in PE, the vascular reactivity of chorionic vessels to 5HT is reduced and it was not due to the altered expression of 5HT(2A) receptors.

Expression regulation of MAO isoforms in monocytic cells in response to Th2 cytokines.

BACKGROUND: Th2-cytokines, such as interleukins-4 and -13 (IL-4, IL-13), have been identified as alternative stimuli of monocytes/macrophages. We have recently profiled the gene-expression pattern of IL-4-treated human peripheral monocytes and found that 15-lipoxygenase-1 (15-LOX1) and monoamine oxidase A (MAO-A) are among the five most strongly upregulated gene products in IL-4-treated cells. Transfection of monocytic cells (U937) with 15-LOX1 also induced MAO-A expression. These data suggested that 15-LOX1 products might play a role in the IL4-induced signaling cascade leading to expression of MAO-A in human monocytes. MATERIAL/METHODS: To test this hypothesis we incubated wild-type and 15-LOX1-transfected U937 cells with different concentrations of either IL-4 or 15-LOX-products [13S-H(p)ODE, 15S-H(p)ETE] and quantified the expression of 15-LOX1, MAO-A, and MAO-B by activity assays and real-time RT-PCR. RESULTS: Wild-type U937 cells express neither MAO-A nor MAO-B, but after three days of IL4 treatment, MAO-A mRNA was detected. A similar isoform-specific expression of MAO-A mRNA was observed when U937 cells were transfected with 15-LOX1 or when the cells were incubated with primary 15-LOX1 products (hydroperoxy fatty acids) or H2O2. In contrast, the corresponding hydroxy fatty acids were ineffective. CONCLUSIONS: These data indicate that increased intracellular peroxide concentrations (oxidative stress) induce MAO-A expression in monocytes/macrophages, which normally do not express the enzyme. Our findings also suggest that IL-4-induced upregulation of MAO-A expression in human peripheral monocytes may proceed via 15-LOX1-dependent and 15-LOX1-independent pathways. The biological role of MAO-A expression for monocyte function is discussed.

Th2 response of human peripheral monocytes involves isoform-specific induction of monoamine oxidase-A.

Monocyte/macrophage function is critically regulated by specific cytokines and growth factors that they are exposed to at inflammatory sites. IL-4 and IL-13 are multifunctional cytokines generated mainly by Th2 lymphocytes that have important biological activities in allergy and inflammation. The Th2 response of human peripheral monocytes is characterized by complex alterations in the gene expression pattern, which involves dominant expression of CD23 cell surface Ag and lipid-peroxidizing 15-lipoxygenase-1 (15-LOX1). In this study, we report that the classical Th2 cytokines IL-4 and IL-13 strongly up-regulate expression of monoamine oxidase A (MAO-A) with no induction of the closely related isozyme, MAO-B. Real-time PCR indicated a >2000-fold up-regulation of the MAO-A transcripts, and immunohistochemistry revealed coexpression of the enzyme with 15-LOX1 in a major subpopulation of monocytes. MAO-A was also induced in lung carcinoma A549 cells by IL-4 in parallel with 15-LOX1. In promyelomonocytic U937 cells, which neither express 15-LOX1 nor MAO-A in response to IL-4 stimulation, expression of MAO-A was up-regulated following transfection with 15-LOX1. This is the first report indicating expression of MAO-A in human monocytes. Its isoform-specific up-regulation in response to Th2 cytokines suggests involvement of the enzyme in modulation of innate and/or acquired immune system.