Chyn-Tair Lan

Melatonin preserves longevity protein (sirtuin 1) expression in the hippocampus of total sleep-deprived rats.

Abstract:  Sleep disorders cause cognitive dysfunction in which impaired neuronal plasticity in the hippocampus may underline the molecular mechanisms of this deficiency. As sirtuin 1 (SIRT1) plays an important role in maintaining metabolic homeostasis and neuronal plasticity, this study is aimed to determine whether melatonin exerts beneficial effects on preserving SIRT1 activation following total sleep deprivation (TSD). TSD was performed by disc on water method for five consecutive days. During this period, animals daily received melatonin at doses of 5, 25, 50 or 100 mg/kg. The cytochrome oxidase (COX) histochemistry, SIRT1 immunohistochemistry together with Morris water maze learning test were performed to examine the metabolic, neurochemical, as well as the behavioral changes in neuronal plasticity, respectively. The results indicate that in normal rats, numerous COX and SIRT1 positive‐labeled neurons with strong staining intensities were found in hippocampal pyramidal and granular cell layers. Following TSD, both COX and SIRT1 reactivities were drastically decreased as revealed by reduced staining pattern and labeling frequency. Behavioral data corresponded well with morphological findings in which spatial memory test in water maze was significantly impaired after TSD. However, in rats receiving different doses of melatonin, both COX and SIRT1 expressions were successfully preserved. Considerably better performance on behavioral testing further strengthened the beneficial effects of melatonin. These findings suggest that melatonin may serve as a novel therapeutic strategy directed for preventing the memory deficits resulting from TSD, possibly by effectively preserving the metabolic function and neuronal plasticity engaged in maintaining cognitive activity.

Sleep deprivation prior to transient global cerebral ischemia attenuates glial reaction in the rat hippocampal formation.

This study was aimed to ascertain the effect of sleep deprivation on subsequent cerebral ischemia in the rat hippocampal formation. Seven days after transient global cerebral ischemia induced by four-vessel occlusion method, most of the pyramidal cells in the hippocampal CA1 subfield underwent disruption and pyknosis as detected by cresyl violet staining. With OX-42, OX-18, OX-6 and ED1 immunohistochemistry, robust microglia/macrophage reactions were observed in the CA1 and dentate hilus. The majority of reactive microglia was rod-shaped, bushy or amoeboidic cells bearing hypertrophic processes. Astrocytes also displayed hypertrophic processes, whose immunostaining for glial fibrillary acidic protein was markedly enhanced. The ischemia-induced neuronal damage and glial reactions, however, were noticeably attenuated in rats subjected to pretreatment with sleep deprivation for five consecutive days. The most drastic effect was the diminution of OX-18, OX-6 and ED1 immunoreactivities, suggesting that the immune potentiality and/or phagocytosis of these cells was suppressed by prolonged sleep deprivation prior to ischemic insult. It is postulated that sleep deprivation may have a preconditioning influence on subsequent lethal cerebral ischemia. Hence, sleep deprivation may be considered as a therapeutic strategy in brain ischemic damage.

Melatonin preserves superoxide dismutase activity in hypoglossal motoneurons of adult rats following peripheral nerve injury.

Abstract:  Peripheral nerve injury (PNI) produces functional changes in lesioned neurons in which oxidative stress is considered to be the main cause of neuronal damage. As superoxide dismutase (SOD) is an important antioxidative enzyme involved in redox regulation of oxidative stress, the present study determined whether melatonin would exert its beneficial effects by preserving the SOD reactivity following PNI. Adult rats subjected to hypoglossal nerve transection were intraperitoneally injected with melatonin at ones for 3, 7, 14, 30 and 60 days successively. The potential neuroprotective effects of melatonin were quantitatively demonstrated by neuronal nitric oxide synthase (nNOS), mitochondrial manganese SOD (Mn‐SOD), and cytosolic copper‐zinc SOD (Cu/Zn‐SOD) immunohistochemistry. The functional recovery of the lesioned neurons was evaluated by choline acetyltransferase (ChAT) immunohistochemistry along with the electromyographic (EMG) recordings of denervation‐induced fibrillation activity. The results indicate that following PNI, the nNOS immunoreactivity was significantly increased in lesioned neurons peaking at 14 days. The up‐regulation of nNOS temporally coincided with the reduction of ChAT and SOD in which the Cu/Zn‐SOD showed a greater diminution than Mn‐SOD. However, following melatonin administration, the nNOS augmentation was successfully suppressed and the activities of Mn‐SOD, Cu/Zn‐SOD, and ChAT were effectively preserved at all postaxotomy periods. EMG data also showed a decreased fibrillation in melatonin‐treated groups, suggesting a potential effect of melatonin in promoting functional recovery. In association with its significant capacity in preserving SOD reactivity, melatonin is suggested to serve as a powerful therapeutic agent for treating PNI‐relevant oxidative damage.

Sleep deprivation predisposes liver to oxidative stress and phospholipid damage: a quantitative molecular imaging study

Sleep disorders are associated with an increased rate of various metabolic disturbances, which may be related to oxidative stress and consequent lipid peroxidation. Since hepatic phosphatidylcholine plays an important role in metabolic regulation, the aim of the present study was to determine phosphatidylcholine expression in the liver following total sleep deprivation. To determine the effects of total sleep deprivation, we used adult rats implanted for polygraphic recording. Phosphatidylcholine expression was examined molecularly by the use of time‐of‐flight secondary ion mass spectrometry, along with biochemical solid‐phase extraction. The parameters of oxidative stress were investigated by evaluating the hepatic malondialdehyde levels as well as heat shock protein 25 immunoblotting and immunohistochemistry. In normal rats, the time‐of‐flight secondary ion mass spectrometry spectra revealed specific peaks (m/z 184 and 224) that could be identified as molecular ions for phosphatidylcholine. However, following total sleep deprivation, the signals for phosphatidylcholine were significantly reduced to nearly one‐third of the normal values. The results of solid‐phase extraction also revealed that the phosphatidylcholine concentration was noticeably decreased, from 15.7 µmol g–1 to 9.4 µmol g–1, after total sleep deprivation. By contrast, the biomarkers for oxidative stress were drastically up‐regulated in the total sleep deprivation‐treated rats as compared with the normal ones (4.03 vs. 1.58 nmol mg–1 for malondialdehyde levels, and 17.1 vs. 6.7 as well as 1.8 vs. 0.7 for heat shock protein 25 immunoblotting and immunoreactivity, respectively). Given that phosphatidylcholine is the most prominent component of all plasma lipoproteins, decreased expression of hepatic phosphatidylcholine following total sleep deprivation may be attributed to the enhanced oxidative stress and the subsequent lipid peroxidation, which would play an important role in the formation or progression of total sleep deprivation‐induced metabolic diseases.

Sleep Deprivation Inhibits Expression of NADPH-d and NOS while Activating Microglia and Astroglia in the Rat Hippocampus

This study investigated the expression of nitric oxide (NO)-synthesizing enzymes and the glial reaction in the rat hippocampal formation following sleep deprivation for 5 days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity was markedly reduced in the hippocampal CA1, CA2 and CA3 sectors as well as in the dentate gyrus, suggesting a suppression of NO production in these areas. Microglial cells were hypertrophic and showed an up-regulation of complement type 3 receptors as determined by antibody OX-42. However, expression of major histocompatibility complex class I and II antigens, and antigen of monocyte/macrophage lineage marked by OX-18, OX-6 and ED1, respectively, was undetected. Astrocytes also displayed hypertrophied processes with enhanced glial fibrillary acidic protein (GFAP) immunoreactivity. Western blots of hippocampal tissues corroborated the above-mentioned morphological findings in that expression of NO-synthase (NOS) was decreased while that of OX-42 and GFAP was increased in the sleep-deprived rats. Since NO is thought to be involved in memory consolidation processes in the hippocampus during sleep, the inhibition of NADPH-d and NOS reactivities may account for the memory decline after long-term sleep deprivation. The concomitant reactions in microglia and astrocytes suggest the involvement of these cells in the deleterious effect of prolonged sleep deprivation.

Neuregulin Facilitates Nerve Regeneration by Speeding Schwann Cell Migration via ErbB2/3-Dependent FAK Pathway

Background Adequate migration of Schwann cells (Sc) is crucial for axon-guidance in the regenerative process after peripheral nerve injury (PNI). Considering neuregulin-erbB-FAK signaling is an essential pathway participating in the regulation of Sc migration during development, the present study is aimed to examine whether neuregulin would exert its beneficial effects on adult following PNI and further determine the potential changes of downstream pathway engaged in neuro-regeneration by both in vitro and in vivo approaches. Methodology and Principal Findings Cultured RSC96 cells treated with neuregulin were processed for erbB2/3 immunofluorescence and FAK immunoblotings. The potential effects of neuregulin on Sc were assessed by cell adherence, spreading, and migration assays. In order to evaluate the functional significance of neuregulin on neuro-regeneration, the in vivo model of PNI was performed by chronic end-to-side neurorrhaphy (ESN). In vitro studies indicated that after neuregulin incubation, erbB2/3 were not only expressed in cell membranes, but also distributed throughout the cytoplasm and nucleus of RSC96 cells. Activation of erbB2/3 was positively correlated with FAK phosphorylation. Neuregulin also increases Sc adherence, spreading, and migration by 127.2±5.0%, 336.8±3.0%, and 80.0±5.7%, respectively. As for in vivo study, neuregulin significantly accelerates the speed of Sc migration and increases Sc expression in the distal stump of injured nerves. Retrograde labeling and compound muscle action potential recordings (CMAP) also showed that neuregulin successfully facilitates nerve regeneration by eliciting noticeably larger CMAP and promoting quick re-innervation of target muscles. Conclusions As neuregulin successfully improves axo-glial interaction by speeding Sc migration via the erbB2/3-FAK pathway, therapeutic use of neuregulin may thus serve as a promising strategy to facilitate the progress of nerve regeneration after PNI.

Sleep deprivation impairs Ca2+ expression in the hippocampus: ionic imaging analysis for cognitive deficiency with TOF-SIMS.

Sleep deprivation causes cognitive dysfunction in which impaired neuronal plasticity in hippocampus may underlie the molecular mechanisms of this deficiency. Considering calcium-mediated NMDA receptor subunit 1 (NMDAR1) and neuronal nitric oxide synthase (nNOS) activation plays an important role in the regulation of neuronal plasticity, the present study is aimed to determine whether total sleep deprivation (TSD) would impair calcium expression, together with injury of the neuronal plasticity in hippocampus. Adult rats subjected to TSD were processed for time-of-flight secondary ion mass spectrometry, NMDAR1 immunohistochemistry, nNOS biochemical assay, cytochrome oxidase histochemistry, and the Morris water maze learning test to detect ionic, neurochemical, bioenergetic as well as behavioral changes of neuronal plasticity, respectively. Results indicated that in normal rats, strong calcium signaling along with intense NMDAR1/nNOS expression were observed in hippocampal regions. Enhanced calcium imaging and neurochemical expressions corresponded well with strong bioenergetic activity and good performance of behavioral testing. However, following TSD, both calcium intensity and NMDAR1/nNOS expressions were significantly decreased. Behavioral testing also showed poor responses after TSD. As proper calcium expression is essential for maintaining hippocampal neuronal plasticity, impaired calcium expression would depress downstream NMDAR1-mediated nNOS activation, which might contribute to the initiation or development of TSD-related cognitive deficiency.

Total sleep deprivation inhibits the neuronal nitric oxide synthase and cytochrome oxidase reactivities in the nodose ganglion of adult rats

Sleep disorders are a form of stress associated with increased sympathetic activity, and they are a risk factor for the occurrence of cardiovascular disease. Given that nitric oxide (NO) may play an inhibitory role in the regulation of sympathetic tone, this study set out to determine the NO synthase (NOS) reactivity in the primary cardiovascular afferent neurons (i.e. nodose neurons) following total sleep deprivation (TSD). TSD was performed by the disc‐on‐water method. Following 5 days of TSD, all experimental animals were investigated for quantitative nicotinamine adenine dinucleotide phosphate‐diaphorase (NADPH‐d, a co‐factor of NOS) histochemistry, neuronal NOS immunohistochemistry and neuronal NOS activity assay. In order to evaluate the endogenous metabolic activity of nodose neurons, cytochrome oxidase (COX) reactivity was further tested. All the above‐mentioned reactivities were objectively assessed by computerized image analysis. The clinical significance of the reported changes was demonstrated by alterations of mean arterial blood pressure (MAP). The results indicated that in normal untreated rats, numerous NADPH‐d/NOS‐ and COX‐reactive neurons were found in the nodose ganglion (NG). Following TSD, however, both the labelling and staining intensity of NADPH‐d/NOS as well as COX reactivity were drastically reduced in the NG compared with normal untreated ganglions. MAP was significantly higher in TSD rats (136 ± 4 mmHg) than in normal untreated rats (123 ± 2 mmHg). NO may serve as an important sympathoinhibition messenger released by the NG neurons, and decrease of NOS immunoexpression following TSD may account for the decrease in NOS content. In association with the reduction of NOS activity, a defect in NOS expression in the primary cardiovascular afferent neurons would enhance clinical hypertension, which might serve as a potential risk factor in the development of TSD‐relevant cardiovascular disturbances.

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on β1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of β1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on β1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of β1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

β-1,4-galactosyltransferase III suppresses extravillous trophoblast invasion through modifying β1-integrin glycosylation

INTRODUCTION Glycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The β-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. METHODS B4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. RESULTS B4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on β1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced β1-integrin degradation. DISCUSSION We propose that B4GALT3-mediated glycosylation change not only enhances β1-integrin binding to laminin, but also attenuates β1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy.