Loss of AMPK activity induces organelle dysfunction and oxidative stress during oocyte aging.

BACKGROUND: Oocyte quality is critical for the mammalian reproduction due to its necessity on fertilization and early development. During aging, the declined oocytes showing with organelle dysfunction and oxidative stress lead to infertility. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which is important for energy homeostasis for metabolism. Little is known about the potential relationship between AMPK with oocyte aging. RESULTS: In present study we reported that AMPK was related with low quality of oocytes under post ovulatory aging and the potential mechanism. We showed the altered AMPK level during aging and inhibition of AMPK activity induced mouse oocyte maturation defect. Further analysis indicated that similar with its upstream regulator PKD1, AMPK could reduce ROS level to avoid oxidative stress in oocytes, and this might be due to its regulation on mitochondria function, since loss of AMPK activity induced abnormal distribution, reduced ATP production and mtDNA copy number of mitochondria. Besides, we also found that the ER and Golgi apparatus distribution was aberrant after AMPK inhibition, and enhanced lysosome function was also observed. CONCLUSIONS: Taken together, these data indicated that AMPK is important for the organelle function to reduce oxidative stress during oocyte meiotic maturation.

[Effect of electroacupuncture on renal vascular microcirculation in diabetic mice based on in vivo two-photon microscopy imaging].

OBJECTIVE: To investigate the protective effect of electroacupuncture (EA) at "Zusanli"(ST36)and "Weiwanxiashu"(EX-B3) on capillary function around the renal tubule and renal tubule structure in diabetic mice based on two-photon microscopy (TPM) imaging, so as to providing visualizable evidence for the regulatory effect of EA on diabetic renal vascular microcirculation. METHODS: Spontaneous type Ⅱ diabetes mellitus mice (db/db) were employed for this study. Twenty db/db mice were randomly divided into model group (n=10) and EA group (n=10), and 10 db/m mice used as the control group. EA was applied to bilateral ST36 and EX-B3 for 20 min/time, 6 times a week for 6 weeks. The body weight was recorded and the fasting blood glucose measured before and after the intervention. The urine production and water consumption of mice in each cage were recorded after EA. The renal in vivo imaging method based on TPM was established to display the morphological structure of renal tubules, and the mouse renal blood flow velocity was detected by injecting 500 kDa dextran-fluorescein into femoral vein after the intervention. RESULTS: Compared with the control group, the proportion of mice with decreased body mass in the model group was increased, accounting for 40%, while that in the control group was 0%; and fasting blood glucose, urine production and water consumption were significantly increased in the model group (P<0.001, P<0.000 1). A renal in vivo imaging method based on TPM was successfully established, which can be applied to quantitatively analyze the renal blood flow and renal tubular diameter of mice. Based on this method, the results showed that compared with the control group, the blood flow velocity of peritubular capillary in the model group was significantly decreased (P<0.000 1, P<0.001), renal tubular cells were slightly exfoliated and the diameter of renal tubular was significantly increased (P<0.000 1). Compared with the model group, EA reduced the body weight loss ratio from 40% to 0%, and significantly decreased the fasting blood glucose, urine production and water consumption (P<0.01, P<0.000 1, P<0.001), and the blood flow velocity of peritubular capillary in the EA group was significantly increased (P<0.001, P<0.05) and tubule dilatation significantly alleviated (P<0.0 1). CONCLUSION: EA at ST36 and EX-B3 can ameliorate renal vascular microcirculation disorder to relieve the renal structure damage and improve renal function in diabetes mice.

Effect of low-dose Levamlodipine Besylate in the treatment of vascular dementia.

Vascular dementia (VaD) is a complex disorder caused by reduced blood flow in the brain. However, there is no effective pharmacological treatment option available until now. Here, we reported that low-dose levamlodipine besylate could reverse the cognitive impairment in VaD mice model of right unilateral common carotid arteries occlusion (rUCCAO). Oral administration of levamlodipine besylate (0.1 mg/kg) could reduce the latency to find the hidden platform in the MWM test as compared to the vehicle group. Furthermore, vehicle-treated mice revealed reduced phospho-CaMKII (Thr286) levels in the hippocampus, which can be partially restored by levamlodipine besylate (0.1 mg/kg and 0.5 mg/kg) treatment. No significant outcome on microglia and astrocytes were observed following levamlodipine besylate treatment. This data reveal novel findings of the therapeutic potential of low-dose levamlodipine besylate that could considerably enhance the cognitive function in VaD mice.

Endogenous Polysialic Acid Based Micelles for Calmodulin Antagonist Delivery against Vascular Dementia.

Clinical treatment for vascular dementia still remains a challenge mainly due to the blood-brain barrier (BBB). Here, a micelle based on polysialic acid (PSA), which is a hydrophilic and endogenous carbohydrate polymer, was designed to deliver calmodulin antagonist for therapy of vascular dementia. PSA was first chemically conjugated with octadecylamine (ODA), and the obtained PSA-ODA copolymer could self-assemble into micelle in aqueous solution with a 120.0 µg/mL critical micelle concentration. The calmodulin antagonist loaded PSA-ODA micelle, featuring sustained drug release behavior over a period of 72 h with a 3.6% (w/w) drug content and a 107.0 ± 4.0 nm size was then fabricated. The PSA-ODA micelle could cross the BBB mainly via active endocytosis by brain endothelial cells followed by transcytosis. In a water maze test for spatial learning, calmodulin antagonist loaded PSA-ODA micelle significantly reduced the escape latencies of right unilateral common carotid arteries occlusion (rUCCAO) mice with dosage significantly reduced versus free drug. The decrease of hippocampal phospho-CaMKII (Thr286/287) and phospho-synapsin I (Ser603) was partially restored in rUCCAO mice following calmodulin antagonist loaded PSA-ODA micelle treatment. Consistent with the restored CaMKII phosphorylation, the elevation of BrdU/NeuN double-positive cells in the same context was also observed. Overall, the PSA-ODA micelle developed from the endogenous material might promote the development of therapeutic approaches for improving the efficacy of brain-targeted drug delivery and have great potential for vascular dementia treatment.

Functional Genomic Analyses Identify Pathways Dysregulated in Animal Model of Autism.

BACKGROUND: Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders that display complicated behavioral symptoms. METHODS: Using gene expressing profiling and the weighted gene co-expression network analysis (WGCNA), we studied genes coregulated by similar factors such as genetic variants or environmental effects in the hippocampus in an animal model of autism. RESULTS: From microarray data, we identified 21,388 robustly expressed genes of which 721 genes were found to be differently expressed in the valproic acid-treated group compared to the control group. WGCNA identified multiple co-expression modules known to associate with cognitive function, inflammation, synaptic, and positive regulation of protein kinase activating. Many of these modules, however, have not been previously linked to autism spectrum disorders which included G-protein signaling, immunity, and neuroactive ligand-receptor interaction pathway. The downregulation of the highly connected (hub) genes Taar7h and Taar7b in neuroactive ligand-receptor interaction pathway was validated by qRT-PCR. Immunoblotting and immunohistochemistry further showed that TAAR7 expression was downregulated not only in valproic acid-treated animals, but also BTBR T+tf/J mice. CONCLUSIONS: This study highlights the advantages of gene microarrays to uncover co-expression modules associated with autism and suggests that Taars and related gene regulation networks may play a significant role in autism.

Valproic Acid Influences MTNR1A Intracellular Trafficking and Signaling in a ß-Arrestin 2-Dependent Manner.

Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled ß-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a ß-arrestin 2-dependent manner.

Calpain-Dependent ErbB4 Cleavage Is Involved in Brain Ischemia-Induced Neuronal Death.

Disturbance of neuregulin-1ß/ErbB4 signaling is considered to be associated with brain ischemia, but the mechanisms of this disruption are largely unknown. In the present study, we provide evidence that degradation of ErbB4 is involved in neuronal cell death in response to ischemia. Our data showed that the application of neuregulin-1ß provided significant protection against oxygen-glucose deprivation (OGD)-induced neuronal death as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide flow cytometry analysis and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining. Furthermore, neuregulin-1ß treatment significantly reduced the infarct volume of ischemic mice, and this result was not seen in the ErbB4 knockout mice. We found that brain ischemia induced the breakdown of ErbB4 in a time-dependent manner in vivo, but not that of ErbB2. In vitro studies further indicated that recombinant calpain induced the cleavage of ErbB4 in a dose-dependent way, whereas the calpain inhibitor significantly reduced the OGD-induced ErbB4 breakdown. Additionally, OGD-induced apoptosis was partially abolished by transfection with the ErbB4E872K mutant. Taken together, neuregulin-1ß elicits its neuroprotective effect in an ErbB4-dependent manner, and the cleavage of ErbB4 by calpain contributes to a neuronal cell death cascade during brain ischemia.

Visualizing peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury.

Accumulating evidence suggests that formation of peroxynitrite (ONOO(-)) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO(-) biology, efficient tools that can realize the real-time tracing of endogenous ONOO(-) fluxes are indispensable. While a few ONOO(-) fluorescent probes have been reported, direct visualization of ONOO(-) fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO(-) imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO(-) both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood-brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO(-) generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO(-) formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.

In vivo two-photon fluorescence microscopy reveals disturbed cerebral capillary blood flow and increased susceptibility to ischemic insults in diabetic mice.

AIMS: Diabetes mellitus increases the risk of stroke, but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia. METHODS: Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later, they were subjected to transient (20 min) MCAO. In vivo blood flow was measured by two-photon laser-scanning microscopy (TPLSM) in cerebral arteries, veins, and capillaries. RESULTS: There was a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice as assessed by TPLSM, yet the regional cerebral blood flow, as assessed by laser Doppler flowmetry, was maintained. Brain capillary flow developed turbulence after MCAO only in diabetic mice. These mice sustained increased neurological deficits after MCAO which were accompanied by an exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation in cerebral tissues indicating disruption of the blood-brain barrier and disturbed cognitive potential. CONCLUSION: Diabetic mice are more susceptible to disturbances of cerebral capillary blood flow which may predispose them to neurovascular defects following ischemia.

Atg5 deficit exaggerates the lysosome formation and cathepsin B activation in mice brain after lipid nanoparticles injection.

The present study was designed to investigate the role of autophagy-lysosome signaling in the brain after application of nanoparticles. Here, lipid nanoparticles (LNs) induced elevations of Atg5, P62, LC3 and cathepsin B in mice brain. The transmission electron microscopy revealed a dramatic elevation of lysosome vacuoles colocalized with LNs cluster inside the neurons in mice brain. Immunoblot data revealed abnormal expression of cathepsin B in brain cortex following LNs injection, whereas its expression was further elevated in Atg5(+/-) mice. The importance of Atg5 in the LNs-induced autophagy-lysosome cascade was further supported by our finding that neurovascular response was exaggerated in Atg5(+/-) mice. In addition, the siRNA knockdown of Atg5 significantly blunted the increasing of LC3 and P62 in LNs-treated Neuro-2a cells. Taken together, we propose that LNs induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway. FROM THE CLINICAL EDITOR: These authors investigated autophagy-lysosome signaling in the mouse brain after application of lipid nanoparticles and report that these nanoparticles induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway.

Targeted therapy of brain ischaemia using Fas ligand antibody conjugated PEG-lipid nanoparticles.

The translation of experimental stroke research from the laboratory to successful clinical practice remains a formidable challenge. We previously reported that PEGylated-lipid nanoparticles (PLNs) effectively transport across the blood-brain barrier along with less inflammatory responses. In the present study, PLNs conjugated to Fas ligand antibody that selectively present on brain ischaemic region were used for therapeutic targeting. Fluorescent analysis of the mice brain show that encapsulated 3-n-Butylphthalide (dl-NBP) in PLNs conjugated with Fas ligand antibody effectively delivered to ipsilateral region of ischaemic brain. Furthermore, the confocal immunohistochemical study demonstrated that brain-targeted nanocontainers specifically accumulated on OX42 positive microglia cells in ischaemic region of mice model. Finally, dl-NBP encapsulated nano-drug delivery system is resulted in significant improvements in brain injury and in neurological deficit after ischaemia, with the significantly reduced dosages versus regular dl-NBP. Overall, these data suggests that PLNs conjugated to an antibody specific to the Fas ligand constituted an ideal brain targeting drug delivery system for brain ischaemia.

Nitrosative stress induces peroxiredoxin 1 ubiquitination during ischemic insult via E6AP activation in endothelial cells both in vitro and in vivo.

AIMS: Although there is accumulating evidence that increased formation of reactive nitrogen species in cerebral vasculature contributes to the progression of ischemic damage, but the underlying molecular mechanisms remain elusive. Peroxiredoxin 1 (Prx1) can initiate the antioxidant response by scavenging free radicals. Therefore, we tested the hypothesis that Prx1 regulates the susceptibility to nitrosative stress damage during cerebral ischemia in vitro and in vivo. RESULTS: Proteomic analysis in endothelial cells revealed that Prx1 was upregulated after stress-related oxygen-glucose deprivation (OGD). Although peroxynitrite upregulated Prx1 rapidly, this was followed by its polyubiquitination within 6 h after OGD mediated by the E3 ubiquitin ligase E6-associated protein (E6AP). OGD colocalized E6AP with nitrotyrosine in endothelial cells. To assess translational relevance in vivo, mice were studied after middle cerebral artery occlusion (MCAO). This was accompanied by Prx1 ubiquitination and degradation by the activation of E6AP. Furthermore, brain delivery of a lentiviral vector encoding Prx1 in mice inhibited blood-brain barrier leakage and neuronal damage significantly following MCAO. INNOVATION AND CONCLUSIONS: Nitrosative stress during ischemic insult activates E6AP E3 ubiquitin ligase that ubiquitinates Prx1 and subsequently worsens cerebral damage. Thus, targeting the Prx1 antioxidant defense pathway may represent a novel treatment strategy for neurovascular protection in stroke.

Melatonin reverses the decreases in hippocampal protein serine/threonine kinases observed in an animal model of autism.

Lower global cognitive function scores are a common symptom of autism spectrum disorders (ASDs). This study investigates the effects of melatonin on hippocampal serine/threonine kinase signaling in an experimental ASD model. We found that chronic melatonin (1.0 or 5.0 mg/kg/day, 28 days) treatment significantly rescued valproic acid (VPA, 600 mg/kg)-induced decreases in CaMKII (Thr286), NMDAR1 (Ser896), and PKA (Thr197) phosphorylation in the hippocampus without affecting total protein levels. Compared with control rats, the immunostaining of pyramidal neurons in the hippocampus revealed a decrease in immunolabeling intensity for phospho-CaMKII (Thr286) in the hippocampus of VPA-treated rats, which was ameliorated by chronic melatonin treatment. Consistent with the elevation of CaMKII/PKA/PKC phosphorylation observed in melatonin-treated rat, long-term potentiation (LTP) was enhanced after chronic melatonin (5.0 mg/kg) treatment, as reflected by extracellular field potential slopes that increased from 56 to 60 min (133.4 ± 3.9% of the baseline, P < 0.01 versus VPA-treated rats) following high-frequency stimulation (HFS) in hippocampal slices. Accordingly, melatonin treatment also significantly improved social behavioral deficits at postnatal day 50 in VPA-treated rats. Taken together, the increased phosphorylation of CaMKII/PKA/PKC signaling might contribute to the beneficial effects of melatonin on autism symptoms.

The effect of lipid nanoparticle PEGylation on neuroinflammatory response in mouse brain.

Nanocarrier-based drug delivery systems have attracted wide interest for the treatment of brain disease. However, neurotoxicity of nanoparticle has limited their therapeutic application. Here we demonstrated that lipid nanoparticles (LNs) accumulated in the brain parenchyma within 3 h of intravenous injection to mice and persisted for more than 24 weeks, coinciding with a dramatic activation of brain microglia. Morphological characteristic of microglial activation also observed in LNs-treated Cx3cr1GFP/+ mice. In vivo study with two-photon confocal microscopy revealed abnormal Ca²âº waves in microglia following LNs injection. The correlated activation of caspase-1, IL-1ß and neurovascular damage following LNs injection was attenuated in P2X7-/- mice. PEGylation of LNs reduced correlated nanoparticles aggregation. Moreover, PEGylation of LNs ameliorated the P2X7/caspase-1/IL-1ß signalling-dependent microglia activation and neurovascular damage. In conclusion, PEGylation of LNs is a promising biomaterial for brain-targeted therapy that inhibits P2X77-dependent neuroinflammatory response.

The disturbance of hippocampal CaMKII/PKA/PKC phosphorylation in early experimental diabetes mellitus.

BACKGROUND: Defining the impact of diabetes and related risk factors on brain cognitive function is critically important for patients with diabetes. AIMS: To investigate the alterations in hippocampal serine/threonine kinases signaling in the early phase of type 1 and type 2 diabetic rats. METHODS: Early experimental diabetes mellitus was induced in rats with streptozotocin or streptozotocin/high fat. Changes in the phosphorylation of proteins were determined by immunoblotting and immunohistochemistry. RESULTS: Our data showed a pronounced decrease in the phosphorylation of Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) in the hippocampi of both type 1 and type 2 diabetic rats compared with age-matched control rats. Unexpectedly, we found a significant increase in the phosphorylation of synapsin I (Ser 603) and GluR1 (Ser 831) in the same experiment. In addition, aberrant changes in hippocampal protein kinase C (PKC) and protein kinase A (PKA) signaling in type 1 and type 2 diabetic rats were also found. Moreover, PP1α and PP2A protein levels were decreased in the hippocampus of type 1 diabetic rats, but significantly up-regulated in type 2 diabetic rats. CONCLUSIONS: The disturbance of CaMKII/PKA/PKC phosphorylation in the hippocampus is an early change that may be associated with the development and progression of diabetes-related cognitive dysfunction.

Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin.

Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S-nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen-glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear-localized Nrf2, as demonstrated through co-immunoprecipitation and immunocytochemical assay. Paralleling the ischemia-induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD-induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S-nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S-nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO-1 expression, paralleled with the restored heme oxygenase-1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia-evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.

The γ-secretase blocker DAPT reduces the permeability of the blood-brain barrier by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia.

BACKGROUND: Tight junction protein degradation is a principal characteristic of the blood-brain barrier (BBB) damage that occurs during brain ischemia. AIMS: We investigated the mechanisms of occludin degradation that underlie permanent middle cerebral artery occlusion (pMCAO) in rats. METHODS AND RESULTS: Western blot and Co-immunoprecipitation data indicated ubiquitination and degradation of occludin in brain after pMCAO, which was consistent with ZO-1 degradation in penumbra regions as observed at 24 h after pMCAO. We further investigated candidate protease(s) responsible for the degradation of occludin during pMCAO. The intraventricular administration of γ-secretase blocker DAPT significantly inhibited the pMCAO-induced neurovascular damage, whereas ALLM and Batimastat, which are inhibitors of calpain and metalloproteinase proteases, respectively, were less effective. Notably, we found that DAPT significantly inhibited BBB disruption in comparison with vehicle treatment, as assessed by Evans blue excretion. Interestingly, the confocal immunostaining revealed that activation of the E3 ubiquitin ligase Itch is associated with degradation of occludin in brain microvessels following ischemia. Furthermore, our data demonstrate that the inhibition of γ-secretase signaling and the itch-mediated ubiquitination of occludin likely underlie the vasoprotective effect of DAPT after pMCAO. CONCLUSION: The γ-secretase blocker DAPT reduces the permeability of the BBB by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia, suggesting that γ-secretase may represent a novel therapeutic target for preventing neurovascular damage.

[Effect of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus].

OBJECTIVE: To investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus. METHODS: SD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry. RESULTS: Compared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals. CONCLUSION: The disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.

P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand.

BACKGROUND: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia. METHODS: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction. RESULTS: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult. CONCLUSION: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.

Regulation of the ischemia-induced autophagy-lysosome processes by nitrosative stress in endothelial cells.

The cellular mechanisms that underlie the diverse nitrosative stress-mediated cellular events associated with ischemic complications in endothelial cells are not yet clear. To characterize whether autophagic elements are associated with the nitrosative stress that causes endothelial damage after ischemia injury, an in vitro sustained oxygen-glucose deprivation (OGD) and an in vivo microsphere embolism model were used in the present study. Consistent with OGD-induced peroxynitrite formation, a rapid induction of microtubule-associated protein 1 light chain 3 (LC3)-I/II conversion and green fluorescent protein-LC3 puncta accumulation were observed in endothelial cells. The Western blot analyses indicated that OGD induced elevations in lysosome-associated membrane protein 2 and cathepsin B protein levels. Similar results were observed in the microvessel insult model, following occlusion of the microvessels using microsphere injections in rats. Furthermore, cultured endothelial cells treated with peroxynitrite (1-50 µm) exhibited a concentration-dependent change in the pattern of autophagy-lysosome signaling. Intriguingly, OGD-induced autophagy-lysosome processes were attenuated by PEP-19 overexpression and by a small-interfering RNA (siRNA)-mediated knockdown of eNOS. The importance of nitrosative stress in ischemia-induced autophagy-lysosome cascades is further supported by our finding that pharmacological inhibition of nitrosative stress by melatonin partially inhibits the ischemia-induced autophagy-lysosome cascade and the degradation of the tight junction proteins. Taken together, the present results demonstrate that peroxynitrite-mediated nitrosative stress at least partially potentiates autophagy-lysosome signaling during sustained ischemic insult-induced endothelial cell damage.