Nan-Nan Lu

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.

P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy.

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.

Melatonin ameliorates hypoglycemic stress-induced brain endothelial tight junction injury by inhibiting protein nitration of TP53-induced glycolysis and apoptosis regulator

Severe hypoglycemia has a detrimental impact on the cerebrovasculature, but the molecular events that lead to the disruption of the integrity of the tight junctions remain unclear. Here, we report that the microvessel integrity was dramatically compromised (59.41% of wild‐type mice) in TP53‐induced glycolysis and apoptosis regulator (TIGAR) transgenic mice stressed by hypoglycemia. Melatonin, a potent antioxidant, protects against hypoglycemic stress‐induced brain endothelial tight junction injury in the dosage of 400 nmol/L in vitro. FRET (fluorescence resonance energy transfer) imaging data of endothelial cells stressed by low glucose revealed that TIGAR couples with calmodulin to promote TIGAR tyrosine nitration. A tyrosine 92 mutation interferes with the TIGAR‐dependent NADPH generation (55.60% decreased) and abolishes its protective effect on tight junctions in human brain microvascular endothelial cells. We further demonstrate that the low‐glucose‐induced disruption of occludin and Caludin5 as well as activation of autophagy was abrogated by melatonin‐mediated blockade of nitrosative stress in vitro. Collectively, we provide information on the detailed molecular mechanisms for the protective actions of melatonin on brain endothelial tight junctions and suggest that this indole has translational potential for severe hypoglycemia‐induced neurovascular damage.

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.

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

UNLABELLED 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.

A fluorescent peptidyl substrate for visualizing peptidyl-prolyl cis/trans isomerase activity in live cells

This communication reports on a fluorescent probe (PPI-P) for imaging active peptidyl-prolyl cis/trans isomerases in live cells. PPI-P is capable of responding to both recombinant and cellular PPIases fluorogenically, and has been shown to specifically image active PPIases in live cells.

Peroxiredoxin 1 participates in ischemia-triggered endothelial polarization

The balance between endothelial cell survival and death plays a pivotal role in brain remodeling and repair after stroke [1]. Extensive changes in endothelial cell behavior have been implicated in angiogenesis and vasculogenesis [2]. Angiogenesis is a prominent feature of ischemic stroke recovery, and increasing endothelial polarity might stimulate early angiogenesis and reflect a beneficial mechanism of revascularization and neuroprotection after stroke [3]. Polarization is important for various aspects of the endothelial response to a variety of stressful stimuli, such as hypoxia and inflammation [4]. However, the precise molecular cues that regulate endothelial polarity and function remain elusive. In the current study, we set out to address the potential role of peroxiredoxin 1 in ischemia-induced endothelial polarization. It is unknown whether peroxiredoxin 1 plays a role in angiogenesis, a process that involves the generation of new blood vessels and relies largely on vascular endothelial cell polarization and proliferation. We previously reported that peroxiredoxin 1 is a pivotal molecule for the protection of endothelial cells and microvessels from ischemia-induced neurovascular damage both in vitro and in vivo [5]. Earlier studies have shown that peroxiredoxin 1 carries out a wide range of cellular functions, including exerting a proliferative effect and playing an antioxidant protective role [5]. It is also known that peroxiredoxin 1 is important for vascular endothelial growth factor (VEGF) expression [6], and it is therefore likely that peroxiredoxin 1 is involved in the induction of angiogenesis following a stroke. Here, we first investigated the involvement of peroxiredoxin 1 in oxygen-glucose deprivation (OGD)-induced endothelial cell polarization. EA.hy926 endothelial cells were exposed to OGD for 6 and 12 h, and the expression of peroxiredoxin 1 was assayed using confocal immunocytochemistry. We defined polarized cells according to the proportion of the longest axis of the cell to that of the shortest axis of the cell [7]. As shown in Figure 1A, peroxiredoxin 1-positive endothelial cells in the OGD 6 h group exhibited polarized characteristics. After 12 h had elapsed, the cells respread, and their elongation became even more pronounced. They exhibited a polarized cell shape with a morphologically defined leading process and a uropod, whereas the control cells displayed oval-shaped morphology. The results of quantitative analyses from three independent experiments are depicted in Figure 1B, indicating that cell elongation significantly increased after OGD, whereas cell ellipsoid decreased (Figure 1C). This elongation might involve directional spreading via a protrusion at the front of the cells mediated by various intracellular signaling mechanisms [7]. These data raise the possibility that peroxiredoxin 1 plays a crucial role in the establishment of endothelial cell polarity in response to ischemic stimuli, which might be related to its proliferative effect and its contributions toward angiogenesis and brain repair. To further ascertain the impact of the upregulation of peroxiredoxin 1 on endothelial polarization in vivo, a mouse transient middle cerebral artery occlusion (tMCAO) model was employed in the present study. Staining brain sections from tMCAO mice for peroxiredoxin 1 and the endothelial marker Tie2 revealed that peroxiredoxin 1 was localized to the endothelial cells of the brain (Figure 2). Peroxiredoxin 1 immunoreactivity was observed predominantly in ipsilateral brain microvessels 12 h after administering tMCAO (Figure 2A) and was accompanied by colocalization with Tie2 immunoreactivity (Figure 2B,C,D). Compared with sham-operated animals, peroxiredoxin 1 expression was increased

Cholinergic Grb2-Associated-Binding Protein 1 Regulates Cognitive Function

Grb2-associated-binding protein 1 (Gab1) is a docking/scaffolding molecule known to play an important role in cell growth and survival. Here, we report that Gab1 is decreased in cholinergic neurons in Alzheimers disease (AD) patients and in a mouse model of AD. In mice, selective ablation of Gab1 in cholinergic neurons in the medial septum impaired learning and memory and hippocampal long-term potentiation. Gab1 ablation also inhibited SK channels, leading to an increase in firing in septal cholinergic neurons. Gab1 overexpression, on the other hand, improved cognitive function and restored hippocampal CaMKII autorphosphorylation in AD mice. These results suggest that Gab1 plays an important role in the pathophysiology of AD and may represent a novel therapeutic target for diseases involving cholinergic dysfunction.

Endothelial ErbB4 deficit induces alterations in exploratory behavior and brain energy metabolism in mice

The receptor tyrosine kinase ErbB4 is present throughout the primate brain and has a distinct functional profile. In this study, we investigate the potential role of endothelial ErbB4 receptor signaling in the brain.