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Myasthenia gravis (MG) is a disease involving neuromuscular transmission that causes fatigue of skeletal muscles and fluctuating weakness. It has been shown that impairment of myogenic differentiation and myofiber maturation may be the underlying cause of MG. In this study, we detected the abnormal expression of circular RNA (circRNA) using next-generation sequencing in patients with MG. We then investigated the regulatory mechanism and the relationship among circRNA, microRNA, and messenger RNA using quantitative reverse-transcription polymerase chain reaction, bioinformatics analysis, and luciferase report analysis. The expression of inflammatory cytokines and regulatory T lymphocytes was shown to be increased. Circ-FBL was significantly increased in MG patients. Bioinformatics and luciferase report analyses confirmed that miR-133 and PAX7 were the downstream targets of circ-FBL. Overexpression of circ-FBL promoted myoblast proliferation by regulation of miR-133/PAX7. Taken together, our study showed that upregulation of circ-FBL promoted myogenic proliferation in patients with MG by regulating miR-133/PAX7.

Energy deprivation activates the cellular energy sensor AMP-activated protein kinase (AMPK), which in turn induces macroautophagy/autophagy. The mitochondrial-associated ER membrane (MAM) plays a key role in mitochondrial division and autophagy, and the mitochondrial fusion protein MFN2 (mitofusin 2) tethers the MAM, but the mechanism by which AMPK and MFN2 regulate autophagy in response to energy stress remains unclear. Here, we found that energy stress not only triggers mitochondrial fission and autophagy, but more importantly increases the number of MAMs, a process that requires AMPK. Interestingly, under energy stress, considerable amounts of AMPK translocate from cytosol to the MAM and the mitochondrion as mitochondrial fission occurs. Unexpectedly, AMPK interacts directly with MFN2. The autophagic ability of mouse embryonic fibroblasts (MEFs) lacking MFN2 (mfn2(−/-)) is significantly attenuated in response to energy stress as compared to wild-type MEFs (WT MEFs), while re-expression of MFN2 in mfn2(−/-) cells rescues the autophagy defects of these cells. The abundance of MAMs is also greatly reduced in MFN2-deficient cells. Functional experiments show that the oxygen consumption rate and the glycolytic function of cells lacking MFN2 but not MFN1 are obviously attenuated, and MFN2 is important for cell survival under energy stress. In conclusion, our study establishes the molecular link between the energy sensor AMPK and the MAM tether MFN2, and reveals the important role of AMPK and MFN2 in energy stress-induced autophagy and MAM dynamics. Abbreviations: ACTB, actin beta; AMPK, AMP-activated protein kinase; BECN1, beclin 1; CANX, calnexin; ER, endoplasmic reticulum; HRP, horseradish peroxidase; EM, electron microscopy; FL, full-length; KD, kinase dead, KO, knockout; MAb, monoclonal antibody; MAMs, mitochondria-associated membranes; MAP1LC3/LC3B, microtubule associated protein 1 light chain 3; MFN2, mitofusin 2; OPA1, OPA1 mitochondrial dynamin like GTPase; PAb, polyclonal antibody; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; SD, standard deviation; TEM, transmission electron microscopy; TOMM20, translocase of outer mitochondrial membrane 20; ULK1, unc-51 like autophagy activating kinase 1; MEF, mouse embryonic fibroblast; WT, wildtype

Alzheimer's disease (AD) is associated with the accumulation and deposition of a beta-amyloid (Αβ) peptide in the brain, resulting in increased neuroinflammation and synaptic dysfunction. Intranasal delivery of targeted drugs to the brain represents a noninvasive pathway that bypasses the blood-brain barrier and minimizes systemic exposure. The aim of this study was to evaluate the therapeutic effect of intranasally delivered 9-cis retinoic acid (RA) on the neuropathology of an AD mouse model. Herein, we observed dramatically decreased Αβ deposition in the brains of amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice (APP/PS1) treated intranasally with 9-cis RA for 4 weeks compared to that in the brains of vehicle-treated mice. Importantly, intranasal delivery of 9-cis RA suppressed Αβ-associated astrocyte activation and neuroinflammation and ultimately restored synaptic deficits in APP/PS1 transgenic mice. These results support the critical roles of Αβ-associated neuroinflammation responses to synaptic deficits, particularly during the deposition of Αβ. Our findings provide strong evidence that intranasally delivered 9-cis RA attenuates neuronal dysfunction in an AD mouse model and is a promising therapeutic strategy for the prevention and treatment of AD.

Background: Atherosclerosis is a complicated disease governed by genetic, environmental and vascular risk factors, while its relationship with specific genes is still unclear. In recent days, it has been discovered that the single-nucleotide polymorphisms of chromosomal region 9p21 were relevant with the genetic susceptibility of Atherosclerosis. We aimed to figure out the relativity between the gene polymorphism of region 9p21 (rs10757274, rs7044859, rs4977574 and rs496892) and carotid plaque. Methods: With polymerase chain reaction-ligase detection reaction, the gene polymorphisms of site rs10757274, rs7044859, rs4977574 and rs496892 in chromosome region 9p21 were analyzed in 764 non-cardiac cerebral infarction (CI) patients (406 with carotid plaque while 358 without). Results: Among the population aged between 45 and 65, compared to patients without carotid plaque, no significant difference were observed on the genotype or allele frequencies distribution in 9p21 genes including rs10757274, rs7044859, rs4977574 and rs496892 in the patients with carotid plaque. There existed strong linkage disequilibrium not only between rs10757274 and rs4977574 but also between rs7044859 and rs496892 as well. Conclusion: In Chinese Han CI population, there was no significant relevance between the polymorphisms of site rs10757274, rs7044859, rs4977574 and rs496892 in region 9p21 and the susceptibility of carotid plaque.

We previously demonstrated that cross-talk between α5β1 integrin and the angiopoietin-1 (Ang1) / Tie2 receptor plays an important role in regulating brain endothelial angiogenic responses in the ischemic penumbra following cerebral ischemic stroke (CIS). However, a recent study suggested that stimulation of the α5β1 integrin also has the potential of increasing blood-brain barrier (BBB) permeability after CIS, raising doubt about whether α5β1 integrin stimulation by itself will protect against ischemic injury. In light of these conflicting roles, the goal of this study was to evaluate the impact of co-overexpression of α5 integrin and Ang1 on vascular remodeling and repair under cerebral ischemic conditions both in vivo following 90 min of ischemia by temporary occlusion of the middle cerebral artery, and in vitro. Our results demonstrate that as compared to mock-transfected controls, overexpression of α5 integrin alone didn't improve the outcomes in neurological score and size of infarct and caused worse BBB breakdown in the ischemic hemisphere, offsetting its beneficial angiogenic effects during the early stages of CIS. However, co-overexpression of α5 integrin with Ang1 led to smaller infarcts and improved neurological deficits, which at the molecular level was underpinned by reduced BBB breakdown and increased expression of endothelial tight junction proteins in the ischemic penumbra during the early stages of CIS. Furthermore, co-overexpression of α5 integrin and Ang1 synergistically promoted BEC proliferation during the early stage of CIS, resulting in increased blood vessel density at later stages. Positive effects of α5 integrin and Ang1 co-overexpression on endothelial proliferation and tight junction protein expression were also confirmed in vitro. Collectively, these data indicate that co-overexpression of Ang-1 and α5 integrin in combination confers synergistic vascular protection against cerebral ischemic injury without the negative side effects on BBB permeability, suggesting a novel combinatorial approach for the treatment of CIS.

With the development of high-throughput screening and bioinformatics technology, natural products with a range of pharmacological targets in multiple diseases have become important sources of new drug discovery. These compounds are derived from various plants, including the dried root of Scutellaria baicalensis Georgi, which is often used as a traditional Chinese herb named Huangqin, a popular medication used for thousands of years in China. Many studies have shown that baicalin, an extract from Scutellaria baicalensis Georgi, exerts various protective effects on liver and gut diseases. Baicalin plays a therapeutic role mainly by mediating downstream apoptosis and immune response pathways induced by upstream oxidative stress and inflammation. During oxidative stress regulation, PI3K/Akt/NRF2, Keap-1, NF-κB and HO-1 are key factors associated with the healing effects of baicalin on NAFLD/NASH, ulcerative colitis and cholestasis. In the inflammatory response, IL-6, IL-1β, TNF-α, MIP-2 and MIP-1α are involved in the alleviation of NAFLD/NASH, cholestasis and liver fibrosis by baicalin, as are TGF-β1/Smads, STAT3 and NF-κB. Regarding the apoptosis pathway, Bax, Bcl-2, Caspase-3 and Caspase-9 are key factors related to the suppression of hepatocellular carcinoma and attenuation of liver injury and colorectal cancer. In addition to immune regulation, PD-1/PDL-1 and TLR4-NF-κB are correlated with the alleviation of hepatocellular carcinoma, ulcerative colitis and colorectal cancer by baicalin. Moreover, baicalin regulates intestinal flora by promoting the production of SCFAs. Furthermore, BA is involved in the interactions of the liver-gut axis by regulating TGR5, FXR, bile acids and the microbiota. In general, a comprehensive analysis of this natural compound was conducted to determine the mechanism by which it regulates bile acid metabolism, the intestinal flora and related signaling pathways, providing new insights into the pharmacological effects of baicalin. The mechanism linking the liver and gut systems needs to be elucidated to draw attention to its great clinical importance.

The vertebrate blood-brain barrier (BBB) creates an obstacle for central nervous system-related drug delivery. Claudin-5 (Cldn5), expressed in large quantities in BBB, plays a vital role in restricting BBB permeability. The C-terminal domain of Clostridium perfringens enterotoxin (cCPE) has been verified as binding to a subset of claudins (Cldns). The Cldn5-binding cCPE194-319 variant cCPEY306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPEY306W/S313H is able to bind specifically to Cldn5 in murine brain vascular endothelial (bEnd.3) cells, and is transported along with Cldn5 from the cell membrane to the cytoplasm, which in turn results in a reduction in transendothelial electrical resistance (TEER). Conversely, this effect can be reversed by removal of cCPEY306W/S313H. In an in vivo experiment, this study estimates the capability of cCPEY306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPEY306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPEY306W/S313H has the capability - both in vitro and in vivo - to modulate BBB permeability temporarily by specific binding to Cldn5.

We have previously demonstrated that in response to cerebral ischemia (CI), the growth factor angiopoietin-1 (Ang1) and α5β1 integrin are both induced in cerebral vessels, which likely provide positive signals driving the endogenous angiogenic response and vascular protection after CI. However, the precise relationship between endothelial Ang1 and α5β1 integrin after CI remains poorly understood. Here, we investigated the effects of the interaction between the Ang1/Tie2 system and α5β1 integrin on brain endothelial cells (BECs) under cerebral ischemic conditions in vivo and in vitro. Immunofluorescence analysis demonstrated that integrin α5β1 co-localized with Tie2/phosphorylated Tie2 on cerebral vessels in the penumbra. The in vitro study showed that oxygen–glucose deprivation/restoration (OGD/R) induced the expression of the Ang1 receptor Tie2 on BECs in a manner similar to that for integrin α5 and Ang1 in response to OGD/R, accompanied by increased activation of Tie2 and its downstream effectors focal adhesion kinase (FAK) and Akt. Knockdown of α5 integrin markedly suppressed OGD/R-induced Tie2 receptor activation in BECs, while in contrast, priming BECs with Ang1 promoted the expression of α5 integrin as well as the Tie2 downstream transcription factor Ets-1 in OGD-treated BECs. In line with this, Ets-1 knockdown significantly attenuated Ang1-mediated upregulation of α5 integrin. Functionally, Ang1 induced cell migration and tube formation of BECs after OGD, but this effect was inhibited by diminishment of the levels of α5 integrin in BECs. Taken together, our data indicate that the Ang1/Tie2 system cross-talks with integrin α5β1 in BECs after CI, which may contribute to the endogenous angiogenic vascular protective response following CI.

PURPOSE We previously demonstrated that 7 days post-ischemia, angiogenic vessels in the ischemic penumbra show strong upregulation of fibronectin (Fn) and its receptors, α5β1 and αvβ3 integrins. The aim of the current study was to precisely define the time-course of angiogenic responses and glial activation following experimental ischemia in the mouse. METHODS Male C57Bl/6 mice were subject to 90 minutes of ischemia by temporary occlusion of the middle cerebral artery followed by reperfusion. Vascular remodeling and glial activation were then examined in the brains of these mice after 0, 1, 2, 4, 7 and 14 days post-ischemia. RESULTS Immunofluorescent studies demonstrated that in the ischemic penumbra, blood vessel density increased up to day 14. In contrast, within the ischemic core, vessel density declined, reaching a low point at day 4, but then started to increase. In the penumbra, expression of Fn and the α5 and β3 integrins peaked at day 7, and this coincided exactly with maximal endothelial proliferation. CONCLUSIONS Our results suggest that upregulation of the Fn-α5β1/αVβ3 integrin axis on blood vessels stimulates BEC proliferation at an early stage of angiogenesis post-ischemia. This could form the basis of novel therapeutic strategies aimed at promoting angiogenesis following cerebral ischemia.

Background Hepatitis B virus (HBV) infection in the body can damage liver cells and cause disorders in blood lipid metabolism. Apolipoprotein C3 (ApoC3) plays an important role in the regulation of lipid metabolism, but no study on the HBV regulation of ApoC3 has been reported. This purpose of this study was to investigate the effect of HBV on ApoC3 expression and its regulatory mechanism. Methods The expression levels of ApoC3 mRNA and protein in the human hepatoma cell lines HepG2 and HepG2.2.15 were determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The HepG2 cells were co-transfected with the ApoC3 gene promoter and either HBV-infected clone pHBV1.3 or its individual genes. The changes in luciferase activity were assayed. The expression levels of ApoC3 mRNA and protein were determined using RT-qPCR and Western blot. The content of ApoC3 in the supernatant of the cultured cells was determined using an enzyme-linked immunosorbent assay (ELISA). The sera were collected from 149 patients with HBV infection and 102 healthy subjects at physical examination as the normal controls. The serological levels of ApoC3 in the HBV group and the normal control group were determined using ELISA. The contents of serum triglyceride (TG) and very-low-density lipoprotein (VLDL) in the HBV patients and the normal control were determined using an automatic biochemical analyser. Results The expression levels of ApoC3 mRNA and protein were lower in the HepG2.2.15 cells than in the HepG2 cells. pHBV1.3 and its X gene could inhibit the activity of the ApoC3 promoter and its mRNA and protein expression. The serum levels of ApoC3, VLDL and TG were 65.39 ± 7.48 μg/ml, 1.24 ± 0.49 mmol/L, and 0.46 ± 0.10 mmol/L in the HBV patients and 41.02 ± 6.88 μg/ml, 0.76 ± 0.21 mmol/L, 0.29 ± 0.05 mmol/L in the normal controls, respectively, statistical analysis revealed significantly lower serum levels of ApoC3, VLDL and TG in HBV patients than in the normal controls (P

The post-stroke angiogenic response is accompanied by changes of tight junctions (TJs) of the blood-brain barrier (BBB). However, the precise dynamic change of TJ proteins (TJPs) in the different stages of stroke-induced vascular remodeling and the molecules mediating these processes have yet to be fully defined. To investigate the temporal relationship between changes in TJPs, the pro-angiogenic factor α5β1 integrin and the anti-permeability factor Ang1 in cerebral vessels following cerebral ischemic stroke, male C57Bl/6 mice were subject to 90min of ischemia by temporary occlusion of the middle cerebral artery followed by reperfusion and their brains analyzed 0, 1, 2, 4, 7 and 14days post-ischemia. Immunofluorescent studies demonstrated that in the ischemic penumbra, TJPs claudin-5 and ZO-1 levels decreased during the early stages of vascular remodeling, but then increased in the later stages. In contrast, within the ischemic core, TJPs levels decreased over the 14-day time-course, plateaued at day 4, and remained at low levels up to day 14. In the penumbra, Ang1 expression was induced, peaking at the same time point as α5β1 expression. Consistent with these findings, oxygen glucose deprivation/reperfusion induced expression of α5β1 and Ang1 on brain endothelial cell (BEC) in a similar manner in vitro, which correlated closely with BEC proliferation and increased expression of TJPs. Our results demonstrate that in the post-ischemic penumbra, a tight temporal correlation exists between the angiogenic markers α5β1 and Ang1 and the TJPs, suggesting a potential role for Ang1 and α5β1 in promoting BBB integrity following ischemic stroke.

The expression of the core autophagy kinase, Unc51-like kinase 1 (ULK1), is regulated transcriptionally and translationally by starvation-induced autophagy. However, how ULK1 is regulated during hypoxia is not well understood. Previously, we showed that ULK1 expression is induced by hypoxia stress. Here, we report a new ULK1-modulating microRNA, miR-93; its transcription is negatively correlated with the translation of ULK1 under hypoxic condition. miR-93 targets ULK1 and reduces its protein levels under hypoxia condition. miR-93 also inhibits hypoxia-induced autophagy by preventing LC3-I to LC3-II transition and P62 degradation; these processes are reversed by the overexpression of an endogenous miR-93 inhibitor. Re-expression of ULK1 without miR-93 response elements restores the hypoxia-induced autophagy which is inhibited by miR-93. Finally, we detected the effects of miR-93 on cell viability and apoptosis in noncancer cell lines and cancer cells. We found that miR-93 sustains the viability of MEFs (mouse embryonic fibroblasts) and inhibits its apoptosis under hypoxia. Thus, we conclude that miR-93 is involved in hypoxia-induced autophagy by regulating ULK1. Our results provide a new angle to understand the complicated regulation of the key autophagy kinase ULK1 during different stress conditions.

Mitophagy receptors mediate the selective recognition and targeting of damaged mitochondria by autophagosomes. The mechanism for the regulation of these receptors remains unknown. Here, we demonstrated that a novel hypoxia-responsive microRNA, microRNA-137 (miR-137), markedly inhibits mitochondrial degradation by autophagy without affecting global autophagy. miR-137 targets the expression of two mitophagy receptors NIX and FUNDC1. Impaired mitophagy in response to hypoxia caused by miR-137 is reversed by re-expression of FUNDC1 and NIX expression vectors lacking the miR-137 recognition sites at their 3' UTR. Conversely, miR-137 also suppresses the mitophagy induced by fundc1 (CDS+3'UTR) but not fundc1 (CDS) overexpression. Finally, we found that miR-137 inhibits mitophagy by reducing the expression of the mitophagy receptor thereby leads to inadequate interaction between mitophagy receptor and LC3. Our results demonstrated the regulatory role of miRNA to mitophagy receptors and revealed a novel link between miR-137 and mitophagy.

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1-binding-deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase-active ULK1 and a phospho-mimicking mutant of FUNDC1 rescue mitophagy in ULK1-null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy.

Background The pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), is expressed in ischemic tissue and is known to modulate angiogenesis; however, the role of the two distinct TNF-α receptors, TNFR1 and TNFR2, in mediating angiogenic signaling after cerebral ischemic stroke is relatively unknown. Methods C57BL6 mice were subject to 90 min of ischemia by temporary occlusion of the middle cerebral artery (MCAO) and given daily intra-cerebroventricular injections of antibodies against TNFR1, TNFR2 or control IgG (doses of 10, 50, and 100 ng/day) for 4 days following 90 min MCAO. Vascular remodeling and α5β1 and αVβ3 integrin expression were then examined in the brains of these mice after 4, 7, and 14 days post-ischemia. In parallel in vitro studies, flow cytometry was used to determine the influence of TNF-α on proliferation and integrin expression of human brain microvascular endothelial cells (HBMECs). Results The post-ischemic cerebral angiogenic response was inhibited by antibodies against TNFR1 but not TNFR2, and this correlated with reduced endothelial proliferation and decreased α5β1 and αVβ3 integrin expression after 4 and 7 days post-ischemia. Consistent with these findings, in vitro studies showed that TNF-α induced endothelial proliferation and upregulation of α5β1 and αVβ3 integrins was abrogated by anti-TNFR1 but not anti-TNFR2 antibodies in cultured HBMECs. In addition, blocking antibodies to α5β1 and αVβ3 integrins significantly inhibited TNF-α-induced HBMEC proliferation. Conclusions Our results suggest that TNFR1-mediated signaling plays a critical role in triggering angiogenic integrins and subsequent angiogenic responses following cerebral ischemia. These novel findings could form a platform for future therapeutic strategies aimed at stimulating angiogenesis following cerebral ischemia. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0697-1) contains supplementary material, which is available to authorized users.

Why certain diseases primarily affect one specific neuronal subtype rather than another is a puzzle whose solution underlies the development of specific therapies. Selective basal forebrain cholinergic (BFC) neurodegeneration participates in cognitive impairment in Alzheimer’s disease (AD), yet the underlying mechanism remains elusive. Here, we report the first recapitulation of the selective BFC neuronal loss that is typical of human AD in a mouse model termed GAP. We created GAP mice by crossing Tg2576 mice that over-express the Swedish mutant human β-amyloid precursor protein gene with G protein-coupled receptor kinase-5 (GRK5) knockout mice. This doubly defective mouse displayed significant BFC neuronal loss at 18 months of age, which was not observed in either of the singly defective parent strains or in the wild type. Along with other supporting evidence, we propose that GRK5 deficiency selectively renders BFC neurons more vulnerable to degeneration.

Summary Propofol (2,6-diisopropylphenol) is an anaesthetic agent with anti-oxidant properties. The aim of the present study was to determine whether propofol can protect pulmonary epithelial (A549) cells against lipopolysaccharide (LPS)-induced cell death and, if so, the mechanisms involved. The effects of LPS alone and in combination with propofol on A549 cell death were investigated. Cell viability was determined using the colourimetric 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptotic A549 cells were detected by flow cytometry, as propidium iodide-negative and annexin-V-positive cells, and terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL). Mitochondrial membrane potential (MMP), caspase 9 activity, Ca2+ concentrations and reactive oxygen species (ROS) were analysed by immunofluorescent methods. Aconitase 2 (ACO2), microtubule-associated light chain 3 (LC3) and beclin-1 levels were evaluated using reverse transcription–polymerase chain reaction and/or western blot analysis. Exposure of A549 cells to 1–50 μg/mL LPS for 3–24 h resulted in the concentration- and time-dependent induction of cell death. Cell apoptosis accounted for approximately 77% of cell death induced by LPS. Propofol (5–150 μmol/L) concentration-dependently inhibited LPS-induced A549 cell death. This protective effect of propofol was accompanied by prevention of LPS-induced mitochondrial dysfunction (reductions in MMP, ACO2 expression and ATP) and was associated with the inhibition of LPS-induced activation of apoptotic signals (caspase 9 activity, ROS overproduction and Ca2+ accumulation). In addition, propofol blocked LPS-induced overexpression of the autophagy-associated proteins LC3 and beclin-1. The data indicate that propofol protects A549 cells against LPS-induced apoptosis, and probably autophagy, by blocking LPS-induced activation of ROS/caspase 9 pathways and upregulation of LC3 and beclin-1, respectively.

Membrane G protein-coupled receptor kinase 5 (GRK5) deficiency is linked to Alzheimer disease, yet its precise roles in the disease pathogenesis remain to be delineated. We have previously demonstrated that GRK5 deficiency selectively impairs desensitization of presynaptic M2 autoreceptors, which causes presynaptic M2 hyperactivity and inhibits acetylcholine release. Here we report that inactivation of one copy of Grk5 gene in transgenic mice overexpressing β-amyloid precursor protein (APP) carrying Swedish mutations (Tg2576 or APPsw) resulted in significantly increased β-amyloid (Aβ) accumulation, including increased Aβ(+) plaque burdens and soluble Aβ in brain lysates and interstitial fluid (ISF). In addition, secreted β-APP fragment (sAPPβ) also increased, whereas full-length APP level did not change, suggesting an alteration in favor of β-amyloidogenic APP processing in these animals. Reversely, perfusion of methoctramine, a selective M2 antagonist, fully corrected the difference between the control and GRK5-deficient APPsw mice for ISF Aβ. In contrast, a cholinesterase inhibitor, eserine, although significantly decreasing the ISF Aβ in both control and GRK5-deficient APPsw mice, failed to correct the difference between them. However, combining eserine with methoctramine additively reduced the ISF Aβ further in both animals. Altogether, these findings indicate that GRK5 deficiency accelerates β-amyloidogenic APP processing and Aβ accumulation in APPsw mice via impaired cholinergic activity and that presynaptic M2 hyperactivity is the specific target for eliminating the pathologic impact of GRK5 deficiency. Moreover, a combination of an M2 antagonist and a cholinesterase inhibitor may reach the maximal disease-modifying effect for both amyloid pathology and cholinergic dysfunction.

Cerebral hypoxia induces a profound angiogenic response in the central nervous system (CNS). Using a mouse model of chronic cerebral hypoxia, we previously demonstrated that angiogenic vessels in the hypoxic CNS show marked upregulation of the extracellular matrix (ECM) protein fibronectin, along with increased expression of its major receptor, α5β1integrin on brain endothelial cells (BEC). As cerebral hypoxia also leads to glial activation, the aim of the current study was to define the temporal relationship between BEC responses and glial cell activation in this model of cerebral hypoxia. This revealed that BEC fibronectin/α5β1 integrin expression and proliferation both reached maximal level after 4 days hypoxia. Interestingly, up to 4 days hypoxia, all dividing cells were BEC, but at later time-points proliferating astrocytes were also observed. GFAP staining revealed that hypoxia induced marked astrocyte activation that reached maximal level between 7–14 days hypoxia. As newly formed cerebral capillaries require ensheathment by astrocyte end-feet in order to acquire mature brain endothelium characteristics, we next examined how expression of astrocyte end-feet adhesion molecules is regulated by hypoxia. This showed that the astrocyte adhesion receptors α6β4 integrin and dystroglycan were both markedly upregulated, with a time-course that closely resembled astrocyte activation. Taken together, this evidence shows that cerebral hypoxia promotes first an endothelial response, in which fibronectin promotes BEC proliferation. This is then followed by an astrocyte response, involving astrocyte activation, proliferation and re-organization of astrocyte end-feet, which correlates with increased expression of astrocyte end-feet adhesion molecules.

Although mutations and variations of several genes have been identified to be involved in Alzheimer's disease (AD), the efforts towards understanding the pathogenic mechanisms of the disease still have a long journey to go. One such effort is to identify the signal transduction deficits, for which previous studies have suggested that the central problems appear to be at the interface between G proteins and their coupled receptors. G protein-coupled receptor kinases (GRKs) are a small family of serine/threonine protein kinases primarily acting at the “receptor-G protein interface””. Recent studies have indicated the possible involvement of GRK, primarily GRK2 and GRK5, dysfunction in the pathogenesis of AD. It seems that mild, soluble, β-amyloid accumulation can lead to a reduced membrane (functional) and an elevated cytosolic GRK2/5. The increased cytosolic GRK2 appears to be colocalized with damaged mitochondria and neurofibrillary tangles. Moreover, the total levels of GRK2, not only in the brain, but also in peripheral blood samples, are increased in a manner inversely correlated with the patient's cognitive levels. The deficiency of GRK5, on the other hand, impairs presynaptic M2 autoreceptor desensitization, which leads to a reduced acetylcholine release, axonal/synaptic degenerative changes, and associated amnestic, mild cognitive impairment. It also promotes an evil cycle to further increase Beta-amyloid accumulation and exaggerates brain inflammation, possibly even the basal forebrain cholinergic degeneration. Therefore, continuous efforts in this direction are necessary before translating the knowledge to any therapeutic strategies.

Recent studies suggested that G protein-coupled receptor kinase 5 (GRK5) deficiency plays a significant role in early Alzheimer's disease (AD) pathogenesis, and that the GRK5 knockout (GRK5KO) mouse displays an early Alzheimer-like cognitive deficit associated with increased hippocampal axonal defects and synaptic degenerative changes. Gender is known to play a role in AD, with females showing more extensive pathologic changes in brain compared to males. Although GRK5 deficiency is linked to AD, it is unknown whether the pathologic changes solely driven by the GRK5 deficiency are gender-dependent. To determine this, extent of the pathologic changes in aged GRK5KO mice was compared between genders. We find that female GRK5KO mice had a 2.5-fold increase in hippocampal swollen axonal clusters compared to male GRK5KO mice. Moreover, hippocampal levels of several synaptic proteins, including synaptophysin, were significantly lower in the female than the male. In addition, although increased Luteinizing hormone (LH) activity is believed to play a significant role in the gender phenomenon in AD, we found that desensitization of LH receptor is not affected by the GRK5 deficiency. Therefore, the worsened pathologic changes in the female mice cannot be attributed to an impaired LH receptor desensitization. Taken together, this study demonstrates a synergistic interaction between GRK5 deficiency and gender in promoting early AD-like pathologic changes in the female GRK5KO mice, although the underlying molecular mechanisms remain to be elucidated.

In hypoxic cells, dysfunctional mitochondria are selectively removed by a specialized autophagic process called mitophagy. The ER-mitochondrial contact site (MAM) is essential for fission of mitochondria prior to engulfment, and the outer mitochondrial membrane protein FUNDC1 interacts with LC3 to recruit autophagosomes, but the mechanisms integrating these processes are poorly understood. Here, we describe a new pathway mediating mitochondrial fission and subsequent mitophagy under hypoxic conditions. FUNDC1 accumulates at the MAM by associating with the ER membrane protein calnexin. As mitophagy proceeds, FUNDC1/calnexin association attenuates and the exposed cytosolic loop of FUNDC1 interacts with DRP1 instead. DRP1 is thereby recruited to the MAM, and mitochondrial fission then occurs. Knockdown of FUNDC1, DRP1, or calnexin prevents fission and mitophagy under hypoxic conditions. Thus, FUNDC1 integrates mitochondrial fission and mitophagy at the interface of the MAM by working in concert with DRP1 and calnexin under hypoxic conditions in mammalian cells.

Fibronectin is a critical regulator of vascular modelling, both in development and in the adult. In the hypoxic adult central nervous system (CNS), fibronectin is induced on angiogenic vessels, and endothelial cells show strong induction of the two fibronectin receptors α5β1 and αvβ3 integrins. In a previous study, we found that the αvβ3 integrin is dispensable for hypoxic-induced cerebral angiogenesis, but a role for the endothelial α5β1 integrin was suggested. To directly investigate the role of endothelial α5 integrin in cerebral angiogenesis, wild-type mice and mice lacking α5 integrin expression in endothelial cells (α5-EC-KO) were subject to hypoxia (8% O[subscript 2]) for 0, 2, 4, 7 or 14 days. Quantification of cerebral vessel density and endothelial-specific proteins claudin-5 and Glut-1 revealed that α5-EC-KO mice displayed an attenuated angiogenic response, which correlated with delayed endothelial proliferation. α5-EC-KO mice showed no defect in the ability to organize a cerebrovascular fibronectin matrix, and no compensatory increase in vascular αvβ3 integrin expression. Consistent with these findings, primary α5KO brain endothelial cells (BEC) in culture exhibited delayed growth and proliferation. Taken together, these studies demonstrate an important angiogenic role for the α5β1 integrin in promoting BEC proliferation in response to cerebral hypoxia., National Institutes of Health (U.S.) (PO1-HL66105), National Institute of General Medical Sciences (U.S.). Cell Migration Consortium (GC11451.126452)

Following focal cerebral ischemia, blood vessels in the ischemic border, or penumbra, launch an angiogenic response. In light of the critical role for fibronectin in angiogenesis, and the observation that fibronectin and its integrin receptors are strongly upregulated on angiogenic vessels in the hypoxic CNS, the aim of this study was to establish whether angiogenic vessels in the ischemic CNS also show this response. Focal cerebral ischemia was established in C57/Bl6 mice by middle cerebral artery occlusion (MCA:O), and brain tissue analyzed seven days following re-perfusion, a time at which angiogenesis is ongoing. Within the ischemic core, immunofluorescent (IF) studies demonstrated vascular expression of MECA-32, a marker of leaky cerebral vessels, and vascular breakdown, defined by loss of staining for the endothelial marker, CD31, and the vascular adhesion molecules, laminin, dystroglycan and α6 integrin. Within the ischemic penumbra, dual-IF with CD31 and Ki67 revealed the presence of proliferating endothelial cells, indicating ongoing angiogenesis. Significantly, vessels in the ischemic penumbra showed strong upregulation of fibronectin and the fibronectin receptors, α5β1 and αvβ3 integrins. Taken together with our recent finding that the α5β1 integrin plays an important role in promoting cerebral angiogenesis in response to hypoxia, these results suggest that stimulation of the fibronectin-α5β1 integrin signalling pathway may provide a novel approach to amplifying the intrinsic angiogenic response to cerebral ischemia.

Background Studies of cerebral ischemia and other neuroinflammatory states have demonstrated a strong association between new vessel formation and microglial recruitment and activation, raising the possibility that microglia may be involved in promoting angiogenesis. As endothelial cell proliferation is a fundamental early step in angiogenesis, the aim of this study was to test this hypothesis by examining the influence of microglial secreted factors on brain endothelial cell (BEC) proliferation using BrdU incorporation. Methods Primary cultures of mouse BEC, microglia and astrocytes were used in this study. Proliferation of BEC was examined by BrdU incorporation. ELISA was used to quantify TNF and TGF-β1 levels within cell culture supernatants. Results Microglia regulated BEC proliferation in a biphasic manner; microglia conditioned medium (MG-CM) from resting microglia inhibited, while that from activated microglia promoted BEC proliferation. A screen of microglial cytokines revealed that BEC proliferation was inhibited by TGF-β1, but promoted by TNF. ELISA showed that TNF and TGF-β1 were both present in MG-CM, and that while TGF-β1 dominated in resting MG-CM, TNF levels were massively increased in activated MG-CM, shifting the balance in favor of TNF. Antibody-blocking studies revealed that the influence of MG-CM to inhibit or promote BEC proliferation was largely attributable to the cytokines TGF-β1 and TNF, respectively. Conclusion This data suggests that microglial activation state might be an important determinant of cerebral angiogenesis; inhibiting BEC proliferation and neovascularization in the normal central nervous system (CNS), but stimulating the growth of new capillaries under neuroinflammatory conditions.

Cerebral angiogenesis is an important adaptive response to hypoxia. As the alpha v beta 3 integrin is induced on angiogenic vessels in the ischemic central nervous system (CNS), and the suggested angiogenic role for this integrin in other systems, it is important to determine whether the alpha v beta 3 integrin is an important mediator of cerebral angiogenesis. alpha v beta 3 integrin expression was examined in a model of cerebral hypoxia, in which mice were subject to hypoxia (8% O(2)) for 0, 4, 7, or 14 days. Immunofluorescence and western blot analysis revealed that in the hypoxic CNS, alpha v beta 3 integrin was strongly induced on angiogenic brain endothelial cells (BEC), along with its ligand vitronectin. In the hypoxia model, beta 3 integrin-null mice showed no obvious defect in cerebral angiogenesis. However, early in the angiogenic process, BEC in these mice showed an increased mitotic index that correlated closely with increased alpha 5 integrin expression. In vitro experiments confirmed alpha 5 integrin upregulation on beta 3 integrin-null BEC, which also correlated with increased BEC proliferation on fibronectin. These studies confirm hypoxic induction of alpha v beta 3 integrin on angiogenic vessels, but suggest distinct roles for the BEC integrins alpha v beta 3 and alpha 5 beta 1 in cerebral angiogenesis, with alpha v beta 3 having a nonessential role, and alpha 5 beta 1 promoting BEC proliferation.

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.

Aims Hippocampal cholinergic hypofunction is known to be involved in the cognitive deficits of Alzheimer's disease, but the detailed mechanisms remain to be elucidated. In order to establish an in vitro hippocampal cholinergic neuronal model for the relevant mechanistic studies, we have characterized a widely used hippocampal neuronal cell line, HT22, a sub-line derived from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture. Main methods Western blot and immunocytochemistry were used to examine expression of cholinergic markers in HT22 cells. High potassium-evoked [3H]ACh release was used to evaluate the cholinergic functional properties of the cells. Key findings We found that HT22 cells express essential cholinergic markers, such as the high affinity choline transporter, choline acetyltransferase, vesicular acetylcholine transporter, and muscarinic acetylcholine receptors. Exposure of HT22 cells to high potassium evoked [3H]ACh release in a dose-dependent manner. In addition, the [3H]ACh release was significantly potentiated when presynaptic autoreceptors were blocked. Significance Our results suggest that HT22 cells possess functional cholinergic properties, and can be used for an in vitro model for defining the mechanisms in cognitive deficits of Alzheimer's disease.

Background Deficiency of membrane G-protein coupled receptor (GPCR) kinase-5 (GRK5) recently has been linked to early AD pathogenesis, and has been suggested to contribute to augmented microglial activation in vitro by sensitizing relevant GPCRs. However, GRK5 deficient mice did not show any signs of microgliosis, except for their moderate increase in axonal defects and synaptic degenerative changes during aging. We have speculated that one possible reason for the absence of microgliosis in these animals might be due to lack of an active inflammatory process involving activated GPCR signaling, since GRKs only act on activated GPCRs. The objective of this study was to determine whether the microgliosis is exaggerated in TgAPPsw (Tg2576) mice also deficient in GRK5, in which fibrillar β-amyloid (Aβ) and an active inflammatory process involving activated GPCR signaling are present. Methods Both quantitative and qualitative immunochemistry methods were used to evaluate the microgliosis and astrogliosis in these animals. Results We found that inactivation of one copy of the GRK5 gene in the TgAPPsw mice resulted in approximately doubled extent of microgliosis, along with significantly exaggerated astrogliosis, in both hippocampus and cortex of the aged animals. Consistent with previous observations, the activated microglia were located primarily near or surrounding the fibrillar Aβ deposits. Conclusion The results demonstrate that GRK5 deficiency in vivo significantly exaggerates microgliosis and astrogliosis in the presence of an inflammatory initiator, such as the excess fibrillar Aβ and the subsequent active inflammatory reactions in the TgAPPsw mice.