Best practices for enhancing surgical research: a perspective from the Canadian Association of Chairs of Surgical Research

Summary: The Canadian Association of Chairs of Surgical Research was created in 2014, with representation from every departmental surgical research committee across Canada, to establish Canadian surgical research as a beacon for health care innovation and to propose solutions for the daily challenges facing surgeon-researchers. Our key mandate has been to identify challenges for surgeons and scientists performing research to prevent further erosion of this vital area of activity that benefits patients, health care service providers and Canadian society. This article outlines the findings of a nationwide survey sent to all members of departments of surgery across Canada, seeking input on current threats and potential solutions. The results suggest that surgical research in Canada is experiencing a decline in funding and an increase in challenges affecting research productivity of academic surgeons, such as pressures to be clinically active, unpredictable surgical schedules, growing administrative demands, and increasing complexity of patient populations. Although surgeons are productive in their research endeavours, institutional changes and sharing of best practices are needed to ensure sustainable growth of research programs.

Neutrophil Infiltration and Matrix Metalloproteinase-9 in Lacunar Infarction.

We use the modified pial vessel disruption rat model to elucidate the cellular and molecular mechanisms of cavitation as it plays a role in lacunar infarction. Here we discuss the similarities between the genesis of pulmonary cavitation in various animal models and lacunar infarction in the cerebral cortex of rats. Both pathological processes involve the creation of a cavity surrounded by fibroblasts or reactive astrocytes. A crucial step in both, the lung and the cerebral cortex, appears to be the migration of neutrophils across the endothelial barrier into the parenchyma. In the lung and cerebral cortex this involves release of matrix metalloproteinase-9 (MMP-9). Inside the parenchyma neutrophils continue to release MMP-9. In both situations batimastat (BB-94) and minocycline reduce release of MMP-9 and prevent cavitation. In the cerebral cortex MMP-9 release by resident microglia plays an additional role. We therefore advance the hypothesis that cavitation in both tissues is driven by MMP-9 originating from invading neutrophils. Therapeutic intervention has to focus on these blood-borne intruder cells and specific MMP actions. Batimastat and its derivatives (marimastat, BB-1101, mCGS-27023-A, ilomastat, GM6001, CTK8G1150) are already in clinical or experimental use in humans for anti-cancer treatment, and these clinically relevant drugs could be repurposed to act as anti-inflammatory to counter neutrophil contribution to lung or cerebral cortex cavitation.

Endothelial LSP1 Modulates Extravascular Neutrophil Chemotaxis by Regulating Nonhematopoietic Vascular PECAM-1 Expression.

During inflammation, leukocyte-endothelial cell interactions generate molecular signals that regulate cell functions. The Ca(2+)- and F-actin-binding leukocyte-specific protein 1 (LSP1) expressed in leukocytes and nonhematopoietic endothelial cells is pivotal in regulating microvascular permeability and leukocyte recruitment. However, cell-specific function of LSP1 during leukocyte recruitment remains elusive. Using intravital microscopy of cremasteric microvasculature of chimeric LSP1-deficient mice, we show that not neutrophil but endothelial LSP1 regulates neutrophil transendothelial migration and extravascular directionality without affecting the speed of neutrophil migration in tissue in response to CXCL2 chemokine gradient. The expression of PECAM-1-sensitive α6ß1 integrins on the surface of transmigrated neutrophils was blunted in mice deficient in endothelial LSP1. Functional blocking studies in vivo and in vitro elucidated that α6ß1 integrins orchestrated extravascular directionality but not the speed of neutrophil migration. In LSP1-deficient mice, PECAM-1 expression was reduced in endothelial cells, but not in neutrophils. Similarly, LSP1-targeted small interfering RNA silencing in murine endothelial cells mitigated mRNA and protein expression of PECAM-1, but not ICAM-1 or VCAM-1. Overexpression of LSP1 in endothelial cells upregulated PECAM-1 expression. Furthermore, the expression of transcription factor GATA-2 that regulates endothelial PECAM-1 expression was blunted in LSP1-deficient or LSP1-silenced endothelial cells. The present study unravels endothelial LSP1 as a novel cell-specific regulator of integrin α6ß1-dependent neutrophil extravascular chemotactic function in vivo, effective through GATA-2-dependent transcriptional regulation of endothelial PECAM-1 expression.

Adenosine A1 and A2A Receptors in the Brain: Current Research and Their Role in Neurodegeneration.

The inhibitory adenosine A1 receptor (A1R) and excitatory A2A receptor (A2AR) are predominantly expressed in the brain. Whereas the A2AR has been implicated in normal aging and enhancing neurotoxicity in multiple neurodegenerative diseases, the inhibitory A1R has traditionally been ascribed to have a neuroprotective function in various brain insults. This review provides a summary of the emerging role of prolonged A1R signaling and its potential cross-talk with A2AR in the cellular basis for increased neurotoxicity in neurodegenerative disorders. This A1R signaling enhances A2AR-mediated neurodegeneration, and provides a platform for future development of neuroprotective agents in stroke, Parkinson's disease and epilepsy.

Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus.

Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7-12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains.

Prolonged adenosine A1 receptor activation in hypoxia and pial vessel disruption focal cortical ischemia facilitates clathrin-mediated AMPA receptor endocytosis and long-lasting synaptic inhibition in rat hippocampal CA3-CA1 synapses: differential regulation of GluA2 and GluA1 subunits by p38 MAPK and JNK.

Activation of presynaptic adenosine A1 receptors (A1Rs) causes substantial synaptic depression during hypoxia/cerebral ischemia, but postsynaptic actions of A1Rs are less clear. We found that A1Rs and GluA2-containing AMPA receptors (AMPARs) form stable protein complexes from hippocampal brain homogenates and cultured hippocampal neurons from Sprague Dawley rats. In contrast, adenosine A2A receptors (A2ARs) did not coprecipitate or colocalize with GluA2-containing AMPARs. Prolonged stimulation of A1Rs with the agonist N(6)-cyclopentyladenosine (CPA) caused adenosine-induced persistent synaptic depression (APSD) in hippocampal brain slices, and APSD levels were blunted by inhibiting clathrin-mediated endocytosis of GluA2 subunits with the Tat-GluA2-3Y peptide. Using biotinylation and membrane fractionation assays, prolonged CPA incubation showed significant depletion of GluA2/GluA1 surface expression from hippocampal brain slices and cultured neurons. Tat-GluA2-3Y peptide or dynamin inhibitor Dynasore prevented CPA-induced GluA2/GluA1 internalization. Confocal imaging analysis confirmed that functional A1Rs, but not A2ARs, are required for clathrin-mediated AMPAR endocytosis in hippocampal neurons. Pharmacological inhibitors or shRNA knockdown of p38 MAPK and JNK prevented A1R-mediated internalization of GluA2 but not GluA1 subunits. Tat-GluA2-3Y peptide or A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine also prevented hypoxia-mediated GluA2/GluA1 internalization. Finally, in a pial vessel disruption cortical stroke model, a unilateral cortical lesion compared with sham surgery reduced hippocampal GluA2, GluA1, and A1R surface expression and also caused synaptic depression in hippocampal slices that was consistent with AMPAR downregulation and decreased probability of transmitter release. Together, these results indicate a previously unknown mechanism for A1R-induced persistent synaptic depression involving clathrin-mediated GluA2 and GluA1 internalization that leads to hippocampal neurodegeneration after hypoxia/cerebral ischemia.

Regulation of methylglyoxal-elicited leukocyte recruitment by endothelial SGK1/GSK3 signaling.

Excessive levels of the glycolysis metabolite methylglyoxal (MG) elicit enhanced expression of adhesion molecules which foster leukocyte-endothelial cell interactions. The signaling mechanisms involved remain elusive. To address this, we investigated the signal transduction of leukocyte- and endothelial-expressed phosphoinositide 3-kinase (PI3K) effector kinases glycogen synthase kinase 3 (GSK3) and serum- and glucocorticoid-inducible kinase 1 (SGK1) in the regulation of MG-elicited leukocyte recruitment. Using intravital microscopy of mouse cremasteric microvasculature, we demonstrate that GSK3 inhibitors lithium and SB216763 mitigate MG-elicited leukocyte recruitment and microvascular hyperpermeability. In SVEC4-10EE2 endothelial cells, but not in neutrophils, MG transiently activates GSK3 by reducing inhibitory phospho-GSK3α/ß (Ser21/9) which parallels decrease of phospho-Akt at early time points (<30min). At later time points (≥1h), MG induces GSK3 deactivation which is dissipated by siRNA silencing of SGK. MG treatment potentiates endothelial SGK1 mRNA, total SGK1, phospho-SGK1 and phospho-NDRG1. The SGK1 inhibitor GSK650394 attenuates MG-elicited leukocyte recruitment. Pharmacological inhibition or silencing endothelial GSK3 or SGK attenuates MG-triggered nuclear factor (NF)-κB activity. Furthermore, silencing SGK blunts MG-triggered redox-sensitive phosphorylation of endothelial transcription factor CREB. Inhibition of SGK1 or GSK3 mitigates the expression of endothelial adhesion molecules P- and E-selectins and ICAM-1. Moreover, SGK1-dependent CREB activation participates in MG-elicited ICAM-1 upregulation. We conclude that temporal activation of endothelial SGK1 and GSK3 is decisive in MG-elicited upregulation of transcription factors, adhesion molecule expression, and leukocyte-vascular endothelium interactions. This novel signaling pathway may link excessive MG levels in vivo to inflammation, thus, unraveling potential therapeutic targets.

The effects of miR-467b on lipoprotein lipase (LPL) expression, pro-inflammatory cytokine, lipid levels and atherosclerotic lesions in apolipoprotein E knockout mice.

Atherosclerosis is a lipid disorder disease characterized by chronic blood vessel wall inflammation driven by the subendothelial accumulation of macrophages. Studies have shown that lipoprotein lipase (LPL) participates in lipid metabolism, but it is not yet known whether post-transcriptional regulation of LPL gene expression by microRNAs (miRNAs) occurs in vivo. Here, we tested that miR-467b provides protection against atherosclerosis by regulating the target gene LPL which leads to reductions in LPL expression, lipid accumulation, progression of atherosclerosis and production of inflammatory cytokines in apolipoprotein E knockout (apoE(-/-)) mice. Treatment of apoE(-/-) mice with intra-peritoneal injection of miR-467b agomir led to decreased blood plasma levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1ß and monocyte chemotactic protein-1 (MCP-1). Using Western blots and real time PCR, we determined that LPL expression in aorta and abdominal cavity macrophages were significantly down-regulated in the miR-467b agomir group. Furthermore, systemic treatment with miR-467b antagomir accelerated the progression of atherosclerosis in the aorta of apoE(-/-) mice. The present study showed that miR-467b protects apoE(-/-) mice from atherosclerosis by reducing lipid accumulation and inflammatory cytokine secretion via downregulation of LPL expression. Therefore, targeting miR-467b may offer a promising strategy to treat atherosclerotic vascular disease.

Growth differentiation factor-15 induces expression of ATP-binding cassette transporter A1 through PI3-K/PKCζ/SP1 pathway in THP-1 macrophages.

OBJECTIVE: The aim of this study was to determine whether ATP-binding cassette transporter A1 (ABCA1) was up-regulated by growth differentiation factor-15 (GDF-15) via the phosphoinositide 3-kinase (PI3K)/protein kinase Cζ (PKCζ)/specificity protein 1 (SP1) pathway in THP-1 macrophages. METHODS AND RESULTS: We investigated the effects of different concentrations of GDF-15 on ABCA1 expression in THP-1 macrophages. The results showed that GDF-15 dramatically increased cholesterol efflux and decreased cellular cholesterol levels. In addition, GDF15 increased ABCA1 mRNA and protein levels. The effects of GDF-15 on ABCA1 protein expression and cellular cholesterol efflux were abolished by wither inhibition or depletion of PI3K, PKCζ and SP1, respectively, suggesting the potential roles of PI3K, PKCζ and SP1 in ABCA1 expression. Taken together, GDF-15 appears to activate PI3K, PKCζ and SP1 cascade, and then increase ABCA1 expression, thereby promoting cholesterol efflux and reducing foam cell formation. CONCLUSION: Our results suggest that GDF-15 has an overall protective effect on the progression of atherosclerosis, likely through inducing ABCA1 expression via the PI3K/PKCζ/SP1 signaling pathway and enhancing cholesterol efflux.

Endothelial Na+/H+ exchanger NHE1 participates in redox-sensitive leukocyte recruitment triggered by methylglyoxal.

BACKGROUND: Excessive levels of methylglyoxal (MG) encountered in diabetes foster enhanced leukocyte-endothelial cell interactions, mechanisms of which are incompletely understood. MG genomically upregulates endothelial serum- and glucocorticoid-inducible kinase 1 (SGK1) which orchestrates leukocyte recruitment by regulating the activation and expression of transcription factors and adhesion molecules. SGK1 regulates a myriad of ion channels and carriers including the Na+/H+ exchanger NHE1. Here, we explored the effect of MG on SGK1-dependent NHE1 activation and the putative role of NHE1 activation in MG-induced leukocyte recruitment and microvascular hyperpermeability. METHODS: Using RT-PCR and immunoblotting, we analyzed NHE1 mRNA and protein levels in murine microvascular SVEC4-10EE2 endothelial cells (EE2 ECs). NHE1 phosphorylation was detected using a specific antibody against the 14-3-3 binding motif at phospho-Ser703. SGK in EE2 ECs was silenced using targeted siRNA. ROS production was determined using DCF-dependent fluorescence. Leukocyte recruitment and microvascular permeability in murine cremasteric microvasculature were measured using intravital microscopy. The expression of endothelial adhesion molecules was determined by immunoblotting and confocal imaging analysis. RESULTS: MG treatment significantly upregulated NHE1 mRNA and dose-dependently increased total- and phospho-NHE1. Treatment with SGK1 inhibitor GSK650394, antioxidant Tempol and silencing SGK all blunted MG-triggered phospho-NHE1 upregulation in EE2 ECs. NHE1 inhibitor cariporide attenuated MG-triggered ROS production, leukocyte adhesion and emigration and microvascular hyperpermeability, without affecting leukocyte rolling. Cariporide treatment did not alter MG-triggered upregulation of P- and E-selectins, but reduced endothelial ICAM-1 expression. CONCLUSION: MG elicits SGK1-dependent activation of endothelial Na+/H+ exchanger NHE1 which participates in MG-induced ROS production, upregulation of endothelial ICAM-1, leukocyte recruitment and microvascular hyperpermeability. Pharmacological inhibition of NHE1 attenuates the proinflammatory effects of excessive MG and may, thus, be beneficial in diabetes-associated inflammation.

Involvement of matrix metalloproteinases-2 and -9 in the formation of a lacuna-like cerebral cavity.

We used a modified pial vessel disruption (PVD) protocol with adult male Wistar rats to mimic small-vessel stroke in the cerebral cortex. Within 3 weeks, this lesion develops into a single lacuna-like cavity, which is fluid-filled and encapsulated by reactive astrocytes. Minocycline treatment that commences 1 hr after lesion and continues for 6 days prevents the cavitation and causes a filling of the lesion with reactive astrocytes and no barrier. Here, we determined whether inhibition of matrix metalloproteinases-2 and -9 (MMPs) mediates this minocycline action. Confocal microscopy revealed increased punctate staining of MMPs inside the lesion sites after 2 days of PVD. Astrocytes lined the lesion border but showed sparse localization inside the lesion. In contrast, increased MMP levels inside the lesion coincided with increased ED1 or OX-42 immunostaining, suggesting that MMP elevation reflected increased secretions from microglia/macrophages. Imaging analyses also revealed that minocycline administered for 2 days before animal euthanasia, significantly decreased MMP levels within the lesion. Moreover, Western blot analysis of cortical tissue extracts showed a significant 30-40% upregulation of MMPs 2 days after lesion. Minocycline administered 2 hr before the lesion significantly inhibited both MMP-9 and MMP-2 levels by ∼40%. In contrast, minocycline administered 1 hr after the lesion only decreased MMP-9 levels by ∼30%. Because MMP inhibition with batimastat injection also prevented cavity formation at 21 days, we conclude that minocycline prevented the creation of a lacuna-like cyst in the cerebral cortex by inhibiting the MMP secretion from microglia in the affected tissue.

Adenosine A1 receptor ligands bind to α-synuclein: implications for α-synuclein misfolding and α-synucleinopathy in Parkinson's disease.

BACKGROUND: Accumulating α-synuclein (α-syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson's disease (PD). Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) could directly interact with α-syn to modulate α-syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN). METHODS: Nanopore analysis and molecular docking were used to test the binding properties of CPA and DPCPX with α-syn in vitro. Sprague-Dawley rats were administered with 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-syn aggregation and neurodegeneration were examined in the SN pars compacta and hippocampal regions using confocal imaging and Western blotting. RESULTS: Using nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-syn, similar to 2-aminoindan, which is expected to promote a "knot" conformation and α-syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-syn, similar to 1-aminoindan, which is expected to promote a "loop" conformation that prevents α-syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Confocal imaging and Western blot analyses revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-syn expression/aggregation and neurodegeneration in both SN pars compacta and hippocampus. In contrast, DPCPX and 1-aminoindan attenuated the CPA-induced α-syn expression/aggregation and neurodegeneration in SN and hippocampus. CONCLUSIONS: The results indicate that A1R agonists and drugs promoting a "knot" conformation of α-syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a "loop" conformation of α-syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in PD.

Adenosine Signaling and Clathrin-Mediated Endocytosis of Glutamate AMPA Receptors in Delayed Hypoxic Injury in Rat Hippocampus: Role of Casein Kinase 2.

Chronic adenosine A1R stimulation in hypoxia leads to persistent hippocampal synaptic depression, while unopposed adenosine A2AR receptor stimulation during hypoxia/reperfusion triggers adenosine-induced post-hypoxia synaptic potentiation (APSP) and increased neuronal death. Still, the mechanisms responsible for this adenosine-mediated neuronal damage following hypoxia need to be fully elucidated. We tested the hypothesis that A1R and A2AR regulation by protein kinase casein kinase 2 (CK2) and clathrin-dependent endocytosis of AMPARs both contribute to APSPs and neuronal damage. The APSPs following a 20-min hypoxia recorded from CA1 layer of rat hippocampal slices were abolished by A1R and A2AR antagonists and by broad-spectrum AMPAR antagonists. The inhibitor of GluA2 clathrin-mediated endocytosis Tat-GluA2-3Y peptide and the dynamin-dependent endocytosis inhibitor dynasore both significantly inhibited APSPs. The CK2 antagonist DRB also inhibited APSPs and, like hypoxic treatment, caused opposite regulation of A1R and A2AR surface expression. APSPs were abolished when calcium-permeable AMPAR (CP-AMPAR) antagonist (IEM or philanthotoxin) or non-competitive AMPAR antagonist perampanel was applied 5 min after hypoxia. In contrast, perampanel, but not CP-AMPAR antagonists, abolished APSPs when applied during hypoxia/reperfusion. To test for neuronal viability after hypoxia, propidium iodide staining revealed significant neuroprotection of hippocampal CA1 pyramidal neurons when pretreated with Tat-GluA2-3Y peptide, CK2 inhibitors, dynamin inhibitor, CP-AMPAR antagonists (applied 5 min after hypoxia), and perampanel (either at 5 min hypoxia onset or during APSP). These results suggest that the A1R-CK2-A2AR signaling pathway in hypoxia/reperfusion injury model mediates increased hippocampal synaptic transmission and neuronal damage via calcium-permeable AMPARs that can be targeted by perampanel for neuroprotective stroke therapy.

HERG channel and cancer: A mechanistic review of carcinogenic processes and therapeutic potential.

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.

Long-term adenosine A1 receptor activation-induced sortilin expression promotes α-synuclein upregulation in dopaminergic neurons.

Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases. However, the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear. In this study, rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) for five weeks. The mobility of rats was evaluated by forced swimming test, while their cognitive capabilities were evaluated by Y-maze test. Expression of sortilin, α-synuclein, p-JUN, and c-JUN proteins in the substantia nigra were detected by western blot analysis. In addition, immunofluorescence staining of sortilin and α-synuclein was performed to detect expression in the substantia nigra. The results showed that, compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (5 mg/kg) + CPA co-treated rats, motor and memory abilities were reduced, surface expression of sortin and α-synuclein in dopaminergic neurons was reduced, and total sortilin and total α-synuclein were increased in CPA-treated rats. MN9D cells were incubated with 500 nM CPA alone or in combination with 10 µM SP600125 (JNK inhibitor) for 48 hours. Quantitative real-time polymerase chain reaction analysis of sortilin and α-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone, but the combination of CPA and SP600125 could inhibit this expression. Predictions made using Jasper, PROMO, and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents. A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction. After sortilin expression was inhibited by sh-SORT1, expression of p-JUN and c-JUN was detected by western blot analysis. Long-term adenosine A1 receptor activation levels upregulated α-synuclein expression at the post-transcriptional level by affecting sortilin expression. The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind to α-synuclein. Co-immunoprecipitation revealed an interaction between sortilin and α-synuclein in MN9D cells. Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression and α-synuclein accumulation, and dramatically improved host cognition and kineticism. This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan (approval No. AUP#20070090) in March 2007 and the Animals Ethics Committee of University of South China (approval No. LL0387-USC) in June 2017.

The Concept of an Epilepsy Brain Bank.

Epilepsy comprises more than 40 clinical syndromes affecting millions of patients and families worldwide. To decode the molecular and pathological framework of epilepsy researchers, need reliable human epilepsy and control brain samples. Brain bank organizations collecting and supplying well-documented clinically and pathophysiologically tissue specimens are important for high-quality neurophysiology and neuropharmacology studies for epilepsy and other neurological diseases. New development in molecular mechanism and new treatment methods for neurological disorders have evoked increased demands for human brain tissue. An epilepsy brain bank is a storage source for both the frozen samples as well as the formaldehyde fixed paraffin embedded (FFPE) tissue from epilepsy surgery resections. In 2014, the University of Saskatchewan have started collecting human epilepsy brain tissues for the first time in Canada. This review highlights the necessity and importance of Epilepsy Brain bank that provides unique access for research to valuable source of brain tissue and blood samples from epilepsy patients.

In situ co-distribution and functional interactions of SAP97 with sinoatrial isoforms of HCN channels.

The sinoatrial node is a region of specialized cardiomyocytes that is responsible for the repetitive activity of the adult heart. The sinoatrial node is heavily innervated compared to the other regions of the heart, and the specialized cardiomyocytes of this region receive neural and hormonal input from the autonomic nervous system, which leads to changes in heart rate. A key regulator of sinoatrial beating frequency in response to autonomic input is the hyperpolarization-activated cyclic nucleotide gated (HCN) channel, a mixed cationic channel whose activity is increased by the binding of cAMP to its cytoplasmic side. HCN channels localize to distinct regions or "hot spots" on the cell surface of sinoatrial myocytes, but how these regions are formed, whether they correspond to specific signaling domains and the specific HCN isoforms and other proteins therein are not known. In this paper, we show that both HCN2 and HCN4 isoforms co-distribute with the adapter protein SAP97, an important component of distinct punctae in the sinoatrial node of the rabbit heart. HCN4, but not HCN2, also co-distributes with the post-synaptic marker beta-catenin, thus identifying diverse organized domains within this tissue. Furthermore, we show, using heterologous expression systems, whole-cell patch clamp electrophysiology and imaging, that SAP97 interacts functionally with HCN in a manner that depends upon the PDZ compatible binding motif of the C-terminus, but that its effects on I(f) behaviour are HCN isoform and context dependent. Together, the data suggest that SAP97 contributes to isoform specific organization of HCN channels within specific domains in the sinoatrial node of the rabbit.

Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

Mitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.

p38 mitogen-activated protein kinase contributes to adenosine A1 receptor-mediated synaptic depression in area CA1 of the rat hippocampus.

Adenosine is arguably the most potent and widespread presynaptic modulator in the CNS, yet adenosine receptor signal transduction pathways remain unresolved. Here, we demonstrate a novel mechanism in which adenosine A1 receptor stimulation leads to p38 mitogen-activated protein kinase (MAPK) activation and contributes to the inhibition of synaptic transmission. Western blot analysis indicated that selective A1 receptor activation [with N6-cyclopentyladenosine (CPA)] resulted in rapid increases in phosphorylated p38 (phospho-p38) MAPK immunoreactivity in membrane fractions, and decreases in phospho-p38 MAPK in cytosolic fractions. Immunoprecipitation with a phospho-p38 MAPK antibody revealed constitutive association of this phosphoprotein with adenosine A1 receptors. Phospho-p38 MAPK activation by A1 receptor stimulation induced translocation of PP2a (protein phosphatase 2a) to the membrane. We then examined the actions of p38 MAPK activation in A1 receptor-mediated synaptic inhibition. Excitatory postsynaptic field potentials evoked in area CA1 of the rat hippocampus markedly decreased in response to adenosine (10 microM), the A1 receptor agonist CPA (40 nM), or a 5 min exposure to hypoxia. These inhibitory responses were mediated by A1 receptor activation because the selective antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (100 nM) prevented them. In agreement with the biochemical analysis, the selective p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] (25 microM) blocked the inhibitory actions of A1 receptor activation, whereas both the inactive analog SB202474 [4-ethyl-2-(p-methoxyphenyl)-5-(4'-pyridyl)-1H-imidazole] (25 microM) and the ERK 1/2 (extracellular signal-regulated kinase 1/2) MAPK inhibitor PD98059 [2'-amino-3'-methoxyflavone] (50 microM) were ineffective. In contrast, the p38 MAPK inhibitors did not inhibit GABA(B)-mediated synaptic depression. These data suggest A1 receptor-mediated p38 MAPK activation is a crucial step underlying the presynaptic inhibitory effect of adenosine on CA3-CA1 synaptic transmission.

C-Jun N-terminal kinase regulates adenosine A1 receptor-mediated synaptic depression in the rat hippocampus.

Adenosine A1 receptors are ubiquitous mediators of presynaptic inhibition of neurotransmission in the central nervous system, yet the signalling pathway linking A1 receptor activation and decreased neurotransmitter release remains poorly resolved. We tested the contribution of c-Jun N-terminal kinase (JNK) to adenosine A1 receptor-mediated depression of field excitatory postsynaptic potentials (fEPSPs) in area CA1 of the rat hippocampus. We found that inhibition of JNK with SP600125 or JNK inhibitor V, but not an inactive analogue, attenuated the depression of fEPSPs induced by adenosine, hypoxia, and the A1 receptor agonist N(6)-cyclopentyladenosine (CPA). In contrast, the JNK inhibitor SP600125 did not inhibit GABA(B)-mediated synaptic depression. In support of our electrophysiological findings, Western blot analysis showed that A1 receptor stimulation resulted in a transient increase in JNK phosphorylation in the membrane fraction of hippocampal lysates. The total amount of JNK in the membrane fraction was unchanged by CPA treatment. The increase in phosphorylated JNK induced by A1 receptor stimulation was blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), indicating that A1 receptors specifically activate JNK in the hippocampus. Together with functional data indicating that JNK inhibition decreased CPA-induced paired pulse facilitation, these results suggest that JNK activation is necessary for adenosine A1 receptor-mediated synaptic depression occurring at a presynaptic locus The adenosine A1 receptor-JNK signalling pathway may represent a novel mechanism underlying inhibition of neurotransmitter release in the CNS.