Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator mediates neuronal detection and adaptation to metabolic stress

doi: 10.1038/jcbfm.2013.124

Figure Lengend Snippet: Glucose deprivation (GD) induces the release of tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons. (A) Representative micrograph of wild-type (Wt) cerebral cortical neurons maintained during 5 minutes under 25 mmol/L (+G) or 0 mmol/L (−G) of glucose and stained with antibodies against the postsynaptic dendritic marker anti-microtubule associated protein (MAP-2) (blue) and tPA (red). Green corresponds to DNA (Hoechst). Magnification × 20 in (a, e) and × 60 in (b–d) and (f–h). (B) Representative micrograph of Wt cerebral cortical neurons maintained during 5 minutes under GD and stained with antibodies against MAP-2 (blue), tPA (red), and synaptophysin (green). Arrows denote the presence of tPA-containing synaptophysin-positive presynaptic vesicles in direct contact with the postsynaptic, MAP-2-positive, dendrite. Magnification × 60. (C) Mean concentration of tPA in the culture medium of Wt cerebral cortical neurons exposed during 0 to 5 minutes to either oxygen deprivation (OD, black circles), or GD (black triangles), or oxygen and glucose deprivation (OGD; black squares), or kept under normal glucose and oxygen concentrations after medium change (baseline; white diamonds). Lines denote s.d. n=10 per condition; *P<0.05 compared with GD and OD; ^P<0.05 compared with OD and OGD; §P<0.05 compared with sister cultures maintained under physiologic conditions and with cultures exposed to either GD or OGD conditions.

Article Snippet: Recombinant murine tPA, proteolytically inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A), human Lys plasmin, an ELISA kit that detects active tPA, and sheep anti-tPA antibodies (Cat # SASMTPA) were acquired from Molecular Innovations (Novi, MI, USA).

Techniques: Staining, Marker, Concentration Assay

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Tissue-type plasminogen activator mediates neuronal detection and adaptation to metabolic stress

doi: 10.1038/jcbfm.2013.124

Figure Lengend Snippet: Tissue-type plasminogen activator (tPA) induces adenosine monophosphate-activated protein kinase (AMPK) activation in the postsynaptic terminal via N-methyl-D-aspartate receptors (NMDARs) activation. (A) Representative western blot analysis of pAMPK expression in non-ischemic wild-type (Wt) cerebral cortical synaptoneurosomes after 5 minutes of incubation with 5 nmol/L of tPA (+) or vehicle (control; −). (B) Representative microphotograph of Wt cerebral cortical neurons incubated during 5 minutes with vehicle (control; a, c, e, and g) or 5 nmol/L of tPA (b, d, f, and h), and stained with antibodies against the dendritic marker anti-microtubule associated protein (MAP-2) (red) and pAMPK (green). Magnification × 40 in (a, b) and × 60 in (c–h). (C) Representative microphotograph of Wt cerebral cortical neurons incubated 5 minutes with 5 nmol/L of tPA and stained with antibodies against synaptophysin (red) and pAMPK (green). Arrows denote examples where presynaptic synaptophysin-positive vesicles are in juxtaposition with postsynaptic pAMPK. Magnification × 100. (D) Representative western blot analysis of pAMPK expression in cerebral cortical synaptoneurosomes prepared from the cerebral cortex of Wt mice after transient middle cerebral artery occlusion (tMCAO) and treatment with either 0.9 mg/kg/IV of recombinant tPA (rtPA) or a comparable volume of saline solution. (E) Representative western blot analysis of the expression NR2A and NR2B subunits of NMDARs in synaptoneurosomes prepared from the cerebral cortex of Wt mice. (F) Representative western blot analysis of pAMPK expression in cerebral cortical synaptoneurosomes prepared from the cerebral cortex of Wt mice 5 minutes after tMCAO and treatment with 0.9 mg/kg/IV of rtPA alone or in combination with 2 μg of MK-801.

Article Snippet: Recombinant murine tPA, proteolytically inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A), human Lys plasmin, an ELISA kit that detects active tPA, and sheep anti-tPA antibodies (Cat # SASMTPA) were acquired from Molecular Innovations (Novi, MI, USA).

Techniques: Activation Assay, Western Blot, Expressing, Incubation, Staining, Marker, Recombinant

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: Inhibitors of proBDNF processing are altered after pilocarpine SE. Representative Western blots of whole hippocampal protein homogenates from WT mice killed 3 h (left panels) and 24 h (right panels) after the induction of SE or time-matched saline controls. Densitometry analysis of abundance of different inhibitor proteins normalized to actin and expressed as the percentage change relative to mean values of the control group (±SEM). A–H , Anti-A2AP (1:2000; A , B ); anti-neuroserpin (1:2000; C , D ); anti-TIMP-1 (1:1000; E , F ); and anti-PAI-1 (1:1000; G , H ). The sample size for 3 h is N = 5 in each group, and for 24 h it is N = 4 in each group. ** p < 0.01, *** p < 0.001; t test.

Article Snippet: The following antibodies and concentrations were used: mouse monoclonal HA.11 clone 16B12 antibody (1:3000; MMS-101P, Covance), mouse monoclonal proBDNF antibody (1:1000; H10001G-MA, GeneCopoeia), rabbit polyclonal to α-2 antiplasmin (1:2000; ab62771, Abcam), rabbit polyclonal furin antibody (1:1000; sc-20801, Santa Cruz Biotechnology), rabbit polyclonal MMP-9 antibody (1:2000; AB13458, Millipore), sheep polyclonal neuroserpin antibody (1:2000; SASMNSP-GF-HT, Molecular Innovations), rabbit polyclonal PAI-1 antibody (1:1000; ASMPAI-GF-HT, Molecular Innovations), rabbit polyclonal plasminogen antibody (1:3000; ASMPLG-GF-HT, Molecular Innovations), sheep polyclonal tPA antibody (1:500; SASTPA-GF-HT, Molecular Innovations), and rabbit polyclonal TIMP-1 antibody (1:1000; AB770, Millipore).

Techniques: Western Blot

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: Schematic representation of different proteins involved in the cleavage of BDNF through extracellular (left panels) and intracellular (right panels) mechanisms. ProBDNF can be cleaved intracellularly within the endoplasmic reticulum by furin and in regulated secretory vesicles by proconvertase enzymes (PC1/3). ProBDNF can also be cleaved extracellularly by MMPs (−3/−7/−9) or by components of the tPA/plasmin proteolytic cascade. The activity of these proteases is tightly regulated by a number of inhibitors, including PAI-1, which inhibits both extracellular and intracellular cleavage; TIMPs, which inhibit MMPs; and neuroserpin and A2AP, which inhibit the tPA/plasmin proteolytic cascade. Red bars indicate inhibition, and green bars indicate activation.

Article Snippet: The following antibodies and concentrations were used: mouse monoclonal HA.11 clone 16B12 antibody (1:3000; MMS-101P, Covance), mouse monoclonal proBDNF antibody (1:1000; H10001G-MA, GeneCopoeia), rabbit polyclonal to α-2 antiplasmin (1:2000; ab62771, Abcam), rabbit polyclonal furin antibody (1:1000; sc-20801, Santa Cruz Biotechnology), rabbit polyclonal MMP-9 antibody (1:2000; AB13458, Millipore), sheep polyclonal neuroserpin antibody (1:2000; SASMNSP-GF-HT, Molecular Innovations), rabbit polyclonal PAI-1 antibody (1:1000; ASMPAI-GF-HT, Molecular Innovations), rabbit polyclonal plasminogen antibody (1:3000; ASMPLG-GF-HT, Molecular Innovations), sheep polyclonal tPA antibody (1:500; SASTPA-GF-HT, Molecular Innovations), and rabbit polyclonal TIMP-1 antibody (1:1000; AB770, Millipore).

Techniques: Activity Assay, Inhibition, Activation Assay