Tissue-type plasminogen activator and the low-density lipoprotein receptor-related protein induce Akt phosphorylation in the ischemic brain

Abstract

Tissue-type plasminogen activator (tPA) is found in the intravascular space and in the central nervous system. The low-density lipoprotein receptor-related protein (LRP) is expressed in neurons and in perivascular astrocytes. During cerebral ischemia, tPA induces the shedding of LRP's extracellular domain from perivascular astrocytes, and this is followed by the development of cerebral edema. Protein kinase B (Akt) is a serine/threonine kinase that plays a critical role not only in cell survival but also in the regulation of the permeability of the blood-brain barrier. We found that, in the early phases of the ischemic insult, the interaction between tPA and LRP induces Akt phosphorylation (pAkt) in perivascular astrocytes and inhibits pAkt in neurons. Coimmunoprecipitation studies indicate that pAkt and LRP's intracellular domain interact in perivascular astrocytes and that this interaction is dependent on the presence of tPA and results in the development of edema. Together, these results indicate that, in the early stages of cerebral ischemia, the interaction between tPA and LRP in perivascular astrocytes induces the activation of a cell signaling event mediated by pAkt that leads to increase in the permeability of the blood-brain barrier.

Figure 1

Effect of tPA deficiency on Akt phosphorylation in the ischemic brain. (A) Representative Western blot analysis for Akt phosphorylated at serine 473 (pAkt) and total Akt in brain extracts from wild-type (WT) and tPA-deficient (tPA^−/−) mice 1 to 24 hours after middle cerebral artery occlusion (MCAO). S denotes sham-operated mice. (B) Mean density of the band of 6 immunoblots for pAkt in wild-type (□) and tPA^−/− mice (■) 1 to 24 hours after MCAO. n = 6. Lines depict SD. *P < .001 relative to either WT or tPA^−/− sham-operated mice. †P < .001 compared with tPA^−/− mice 1 hour after MCAO. ‡P < .001 relative to WT mice 24 hours after MCAO. (C) Representative Western blot analysis for pAkt in brain extracts from tPA^−/− mice 1 hour after middle cerebral artery occlusion (MCAO) and either no treatment (NT) or the intracerebral injection of tPA (tPA).

Figure 2

Effect of tPA deficiency on ischemic cell death. (A-D) Immunohistochemical analysis of neuronal pAkt in the area of interest-2 (AOI-2) 1 (A,B) and 24 (C,D) hours after middle cerebral artery occlusion (MCAO) in wild-type (A,C) and tPA^−/− (B,D) mice. Blue indicates 4′,6-diamidino-2-phenylindole and red indicates pAkt in panels A and B and NeuN in panels C and D. Green indicates GFAP in panels A and B and pAkt in panels C and D. Original magnification, A-D, 40×. Images were visualized using a Leica DMRBE microscope (Leica, Houston, TX) equipped with a 100×/1.30 numeric aperture (NA) and a LeicaDC500 camera. Images were processed using software provided by the camera manufacturer. (E) Representative Western blot analysis of PARP-1 cleavage in brain extracts from wild-type (WT) and tPA^−/− mice 6 and 24 hours after MCAO. C denotes a control WT animal. The arrow indicates an approximately 89-kDa PARP-1 cleavage product. Each observation was repeated 4 times. (F) Mean percentage of TUNEL-positive cells in the area of ischemic penumbra in WT and tPA^−/− mice 24 hours after MCAO; n = 7. *P < .005 compared with WT mice.

Figure 3

tPA induces Akt phosphorylation in perivascular astrocytes. (A) Representative micrographs of immunogold electron microscopy analysis for pAkt in perivascular astrocytes in the area of interest-2 (AOI-2) of wild-type (i) and tPA^−/− mice (ii) 1 hour after MCAO. Bv indicates blood vessel; and bm, basement membrane. Each observation was repeated 4 times. (B) Mean Evans blue dye extravasation in wild-type mice 6 hours after reperfusion and the intraventricular injection of either vehicle (control, ■) or wortmannin (□). Results are given as a percentage compared with control-treated mice. Lines depict SD; n = 8. *P < .005 compared with control-treated mice.

Figure 4

tPA induces Akt phosphorylation via a plasminogen-independent mechanism. (A,B) Representative Western blot analysis of pAkt and total Akt in brain extracts from Plg^−/− mice (A) and tPA^−/− animals (B) 1 hour after MCAO. A subset of tPA^−/− mice was either left untreated (NT) or injected directly into the ischemic tissue with murine tPA (tPA) or a combination of tPA and RAP (tPA + RAP). S denotes sham-operated animal. Each observation was repeated 3 times.

Figure 5

The effect of the interaction between tPA and LRP on pAkt is cell type– specific. (A,B) Representative Western blot analysis of pAkt and total Akt in extracts from astrocytic (A) and neuronal (B) cultures after 1 hour of exposure to oxygen-glucose deprivation (OGD) conditions and incubation with either tPA or a combination of tPA and the receptor-associated protein (RAP). RAP is a molecular chaperone that inhibits the binding of LRP to its ligands. NT indicates no treatment. Each observation was repeated 4 times.

Figure 6

LRP's intracellular domain and pAkt interact in perivascular astrocytes. (A) Coimmunoprecipitation studies in brain extracts from Wt and tPA^−/− mice 1 hour after MCAO. tPA^−/− were either left untreated (−tPA) or injected directly into the ischemic tissue with tPA (1 μM;+tPA). (B) Coimmunoprecipitation studies in brain extracts from Wt mice 1 hour after MCAO. Animals were either left untreated (MCAO) or injected into the ischemic tissue with RAP (9 μM; MCAO + RAP).