Background-: Adiponectin is a hormone produced in and released from adipose cells, which has been shown to have anti-diabetic and anti-inflammatory actions in peripheral cells. Two cell surface adiponectin receptors (ADRs) mediate the majority of the known biological actions of adiponectin. Thus far, ADR expression in the brain has been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. Recent findings suggest that levels of circulating adiponectin increase after an ischemic stroke, but the role of adiponectin receptor activation in stroke pathogenesis and its functional outcome is unclear. Methods-: Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion. Primary cortical neuronal cultures were established from individual embryonic neocortex. For glucose deprivation (GD), cultured neurons were incubated in glucose-free Locke's medium for 6, 12 or 24 h. For combined oxygen and glucose deprivation (OGD), neurons were incubated in glucose-free Locke's medium in an oxygen-free chamber with 95% N2/5% CO₂ atmosphere for either 3, 6, 9, 12 or 24 h. Primary neurons and brain tissues were analysed for Adiponectin and ADRs using reverse transcriptase polymerase chain reaction (RT-PCR), immunoblot and immunochemistry methods. Results-: Cortical neurons express ADR1 and ADR2, and that the levels of ADR1 are increased in neurons in response to in vitro or in vivo ischemic conditions. Neurons treated with either globular or trimeric adiponectin exhibited increased vulnerability to oxygen and glucose deprivation which was associated with increased activation of a pro-apoptotic signaling cascade involving p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK). Conclusions-: This study reveals a novel pathogenic role for adiponectin and adiponectin receptor activation in ischemic stroke. We show that cortical neurons express ADRs and reveal a pro-apoptotic role for ADR1 activation in neurons, which may render them vulnerable to ischemic death.

Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II, was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins.