Abstract
The aim of this study was to assess the functional role of peptidase neurolysin (Nln) in the brain after ischemic stroke. Nln is known to inactivate bradykinin, substance P, neurotensin, and angiotensin II, which are cerebrotoxic in the ischemic brain, and to generate angiotensin-(1-7) and Met/Leu-enkephalins, which are cerebroprotective/restorative peptides. To study the functional significance of Nln upregulation in the post-stroke brain we utilized a specific inhibitor of Nln, Agaricoglyceride A (AgaA), in a mouse MCAO model of stroke. Administration of AgaA (10 and 50 mg/kg, i.p.) to mice 1 h after reperfusion (1 h occlusion) aggravated stroke injury measured by increased infarct and edema volume (p < 0.01) and neurological deficit (p < 0.01), in a dose-dependent manner at 24 h after stroke. In this setting, AgaA resulted in a dose-dependent inhibition of Nln in the ischemic but not in the contralateral hemisphere, which, based on preliminary experiments, was also accompanied by increased levels of Nln substrate neuropeptides. In a reverse approach, we developed an adeno-associated virus serotype 2/5 vector encoding mouse Nln driven by cytomegalovirus enhancer/chicken β-actin hybrid (CAG) promoter (AAV2/5-CAG-Nln) to overexpress Nln in the mouse brain prior to stroke. Applicability of AAV2/5-CAG-Nln to transduce catalytically active Nln was confirmed in mouse primary cortical neurons and in mice. In the later experiment, overexpression of Nln in the mouse brain was also accompanied by decreased levels of Nln substrate neuropeptides. Two weeks after
in vivo
transduction using the AAV5-CAG-Nln, or a control AAV5-CAG-eGFP vector (intrastriatal administration; 1.5 x 10
11
genome copies) mice were subjected to MCAO (1 h occlusion followed by 72 h reperfusion). The results of these experiments revealed that abundance of Nln in the brain protects animals from stroke injury measured by reduced infarct and edema volume (p < 0.01 and 0.05, respectively), and improved neurological deficit (p < 0.05). Based on these observations and the knowledge about endogenous substrates of Nln, we view this peptidase as one of brain’s self-protective mechanisms directed towards preservation and recovery of brain after stroke.