In this study, we provide evidence for the first time that membrane-bound endopeptidase neurolysin is up-regulated in different parts of mouse brain affected by focal ischemiareperfusion in a middle cerebral artery occlusion model of stroke. Radioligand binding, enzymatic and immunoblotting experiments in membrane preparations of frontoparietal cortex, striatum, and hippocampus isolated from the ischemic hemisphere of mouse brain 24 h after reperfusion revealed statistically significant increase (≥ twofold) in quantity and activity of neurolysin compared with sham-operated controls. Cerebellar membranes isolated from the ischemic hemisphere served as negative control supporting the observations that up-regulation of neurolysin occurs in post-ischemic brain regions. This study also documents sustained functional upregulation of neurolysin in frontoparietal cortical membranes for at least 7 days after stroke, which appears not to be transcriptionally or translationally regulated, but rather depends on translocation of cytosolic neurolysin to the membranes and mitochondria. Considering diversity of endogenous neurolysin substrates (neurotensin, bradykinin, angiotensins I/II, substance P, hemopressin, dynorphin A(1-8), metorphamide, somatostatin) and the well-documented role of these peptidergic systems in pathogenesis of stroke, resistance to ischemic injury and/or post-stroke brain recovery, our findings suggest that neurolysin may play a role in processes modulating the brain's response to stroke and its recovery after stroke.

Background: Angiotensin II, the renin-angiotensin system effector peptide, interacts with a recently discovered binding site that is distinctly different from its classic receptors. Results: A radioiodinated angiotensin II photoprobe bound to a ϳ75-kDa membrane protein, enabling its isolation and identification. Conclusion: Membrane-bound metalloendopeptidase neurolysin (EC 3.4.24.16) is the novel angiotensin-binding protein.Significance: This metalloendopeptidase may be a crucial component of the renin-angiotensin system.

Previous studies documented up‐regulation of peptidase neurolysin (Nln) after brain ischemia, however, the significance of Nln function in the post‐stroke brain remained unknown. The aim of this study was to assess the functional role of Nln in the brain after ischemic stroke. Administration of a specific Nln inhibitor Agaricoglyceride A (AgaA) to mice after stroke in a middle cerebral artery occlusion model, dose‐dependently aggravated injury measured by increased infarct and edema volumes, blood–brain barrier disruption, increased levels of interleukin 6 and monocyte chemoattractant protein‐1, neurological and motor deficit 24 h after stroke. In this setting, AgaA resulted in inhibition of Nln in the ischemic hemisphere leading to increased levels of Nln substrates bradykinin, neurotensin, and substance P. AgaA lacked effects on several physiological parameters and appeared non‐toxic to mice. In a reverse approach, we developed an adeno‐associated viral vector (AAV2/5‐CAG‐Nln) to overexpress Nln in the mouse brain. Applicability of AAV2/5‐CAG‐Nln to transduce catalytically active Nln was confirmed in primary neurons and in vivo. Over‐expression of Nln in the mouse brain was also accompanied by decreased levels of its substrates. Two weeks after in vivo transduction of Nln using the AAV vector, mice were subjected to middle cerebral artery occlusion and the same outcome measures were evaluated 72 h later. These experiments revealed that abundance of Nln in the brain protects animals from stroke. This study is the first to document functional significance of Nln in pathophysiology of stroke and provide evidence that Nln is an endogenous mechanism functioning to preserve the brain from ischemic injury.

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