Abstract P148: Role of Endothelin B Receptors in Neuronal Progenitor Cell Differentiation and Mitochondrial Function in Ischemic Stroke

Abstract

Neuronal progenitor cells (NPCs) and mitochondria are well known to play roles in regeneration and functions of the brain, respectively. However, stimulation of endothelin-B receptors (ETBR) using an agonist, sovateltide (IRL-1620) on their fate in the brain after stroke remains elusive. We evaluated the effect of sovateltide mediated ETBR stimulation on differentiation of NPCs and fate of mitochondria after stroke. Ischemic stroke was induced by performing permanent right middle cerebral artery occlusion (MCAO) in rats. Sovateltide (5 μg/kg) or saline (equal volume) was injected intravenously; neurological and motor function evaluation was done at 24 hrs and day 7 post MCAO. Brain tissues were analyzed for NPCs differentiation and mitochondrial fate using techniques such as western blots, immunofluorescence, transmission electron microscopy and in situ PCR. In vitro hypoxia experiment was carried out to confirm sovateltide mediated neuronal differentiation. Neurological and motor functions were significantly improved in sovateltide treated rats at 24 hrs and day 7 post MCAO. Differentiation of NPCs was evident with upregulation of neuronal differentiation markers Neuro D1 (p=0.00002) as well as HuC/HuD (p=0.0037) along with neuronal marker Doublecortin (DCX) (p=0.00011) at 24 hrs post MCAO. However, significant upregulation only in HuC/HuD (p=0.043) was observed at day 7. Better preserved mitochondrial fate was observed in sovateltide rat brains with downregulation of mitochondrial fission marker, DRP1 (p<0.001), increase in fusion marker, MFN2 (p<0.0001) and increase in cross-sectional area x number (p<0.05) as well as mitochondrial/tissue area (p<0.05) at 24 hrs and day 7 post MCAO. In situ PCR analysis showed increased mitochondrial DNA (p=0.0418), indicating better mitochondrial biogenesis at day 7 post MCAO. In vitro exposure of sovateltide and hypoxia to cultured NPCs showed higher NeuroD1 and NeuN (a mature neuronal marker) expression confirming NPCs differentiation. Sovateltide mediated ETBR stimulation promotes differentiation of NPCs and mitochondrial fusion as well as biogenesis and helps in neuronal regeneration and function restoration in acute ischemic brains.