RTN4/Nogo-A-S1PR2 negatively regulates angiogenesis and secondary neural repair through enhancing vascular autophagy in the thalamus after cerebral cortical infarction.

Cerebral infarction induces angiogenesis in the thalamus and influences functional recovery. The mechanisms underlying angiogenesis remain unclear. This study aimed to investigate the role of RTN4/Nogo-A in mediating macroautophagy/autophagy and angiogenesis in the thalamus following middle cerebral artery occlusion (MCAO). We assessed secondary neuronal damage, angiogenesis, vascular autophagy, RTN4 and S1PR2 signaling in the thalamus. The effects of RTN4-S1PR2 on vascular autophagy and angiogenesis were evaluated using lentiviral and pharmacological approaches. The results showed that RTN4 and S1PR2 signaling molecules were upregulated in parallel with angiogenesis in the ipsilateral thalamus after MCAO. Knockdown of Rtn4 by siRNA markedly reduced MAP1LC3B-II conversion and levels of BECN1 and SQSTM1 in vessels, coinciding with enhanced angiogenesis in the ipsilateral thalamus. This effect coincided with rescued neuronal loss of the thalamus and improved cognitive function. Conversely, activating S1PR2 augmented vascular autophagy, along with suppressed angiogenesis and aggravated neuronal damage of the thalamus. Further inhibition of autophagic initiation with 3-methyladenine or spautin-1 enhanced angiogenesis while blockade of lysosomal degradation by bafilomycin A1 suppressed angiogenesis in the ipsilateral thalamus. The control of autophagic flux by RTN4-S1PR2 was verified in vitro. Additionally, ROCK1-BECN1 interaction along with phosphorylation of BECN1 (Thr119) was identified in the thalamic vessels after MCAO. Knockdown of Rtn4 markedly reduced BECN1 phosphorylation whereas activating S1PR2 increased its phosphorylation in vessels. These results suggest that blockade of RTN4-S1PR2 interaction promotes angiogenesis and secondary neural repair in the thalamus by suppressing autophagic activation and alleviating dysfunction of lysosomal degradation in vessels after cerebral infarction.Abbreviations: 3-MA: 3-methyladenine; ACTA2/ɑ-SMA: actin alpha 2, smooth muscle, aorta; AIF1/Iba1: allograft inflammatory factor 1; BafA1: bafilomycin A1; BMVECs: brain microvascular endothelial cells; BrdU: 5-bromo-2'-deoxyuridine; CLDN11/OSP: claudin 11; GFAP: glial fibrillary acidic protein; HUVECs: human umbilical vein endothelial cells; LAMA1: laminin, alpha 1; MAP2: microtubule-associated protein 2; MBP2: myelin basic protein 2; MCAO: middle cerebral artery occlusion; PDGFRB/PDGFRß: platelet derived growth factor receptor, beta polypeptide; RECA-1: rat endothelial cell antigen-1; RHOA: ras homolog family member A; RHRSP: stroke-prone renovascular hypertensive rats; ROCK1: Rho-associated coiled-coil containing protein kinase 1; RTN4/Nogo-A: reticulon 4; RTN4R/NgR1: reticulon 4 receptor; S1PR2: sphingosine-1-phosphate receptor 2; SQSTM1: sequestosome 1.

Blockade of Nogo-A/Nogo-66 receptor 1 (NgR1) Inhibits Autophagic Activation and Prevents Secondary Neuronal Damage in the Thalamus after Focal Cerebral Infarction in Hypertensive Rats.

Focal cerebral infarction leads to autophagic activation, which contributes to secondary neuronal damage in the ipsilateral thalamus. Although Nogo-A deactivation enhances neuronal plasticity, its role in autophagic activation in the thalamus after ischemic stroke remains unclear. This study aimed to investigate the potential roles of Nogo-A/Nogo-66 receptor 1 (NgR1) in autophagic activation in the ipsilateral thalamus after cerebral infarction. Focal neocortical infarction was established using the middle cerebral artery occlusion (MCAO) method. Secondary damage in the ipsilateral thalamus was assessed by Nissl staining and immunostaining. The expression of Nogo-A, NgR1, Rho-A and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) as well as autophagic flux were evaluated by immunofluorescence and immunoblotting. The roles of Nogo-A-NgR1 signaling in autophagic activation were determined by intraventricular delivery of an NgR1 antagonist peptide, NEP1-40, at 24 h after MCAO. The results showed that Nogo-A and NgR1 overexpression temporally coincided with marked increases in the levels of Beclin1, LC3-II and sequestosome 1 (SQSTM1)/p62 in the ipsilateral thalamus at seven and fourteen days after MCAO. In contrast, NEP1-40 treatment significantly reduced the expression of Rho-A and ROCK1 which was accompanied by marked reductions of LC3-II conversion as well as the levels of Beclin1 and SQSTM1/p62. Furthermore, NEP1-40 treatment significantly reduced neuronal loss and gliosis in the ipsilateral thalamus, and accelerated somatosensory recovery at the observed time-points after MCAO. These results suggest that blockade of Nogo-A-NgR1 signaling inhibits autophagic activation, attenuates secondary neuronal damage in the ipsilateral thalamus, and promotes functional recovery after focal cerebral cortical infarction.