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

Autophagy. 2022 Nov;18(11):2711-2730. doi: 10.1080/15548627.2022.2047344. Epub 2022 Mar 9.

Abstract

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/PDGFRβ: 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.

Keywords: Angiogenesis; RTN4; autophagy; cerebral infarction; secondary neurodegeneration; thalamus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy* / physiology
  • Endothelial Cells / metabolism
  • Humans
  • Infarction, Middle Cerebral Artery* / complications
  • Neovascularization, Pathologic / metabolism
  • Nogo Proteins* / metabolism
  • Nogo Proteins* / pharmacology
  • Rats
  • Sequestosome-1 Protein / metabolism
  • Sphingosine-1-Phosphate Receptors*
  • Thalamus / metabolism
  • rho-Associated Kinases / metabolism
  • rho-Associated Kinases / pharmacology

Substances

  • bafilomycin A
  • Nogo Proteins
  • rho-Associated Kinases
  • Sequestosome-1 Protein
  • Sphingosine-1-Phosphate Receptors
  • Rtn4 protein, rat
  • sphingosine-1-phosphate receptor-2, rat

Grants and funding

This work was supported by the National Key R&D Program of China [2017YFC1307500]; the National Natural Science Foundation of China [81971118, 82171281]; the Natural Science Foundation of Guangdong Province, China [2021A1515010094]; the Science and Technology Planning Project of Guangzhou, China [201904010313];; the Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases [2015B050501003]; Guangdong Provincial Engineering Center for Major Neurological Disease Treatment; Guangdong Provincial Translational Medicine Innovation Platform for Diagnosis and Treatment of Major Neurological Disease; Guangdong Provincial Clinical Research Center for Neurological Diseases, China.