Dataset Information

Autophagy protein NRBF2 attenuates endoplasmic reticulum stress-associated neuroinflammation and oxidative stress via promoting autophagosome maturation by interacting with Rab7 after SAH.

ABSTRACT:

Background

Neuroinflammation and oxidative stress plays an important role in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). This study is the first to show that activation of autophagy protein nuclear receptor binding factor 2 (NRBF2) could reduce endoplasmic reticulum stress (ERS)-associated inflammation and oxidative stress after SAH.

Methods

Male C57BL/6J mice were subjected to endovascular perforation to establish a model of SAH. NRBF2 overexpression adeno-associated virus (AAV), NRBF2 small interfering RNAs (siRNA), lysosomal inhibitor-chloroquine (CQ), and late endosome GTPase Rab7 receptor antagonist-CID1067700 (CID) were used to investigate the role of NRBF2 in EBI after SAH. Neurological tests, brain water content, western blotting and immunofluorescence staining were evaluated.

Results

Our study found that the level of NRBF2 was increased after SAH and peaked at 24 h after SAH. In addition, we found that the overexpression of NRBF2 significantly improved neurobehavioral scores and reduced ERS, oxidative stress, and neuroinflammation in SAH, whereas the inhibition of NRBF2 exacerbated these phenotypes. In terms of mechanism, NRBF2 overexpression significantly promoted autophagosome maturation, with the downregulation of CHOP, Romo-1, TXNIP, NLRP3, TNF-α, and IL-1β expression through interaction with Rab7. The protective effect of NRBF2 on ERS-associated neuroinflammation and oxidative stress after SAH was eliminated by treatment with CQ. Meanwhile, it was also reversed by intraperitoneal injection of CID. Moreover, the MIT domain of NRBF2 was identified as a critical binding site that interacts with Rab7 and thereby promotes autophagosome maturation.

Conclusion

Our data provide evidence that the autophagy protein NRBF2 has a protective effect on endoplasmic reticulum stress-associated neuroinflammation and oxidative stress by promoting autophagosome maturation through interactions with Rab7 after SAH.

SUBMITTER: Zeng H

PROVIDER: S-EPMC8447596 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:NRBF2 is a component of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex. Our previous study has revealed its role in regulating ATG14-associated PtdIns3K activity for autophagosome initiation. In this study, we revealed an unknown mechanism by which NRBF2 modulates autophagosome maturation and APP-C-terminal fragment (CTF) degradation. Our data showed that NRBF2 localized at autolysosomes, and loss of NRBF2 impaired autophagosome maturation. Mechanistically, NRBF2 colocalizes with RAB7 and is required for generation of GTP-bound RAB7 by interacting with RAB7 GEF CCZ1-MON1A and maintaining the GEF activity. Specifically, NRBF2 regulates CCZ1-MON1A interaction with PI3KC3/VPS34 and CCZ1-associated PI3KC3 kinase activity, which are required for CCZ1-MON1A GEF activity. Finally, we showed that NRBF2 is involved in APP-CTF degradation and amyloid beta peptide production by maintaining the interaction between APP and the CCZ1-MON1A-RAB7 module to facilitate the maturation of APP-containing vesicles. Overall, our study revealed a pivotal role of NRBF2 as a new RAB7 effector in modulating autophagosome maturation, providing insight into the molecular mechanism of NRBF2-PtdIns3K in regulating RAB7 activity for macroautophagy/autophagy maturation and Alzheimer disease-associated protein degradation..Abbreviations: 3xTg AD, triple transgenic mouse for Alzheimer disease; Aβ, amyloid beta peptide; Aβ1-40, amyloid beta peptide 1-40; Aβ1-42, amyloid beta peptide 1-42; AD, Alzheimer disease; APP, amyloid beta precursor protein; APP-CTFs, APP C-terminal fragments; ATG, autophagy related; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG14, autophagy related 14; CCD, coiled-coil domain; CCZ1, CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; CHX, cycloheximide; CQ, chloroquine; DAPI, 4',6-diamidino-2-phenylindole; dCCD, delete CCD; dMIT, delete MIT; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; FYVE, Fab1, YGL023, Vps27, and EEA1; GAP, GTPase-activating protein; GDP, guanine diphosphate; GEF, guanine nucleotide exchange factor; GTP, guanine triphosphate; GTPase, guanosine triphosphatase; HOPS, homotypic fusion and vacuole protein sorting; ILVs, endosomal intralumenal vesicles; KD, knockdown; KO, knockout; LAMP1, lysosomal associated membrane protein 1; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MLVs, multilamellar vesicles; MON1A, MON1 homolog A, secretory trafficking associated; NRBF2, nuclear receptor binding factor 2; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RILP, Rab interacting lysosomal protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62, sequestosome 1; UVRAG, UV radiation resistance associated; VPS, vacuolar protein sorting; WT, wild type.