Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to pyruvate kinase M2 (PKM2)-dependent conventional caspase-3/gasdermin E (GSDME) cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-programmed death-1 (anti-PD-1). In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.
Pyroptosis/immunology* ; Humans ; Endoplasmic Reticulum/immunology* ; Animals ; Nogo Proteins/antagonists & inhibitors* ; Mice ; Cell Line, Tumor ; Xanthones/pharmacology* ; Neoplasms/pathology* ; Mice, Nude

OBJECTIVENogo-A antibody IN-1 can neutralize Nogo-A, a neurite growth inhibitory protein, promoting axonal regeneration following lesions of the central nervous system (CNS) in adult rats. This study aimed to examine the effect of ventricle injection of Nogo-A antibody on neuronal regeneration in neonatal rats following hypoxic-ischemic brain damage (HIBD).

METHODSA model of neonatal HIBD was prepared by the ligation of the left common carotid artery, followed by 8% hypoxia exposure. Forty HIBD rats were randomly given a ventricle injection of 10 microL Nogo-A antibody IN-1 (IN-1 group) or 10 microL artificial cerebrospinal fluid (artificial CSF group) (n=20 each). Another 20 neonatal rats were sham-operated, without hypoxia-ischemia, and were used as the controls. The levels of Nogo-A and GAP-43 protein in the brain were measured by immunohistochemistry.

RESULTSThe number of immunohistory positive cells of Nogo-A in the brain in the IN-1 group (28.61+/-1.70) was obviously less than that in the artificial CSF (39.52 +/-1.40) and the sham-operated groups (32.78 +/- 1.87) (both P < 0.01). There were significant differences in the Nogo-A protein expression between the artificial CSF and the sham-operated groups (P < 0.01). The GAP-43 protein expression in the IN-1 group (31.14 +/- 1.88) was noticeably higher than that in the artificial CSF group (27.73 +/- 1.43 ) (P < 0.01). Both the IN-1 and the artificial CSF groups showed lower GAP-43 protein levels than the sham-operated groups (33.64 +/- 1.24) (both P < 0.01).

CONCLUSIONSNogo-A antibody can reduce the expression of Nogo-A protein in the brain and thus promote neuronal regeneration in neonatal rats following HIBD. An increased GAP-43 protein expression in the brain after Nogo-A antibody administration shows an enhanced neuronal regeneration in the neonatal rats following HIBD.

Animals ; Animals, Newborn ; Antibodies ; administration & dosage ; Brain Chemistry ; Female ; GAP-43 Protein ; analysis ; Hypoxia-Ischemia, Brain ; metabolism ; physiopathology ; therapy ; Immunohistochemistry ; Injections, Intraventricular ; Male ; Myelin Proteins ; analysis ; antagonists & inhibitors ; immunology ; Nerve Regeneration ; Nogo Proteins ; Rats ; Rats, Sprague-Dawley

Biomedical Research ; trends ; Computers ; Humans ; Implants, Experimental ; Myelin Proteins ; antagonists & inhibitors ; immunology ; Nerve Regeneration ; immunology ; physiology ; Nogo Proteins ; Patient Education as Topic ; Spinal Cord Injuries ; therapy