Fig 1: p62 mediated myelin excretion in peripheral demyelination. a Binding of recombinant UBA domain of p62 with myelin basic protein (MBP). G-A: glutathione-agarose bead, p62-A: p62-agarose bead. b Purified myelin fraction was immunoblotted with indicated antibodies. c ELISA results showing the secretion of p62 in the sciatic nerve explant culture medium. Nocut + 6 h: 6 h explant cultures with uncut nerves. 3 DPI + 6 h: 6 h explant culture with preinjured nerves at 3 days before the culture. d ELISA results showing the secretion of p62 in the medium of sciatic nerve explant culture for 3 days. 3MA: 3-methyladenine. e Quantification of the number of pre-SP clusters in DSCs. f Quantification of exocytosed myelinosomes in WT and p62 KO mice at 3DPI. Each dot indicates the percentage of exocytosed myelinosome in each DSC. **P < 0.01. g Sciatic nerve lysates from experimental autoimmune neuritis (EAN) rats were immunoblotted for the indicated antibodies. h Representative IF staining on p62 and MBP in EAN nerves. Arrow: SCs. Scale bar = 20 μm. i, j ELISA results showing p62 levels in the serum of rat EAN (i) and patients with acute inflammatory demyelinating polyradiculoneuropathy (AIDP, j). HC healthy controls. *P < 0.05, **P < 0.01, ns non-significant. Data are represented as mean ± standard error of mean. k Model of autophagy reprogramming from digestive to secretory autophagy for myelin clearance in SC after nerve injury. l Connected secretory autophagy model. AP autophagosome, AL autolysosome, Ly lysosome
Fig 2: Identification of MDBs in vitro. (A) Representative images of HE staining and immunofluorescence staining. Eosinophilic bodies and CK8/18 aggregates were observed in glucotoxicity, lipotoxicity, and glucolipotoxicity groups. However, p62-positive bodies were identified only in the glucotoxicity and glucolipotoxicity groups. Red arrows indicate MDBs. Scale bar, 100 μm in normal views. Scale bar, 50 μm in enlarged views. HE: Hematoxylin–eosin, CK: Cytokeratin. (B) p62 protein quantification in PHH/HSC sheets by ELISA. p62 levels were significantly higher in the lipotoxicity and glucolipotoxicity groups compared to control and glucotoxicity groups. n = 4, 3 independent experiments, **p < 0.01, *p < 0.05. (C) Representative images of TEM. Red arrows indicate MDBs. MDBs were observed in the glucotoxicity and glucolipotoxicity groups. Scale bar, 10 μm. N: nuclear. (D) Representative images of TEM. Giant mitochondria was observed in the glucolipotoxicity group. Scale bar, 2 μm. M: mitochondria, GM: giant mitochondria with crystalline inclusion.
Fig 3: The suppression of canonical autophagy in DSCs. a Representative images of fluorescent autophagosomes in the perinuclear areas of DSC in the teased nerve fibers of autophagy reporter mice at adulthood. Arrowheads: SC nuclei. Scale bars in a = 20 μm. b Graphs indicate the numbers of RFP(+)/GFP(−) and RFP(+)/GFP(+) puncta in the perinuclear area and extranuclear internode. DPI: days postinjury. c The BODIPY FL-Pepstatin A (BPA) staining in teased sciatic nerves at postnatal 2 weeks (P2W), and before and 3 days after injury at adulthood (P7W: postnatal 7 weeks). Arrowheads: SC nuclei, Arrows macrophages. The graph indicating the number of BPA-positive puncta (green) in the perinuclear area. Scale bars in c = 10 μm. d Western blot analysis showing the levels of p62 in the sciatic nerve after injury and explant culture (DIV: days in vitro). Quantification of p62 levels in the sciatic nerves was shown in the graph. e IF images of teased nerve fibers showing p62 immunostaining. Asterisks: degenerating myelin chambers, Arrows: macrophages. f Representative images of teased nerve fibers of autophagy reporter mice at P2W and P7W. Scale bars in e, f = 20 μm. g Quantification of the numbers of perinuclear LC3-RFP dots in the teased nerve fiber from the sciatic nerve explant culture for 3 days from the autophagy reporter mice. h Western blot analysis showing the levels of p62 in the sciatic nerve explant culture with quantification. *P < 0.05, **P < 0.01, ns non-significant
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