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Impairment of the autophagy-lysosomal pathway and activation of pyroptosis in macular corneal dystrophy.

ABSTRACT: Macular corneal dystrophy (MCD) is ascribed to mutations in the carbohydrate sulfotransferase (CHST6) gene affecting keratan sulfate (KS) hydrophilicity and causing non-sulfated KS to precipitate in keratocytes and the corneal stroma. We investigated roles for inflammatory responses in MCD pathogenesis by examining the lysosomal-autophagy pathway and activation of pyroptosis in MCD keratocytes. Normal and lesioned keratocytes were obtained from MCD patients undergoing corneal transplantation. The keratocytes were subjected to gene sequencing, RT-PCR, western blotting, transmission electron microscopy, histological staining, induction and inhibition assays of autophagy and pyroptosis, CCK-8 and LysoTracker Green DND-26 labeling, and flow cytometry. A novel homozygous MCD mutation was identified in a family from Northeast China; the mutation was distinguished by cytoplasmic vacuolation, cell membrane disruption, electron dense deposits, and deposition of a band of Periodic acid-Schiff and Alcian blue-positive material in the keratocytes and stroma layer. KS protein levels were decreased, expression of p62 and LC3-II proteins was enhanced, cathepsin D expression was declined and the LysoTracker Green DND-26 signal was dramatically reduced in MCD keratocytes. Bafilomycin-A1 treatment significantly increased caspase-1 and Pro-IL-1β expression in normal and MCD keratocytes. Nod-like receptors pyrins-3 (NLRP3), caspase-1, Pro-IL-1β, and IL-1β levels were pronouncedly elevated in cells exposed to H₂O₂. Ac-YVAD-CMK treatment reversed this expression in normal and MCD keratocytes. Suppression of the autophagic degradation of non-sulfated KS by impaired autophagic flux in MCD keratocytes triggers pyroptosis. Amelioration of impaired autophagy and restraint of pyroptosis may, therefore, have therapeutic efficacy in the treatment of MCD.

SUBMITTER: Zheng T

PROVIDER: S-EPMC7487068 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The autophagy-lysosomal pathway plays an essential role in cellular homeostasis as well as a protective function against a variety of diseases including neurodegeneration. Conversely, inhibition of autophagy, for example due to lysosomal dysfunction, can lead to pathological accumulation of dysfunctional autophagosomes and consequent neuronal cell death. We previously reported that autophagy is inhibited and contributes to neuronal cell death following spinal cord injury (SCI). In this study, we examined lysosomal function and explored the mechanism of lysosomal defects following SCI. Our data demonstrated that expression levels and processing of the lysosomal enzyme cathepsin D (CTSD) are decreased by 2 h after SCI. Enzymatic activity levels of CTSD and another lysosomal enzyme, N-acetyl-alpha-glucosaminidase, are both decreased 24 h post injury, indicating general lysosomal dysfunction. Subcellular fractionation and immunohistochemistry analysis demonstrated that this dysfunction is due to lysosomal membrane permeabilization and leakage of lysosomal contents into the cytosol. To directly assess extent and mechanisms of damage to lysosomal membranes, we performed mass spectrometry-based lipidomic analysis of lysosomes purified from SCI and control spinal cord. At 2 h post injury our data demonstrated increase in several classes of lysosophospholipids, the products of phospholipases (PLAs), as well as accumulation of PLA activators, ceramides. Phospholipase cPLA2, the main PLA species expressed in the CNS, has been previously implicated in mediation of secondary injury after SCI, but the mechanisms of its involvement remain unclear. Our data demonstrate that cPLA2 is activated within 2 h after SCI preferentially in the lysosomal fraction, where it colocalizes with lysosomal-associated membrane protein 2 in neurons. Inhibition of cPLA2 in vivo decreased lysosomal damage, restored autophagy flux, and reduced neuronal cell damage. Taken together our data implicate lysosomal defects in pathophysiology of SCI and for the first time indicate that cPLA2 activation leads to lysosomal damage causing neuronal autophagosome accumulation associated with neuronal cell death.

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Impairment of the autophagy-lysosomal pathway and activation of pyroptosis in macular corneal dystrophy.

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