Dataset Information

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma.

ABSTRACT: Lysosomal membrane permeabilization (LMP) is observed under many pathological conditions, leading to cellular dysfunction and death. However, the mechanisms by which lysosomal membranes become leaky in vivo are not clear. Our data demonstrate that LMP occurs in neurons following controlled cortical impact induced (CCI) traumatic brain injury (TBI) in mice, leading to impaired macroautophagy (autophagy) and neuronal cell death. Comparison of LC-MS/MS lysosomal membrane lipid profiles from TBI and sham animals suggested a role for PLA2G4A/cPLA2 (phospholipase A2, group IVA [cytosolic, calcium-dependent]) in TBI-induced LMP. Activation of PLA2G4A caused LMP and inhibition of autophagy flux in cell lines and primary neurons. In vivo pharmacological inhibition of PLA2G4A attenuated TBI-induced LMP, as well as subsequent impairment of autophagy and neuronal loss, and was associated with improved neurological outcomes. Inhibition of PLA2G4A in vitro limited amyloid-β-induced LMP and inhibition of autophagy. Together, our data indicate that PLA2G4A -mediated lysosomal membrane damage is involved in neuronal cell death following CCI-induced TBI and potentially in other neurodegenerative disorders.Abbreviations: AACOCF₃, arachidonyl trifluoromethyl ketone; ACTB/β-actin, actin, beta; AD, Alzheimer disease; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG12, autophagy related 12; BECN1, beclin 1, autophagy related; C1P, ceramide-1-phosphate; CCI, controlled cortical impact; CTSD, cathepsin D; CTSL, cathepsin L; GFP, green fluorescent protein; IF, immunofluorescence; LAMP1, lysosomal-associated membrane protein 1; LAMP2, lysosomal-associated membrane protein 2; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LMP, Lysosomal membrane permeabilization; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MAP1LC3/LC3, microtuble-associated protein 1 light chain 3; NAGLU, alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); PC, diacyl glycerophosphatidylcholine; PE, diacyl glycerophosphatidylethanolamine; PE-O, plasmanyl glycerophosphatidylethanolamine; PE-P, plasmenyl glycerophosphatidylethanolamine; PLA2G4A/cPLA2, phospholipase A2, group IVA (cytosolic, calcium-dependent); RBFOX3, RNA binding protein, fox-1 homolog (C. elegans) 3; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUBA1/α-tubulin, tubulin, alpha; TBI, traumatic brain injury; TFEB, transcription factor EB; ULK1, unc-51 like kinase 1.

SUBMITTER: Sarkar C

PROVIDER: S-EPMC6999646 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Necrosis that appears at the ischemic distal end of random-pattern skin flaps increases the pain and economic burden of patients. Necroptosis is thought to contribute to flap necrosis. Lysosomal membrane permeabilization (LMP) plays an indispensable role in the regulation of necroptosis. Nonetheless, the mechanisms by which lysosomal membranes become leaky and the relationship between necroptosis and lysosomes are still unclear in ischemic flaps. Based on Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry (LC-MS) analysis results, we found that LMP was presented in the ischemic distal portion of random-pattern skin flaps, which leads to disruption of lysosomal function and macroautophagic/autophagic flux, increased necroptosis, and aggravated necrosis of the ischemic flaps. Moreover, bioinformatics analysis of the LC-MS results enabled us to focus on the role of PLA2G4E/cPLA2 (phospholipase A2, group IVE) in LMP of the ischemic flaps. In vivo inhibition of PLA2G4E with an adeno-associated virus vector attenuated LMP and necroptosis, and promoted flap survival. In addition, microRNA-seq helped us determine that Mir504-5p was differentially expressed in ischemic flaps. A string of in vitro and in vivo tests was employed to verify the inhibitory effect of Mir504-5p on PLA2G4E, LMP and necroptosis. Finally, we concluded that the inhibition of PLA2G4E by Mir504-5p reduced LMP-induced necroptosis, thereby promoting the survival of random-pattern skin flaps.Abbreviations: AAV: adeno-associated virus; ACTA2/α;-SMA: actin alpha 2, smooth muscle, aorta; ALOX15/12/15-LOX: arachidonate 15- lipoxygenase; c-CASP8: cleaved caspase; c-CASP3: cleaved caspase 3; CTSD: cathepsin D; CTSB: cathepsin B; CTSL: cathepsin L; DMECs: primary mouse dermal microvascular endothelial cells; ELISA: enzyme-linked immunosorbent assay; F-CHP: 5-FAM-conjugated collagen hybridizing peptide; FISH: fluorescence in situ hybridization; HUVECs: human umbilical vein endothelial cells; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC-MS: liquid chromatography-mass spectrometry; LDBF: laser doppler blood flow; LMP: lysosomal membrane permeabilization; LPE: lysophosphatidylethanolamine; LPC: lysophosphatidylcholine; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MLKL: mixed lineage kinase domain-like; NDI: N-dodecylimidazole; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; PLA2G4A/cPLA2: phospholipase A2, group IVA (cytosolic, calcium-dependent); PLA2G4E/cPLA2: phospholipase A2, group IVE; qPCR: quantitative real-time polymerase chain reaction; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RISC: RNA-induced silencing complex; ROS: reactive oxygen species; shRNA: short hairpin RNA; SQSTM1: sequestosome 1; TBHP: tert-butyl hydroperoxide; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

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.

Dataset Information

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets