Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

ABSTRACT

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations ACTB actin, beta; Ad-GFP adenovirus-green fluorescent protein; Ad-LCN2 adenovirus-lipocalin 2; Ad-LCN2-GFP adenovirus-LCN2-green fluorescent protein; LCN2AKT2 AKT serine/threonine kinase 2; AMBRA1 autophagy and beclin 1 regulator 1; AMD age-related macular degeneration; ARPE19 adult retinal pigment epithelial cell line-19; Asp278 aspartate 278; ATG4B autophagy related 4B cysteine peptidase; ATG4C autophagy related 4C cysteine peptidase; ATG7 autophagy related 7; ATG9B autophagy related 9B; BLOC-1 biogenesis of lysosomal organelles complex 1; BLOC1S1 biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J C57 black 6J; CGAS cyclic GMP-AMP synthase; ChQ chloroquine; cKO conditional knockout; Cys74 cysteine 74; Dab2 DAB adaptor protein 2; Def deferoxamine; DHE dihydroethidium; DMSO dimethyl sulfoxide; ERG electroretinography; FAC ferric ammonium citrate; Fe2+ ferrous; FTH1 ferritin heavy chain 1; GPX glutathione peroxidase; GST glutathione S-transferase; H2O2 hydrogen peroxide; His280 histidine 280; IFNL/IFNλ interferon lambda; IL1B/IL-1ß interleukin 1 beta; IS Inner segment; ITGB1/integrin ß1 integrin subunit beta 1; KO knockout; LC3-GST microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; LCN2 lipocalin 2; mAb monoclonal antibody; MDA malondialdehyde; MMP9 matrix metallopeptidase 9; NLRP3 NLR family pyrin domain containing 3; NOD-SCID nonobese diabetic-severe combined immunodeficiency; OS outer segment; PBS phosphate-buffered saline; PMEL/PMEL17 premelanosome protein; RFP red fluorescent protein; rLCN2 recombinant LCN2; ROS reactive oxygen species; RPE SM retinal pigmented epithelium spent medium; RPE retinal pigment epithelium; RSL3 RAS-selective lethal; scRNAseq single-cell ribonucleic acid sequencing; SD-OCT spectral domain optical coherence tomography; shRNA small hairpin ribonucleic acid; SM spent medium; SOD1 superoxide dismutase 1; SQSTM1/p62 sequestosome 1; STAT1 signal transducer and activator of transcription 1; STING1 stimulator of interferon response cGAMP interactor 1; TYR tyrosinase; VCL vinculin; WT wild type.