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-1β:
interleukin 1 beta; IS: Inner segment; ITGB1/
integrin β1:
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.