Erucamide targets microglia to regulate the retinal angiogenic microenvironment and function as a neurotrophic factor

Abstract

Purpose : Erucamide, a highly hydrophobic fatty acid amide synthesized by photoreceptors, maintains the structure and function of retinas, but its mechanism of action remains unknown. Here we used multiple biochemical techniques to identify erucamide’s target cells, binding proteins, and potential mechanism of neurotrophic action.

Methods : Surface functionalized porous silicon nanoparticles (pSiNPs) were used to efficiently deliver erucamide into the retina. BODIPY-erucamide probes were synthesized to facilitate visualization of erucamide in vivo. Photo affinity labeling proteomics were performed to identify the binding proteins of erucamide. Gene profiling experiments were conducted to elucidate molecular pathways in the target cells.

Results : We used silane-modified pSiNPs to overcome the chanllage of delivering hydrophobic erucamide in vivo. Both intravitreal and subretinal injection of erucamide-pSiNPs resulted in wide-spread activation of GS-Lectin positive cells that were subsequently found to be CD11b+ microglia. BODIPY-labelled erucamide was taken up by CD11b+ cells three days after injection, suggesting that microglia may be directly targeted by erucamide. Angiogenic cytokines like VEGF, PDGFa, FGF, IL-6, TNFa were upregulated after erucamide treatment in both microglia sorted from injected mouse retinas or human iPSC derived microglia. Photo sensitive probes of erucamide were designed, synthesized and used for photo affinity labeling proteomics of the human microglial cell line HMC3. Results from these experiments were used to identify potential binding targets of erucamide. Several novel binding protein candidates were subsequently found to be essential for activation of microglia regulated by erucamide.

Conclusions : We hypothesize that, as a known angiogenic factor itself, erucamide may also work in a paracrine fashion to regulate and maintain a neurotrophic microenvironment in the retina. Microglia can be a direct target of erucamide both in vitro and in vivo. This neurotrophic paracrine activity may be mediated by a number of novel microglial-associated erucamide-binding proteins that are responsible for the activation of, and angiogenic cytokine secretion from, microglia. Modulation of these pathways may represent novel targets for drug discovery in the treatment of neurovasculoglial degenerative retinal diseases.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.