Spatial proteomic mapping of the human and mouse retina using IBEX.

Area of Science:

• Ophthalmology
• Neuroscience
• Cell Biology

Background:

• Understanding retinal cell organization and the outer limiting membrane (OLM) is crucial for eye health.
• Existing techniques have limitations in simultaneously visualizing multiple proteins in their native spatial context.

Purpose of the Study:

• To generate a comparative spatial proteomic atlas of the human and mouse retina.
• To refine the iterative bleaching extends multiplexity (IBEX) technique for enhanced multiplexing and cross-species compatibility.
• To investigate the structural components and molecular organization of the OLM and its role in retinal integrity.

Main Methods:

• Developed an enhanced iterative bleaching extends multiplexity (IBEX) workflow with antibody dissociation and chemical bleaching.
• Applied super-resolution imaging with deconvolution microscopy to human and mouse retinal tissue.
• Performed cross-species comparison with single-cell RNA sequencing (scRNA-seq) data.

Main Results:

• Generated spatial proteomic atlases imaging over 25 protein markers in human and mouse retinas.
• Identified CD44 as a core structural component of the OLM, colocalized with an F-actin belt in Müller glial endfeet.
• Observed OLM structural failure and rosette formation in the Crb1rd8 mouse model, associated with disruption of the CD44 complex.

Conclusions:

• Spatial proteomic atlases provide novel insights into retinal cell arrangement and OLM ultrastructure.
• CD44 and F-actin are key components of the OLM, essential for its structural integrity.
• Disruption of the OLM complex contributes to retinal pathology, as demonstrated in the Crb1rd8 mouse model.