NEI study: New take on old marker of cell death

Illustration shows how axons from ganglion cells in the retina join together to form the optic nerve, which extends into the brain. Created with BioRender.

New findings from a National Eye Institute-led study add a twist on how a widely used cell death marker, annexin-V, can be interpreted in the lab and the clinic for tracking retinal cell death in eye diseases such as glaucoma. The paper published online in the International Journal of Biological Sciences.

Annexin-V has long been used as a marker of apoptosis (cell death) in retinal ganglion cells, which connect the light-sensing retina to the brain. Loss of these neurons is what causes vision loss in glaucoma.

"Now we discovered that annexin-V also binds to immune cells, which complicates our interpretation of findings when using this biomarker," said the study's first co-author, Kiyoharu Miyagishima, Ph.D., staff scientist in the NEI Retinal Neurophysiology Section.

Annexin-V became a widely used marker of cell death because it binds with a strong attraction to phosphatidyl serine, a lipid that moves to the surface of a cell in the early stages of apoptosis.

Unlike necrosis, an unnatural death that sets off an inflammatory response, apoptosis is a process of programmed cell death. Through apoptosis, individual cells and their contents shrink and are engulfed by immune cells called macrophages that clear away the cell body without setting off an inflammatory response. For macrophages, exposed phosphatidyl serine on the cell surface is a kind of "eat me" signal that prompts them to clear away damaged cells.

Annexin-V can be fluorescently labeled, which enables researchers to non-invasively visualize and track apoptosis of retinal ganglion cells in animals and people using an imaging technique known as DARC (detection of apoptosing retinal cells). Over the past two decades, advances in biochemistry and artificial intelligence have established annexin-V DARC imaging as a widely used technique for investigating various eye diseases in basic research and clinical trial settings.

But even as annexin-V has become a widely used biomarker, there are still unexpected findings. For example, annexin-V labeling appears at the optic nerve head, a disc-shaped region that itself is devoid of retinal ganglion cell somas (the main part of the neuron), though it is the point where axons from retinal ganglion cells exit the eye and form the optic nerve that extends into the brain

Spectral-domain optical coherence tomography (SD-OCT) shows annexin-V labeling of apoptosis at the optic nerve head (OHN) in a control retina (left). Immunostaining of retinal ganglion cells shown surrounding the OHN (middle). On SD-OCT microglial cells are labeled green, and subset are also labeled with annexin-V.

"That annexin-V labeling appears in unexpected locations is a mystery that inspired us to check its accuracy," said Francisco Nadal- Nicolás, Ph.D., a postdoctoral fellow in the NEI Retinal Neurophysiology Section.

The team used an optic nerve crush model to validate DARC imaging of apoptosis since it is a well-established model in the field and there is a firm understanding of the time course of retinal ganglion cell death. When just the axons of retinal ganglion cells are damaged, it triggers apoptosis, as opposed to necrosis.

Unexpectedly, they found that in addition to apoptotic retinal ganglion cells, annexin-V also binds to immune cells and to a subset of microglial cells in the retina, suggesting that it has a role to play in detecting early inflammatory responses. Microglia are the first cells to respond to damage and changes in their environment.

Annexin-V labeling of immune cells was also brighter, masking the weaker labeling associated with apoptosis. After using a drug to deplete immune cells, the researchers could observe the apoptotic annexin-V labeling of retinal ganglion cells.

"These findings challenge the interpretation of DARC imaging counts of fluorescing annexin-V, and the role annexin-V should play in the diagnosis of retinal diseases," the authors wrote.

At the same time, the findings present some interesting opportunities. The fact that annexin-V binds to retinal immune cells in response to injury or disease provides an opportunity to monitor potential therapeutics targeting microglial activation and neuroinflammation in the retina. This could be of value in studying treatments for neurodegenerative diseases like age-related macular degeneration, glaucoma, and retinitis pigmentosa.

This work was supported in part by the Intramural Research Program of the NEI and also by the Office of the Assistant Secretary of Defense for Health Affairs and the Defense Health Agency J9, Research and Development Directorate, through the Vision Research Program under Award No. (CDMRPL-18-0-VR180205).

Reference

Miyagishima KJ, Nadal-Nicolás FM, Ma W, Li W. Annexin-V binds subpopulation of immune cells altering its interpretation as an in vivo biomarker for apoptosis in the retina. Int J Biol Sci 2024; 20(15):6073-6089. doi:10.7150/ijbs.102551. https://www.ijbs.com/v20p6073.htm

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