Abstract
Purpose :
Zebrafish have the innate ability to regenerate lost or damaged retinal neurons, a process that mammals are unable to recapitulate even though they possess similar cellular populations. Recently, we identified a subset of immune cells that become senescent after damage in the retina. These cells show a transient appearance and clearance during retina regeneration that correlates with the pro- to anti-inflammatory changeover that is observed in zebrafish. It is unknown what factors influence the genesis of these senescent-immune cells, or how interrupting the SASP of these cells influences regeneration.
Methods :
We injected wild type AB zebrafish with NMDA to selectively damage retinal ganglion cells (RGCs) and then treated those fish either with the senolytics Metformin and ABT-263, or the pharmacologic agents RTA-408 (an Nrf2 activator) or Betulinic Acid (an Nf-κb activator). We then immunostained for senescence associated β-galactosidase (SA-βGal) to identify senescent cells, 4c4 antibodies to identify immune cells, HuC/D antibodies to identify RGCs, and EdU to identify proliferating cells. This allowed us to assay the changes in senescent cells when influenced by senolytics or inflammatory pathway agonists.
Results :
Intravitreal injection of NMDA resulted in transient detection of senescent cells starting at 2dpi that co-localized with 4c4+ immune cells. Senolytic treatment showed a prolonged appearance of gaps in the RGC layer, indicating a truncation in regeneration. Nrf2 pathway agonists showed an increase in senescent cells without the induction of damage, and displayed an additive effect on senescent cell genesis when combined with NMDA damage. Nf-κb pathway agonism delayed senescent cell formation, and after drug clearance the number of senescent cells increased.
Conclusions :
Senescent immune cells play a pro-regenerative role and their premature clearance truncated retina regeneration. This supports the hypothesis that modulation of the immune response is needed for proper retina regeneration in zebrafish. The activation of the anti-inflammatory Nrf2 pathway was sufficient to induce senescence in the retina, and induced additional senescence in NMDA damaged eyes. Conversely, activation of the pro-inflammatory Nf-kb pathway suppressed senescent cell appearance. The data indicate a tight connection between inflammation, senescent cell regulation, and regeneration in the retina.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.