Abstract
Purpose :
Extracellular matrix (ECM) is an important source of developmental and homeostatic molecular cues. Although much is known regarding the molecular mechanisms of retinal regeneration, our understanding of the ECM’s role in this context remains unclear. This study investigates the role of the ECM molecule nephronectin (Npnt) in regulating neuroprotection in the light-damaged zebrafish retina, as well as Muller glia (MG) and neural progenitor cell (NPC) proliferation during regeneration.
Methods :
We used in vivo morpholino-mediated knockdown of Npnt expression in the adult zebrafish dorsal retina. Anti-PCNA immunolabeling of Tg(gfap:egfp) zebrafish retinal sections was used to visualize proliferating MG and NPCs. TUNEL labeling was used to assess cell death. qRT-PCR and FISH were used to examine expression of target genes. Protein expression levels and subcellular distribution were assessed using protein immunoblotting of samples enriched from either cytosolic or nuclear dorsal retina fractions.
Results :
npnt is expressed by HuC/D+ amacrine and ganglion cells in adult zebrafish retinas and is upregulated after light-induced damage. Knockdown of Npnt expression results in decreased NPC proliferation and a significant increase in the number of dying inner retinal neurons via repression of the GDNF pathway. Protein immunoblotting revealed a novel pattern of Npnt processing and translocation. Under normal conditions, Npnt is observed as a 60 kDa protein, with a percentage cleaved into 20 and 40 kDa fragments. Upon light damage, all the Npnt is cleaved into the two fragments. Furthermore, the 40 kDa fragment is translocated into the nucleus by a mechanism involving Itga11a receptor, suggesting it may directly regulate expression of target genes.
Conclusions :
These findings demonstrate that the ECM provides regulatory cues guiding zebrafish retinal regeneration. Npnt appears to regulate retinal neuron survival and NPC proliferation. Interestingly, our data suggest that upon damage, Npnt is transported into the nucleus, where it may directly regulate expression of genes that govern the regeneration response and neuron survival. These findings suggest a novel mechanism for an ECM molecule and expand our understanding of the role of the microenvironment in retinal regeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.