Abstract
Purpose :
Neurodegenerative diseases affecting both the brain and retina target visual neurons, such as the retinal ganglion cells (RGC). While glaucoma's impact on RGC loss is well-studied, limited research explores brain neurodegeneration's consequences on RGC loss. Prior research indicates that certain RGC subtypes exhibit resistance or susceptibility to various forms of neurodegeneration, suggesting a subtype-specific response on RGC apoptosis. Thus, understanding RGC subtype loss and exploring the mechanisms of various disease etiologies is crucial for developing targeted therapeutics.
Methods :
Human induced pluripotent stem cells (hiPSCs) were generated from donor control and glaucoma corneal fibroblasts (N=4), as well as donors with comorbidities of Parkinson's or Alzheimer's with glaucoma using the CytoTune Sendai reprogramming kit. Characterization of fibroblasts (Keratocan, α-SMA) and hiPSCs (TRA-1-60, OCT4, SOX2, NANOG, C-MYC, KLF4) was performed through protein assays. Karyotyping assessed the lack of chromosomal abnormalities in hiPSCs. The hiPSCs were differentiated to 3D retinal organoids (RO) with downstream RGC generation. The RGCs were characterized using protein (RBPMS) and gene expression (THY-1, RBPMS) assays. Further, hiPSC-derived RGCs were analyzed using a custom RGC subtype-specific TaqMan array (CDH6, MMP17, RBPMS2, FABP4, SPP1, OPN4, SDK2, JAM2, PVALB, FOXP1). To investigate the correlation of diseased proteins with RGC loss, the RGCs were stained with amyloid beta and synuclein. RGC apoptosis was evaluated by immunostaining with cleaved CASP-3.
Results :
Fibroblasts and hiPSCs were successfully generated with no observed chromosomal abnormalities. The hiPSCs were successfully differentiated into ROs with subsequent RGC generation. Seven different iPSC-RGC subtypes were identified across groups. Significant loss of FOXP1(p<0.05) (hRGC7 subtype) and JAM2 (JRGC subtype) (p<0.05) was observed in glaucoma compared to controls, while compromised levels of OPN4 (ipRGCs subtype) observed in Parkinson’s etiologies with glaucoma. A correlation was observed between amyloid beta and synuclein levels with apoptotic marker CASP3 in hiPSC-derived RGCs, suggesting their potential involvement in RGC loss.
Conclusions :
The study confirmed RGC subtype loss in neurodegenerative diseases, highlighting the need to unravel mechanisms for targeted therapeutics in conditions affecting RGCs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.