PDZD2 and primary open-angle glaucoma : study of rs72759609 in human NT2 cells and glaucoma-like phenotypes in Pdzd2-deficient mice

Abstract

Glaucoma, of which primary open-angle glaucoma (POAG) is the most common type, is a major cause of irreversible blindness worldwide. The global prevalence of POAG on population over 40 years old is 2.4% for the last 20 years. It is characterized by degeneration of retinal ganglion cells (RGCs), resulting in optic nerve cupping and eventually, vision impairment. In 2017, a single nucleotide polymorphism (SNP) rs72759609, statistically significantly related to POAG endophenotypes, was found within a highly conserved non-coding region (~900-bp) of the PDZD2 locus. PDZD2 is a multi-PDZ domain containing protein that can be proteolytically cleaved at its C-terminus to generate a secreted peptide named secreted PDZD2 (sPDZD2), which can serve as an extracellular signaling molecule in Hedgehog (Hh) signaling pathway that is vital for retinal cell functions.

In my study, glaucoma mimicked in the human neuronal precursor cells NT2 under respective conditions of elevated pressure and extremely high glucose concentration increased PDZD2 expression and promoted Hh signaling. PDZD2, which played a pivotal role in NT2 cell viability, was further demonstrated to be a positive modulator of Hh signaling in both NT2 cells and mouse retinal ganglion precursor-like 661W cells via administration of recombinant sPDZD2 protein and gene silencing by short hairpin RNAs (shRNAs) targeting various PDZD2 exons respectively. Moreover, deletion of the ~900-bp conserved intronic region, in which rs72759609 lies, was shown to upregulate PDZD2 expression (and Hh signaling) in human NT2 cells, demonstrating its role as a silencer. Interestingly, luciferase reporter assay revealed that the alternative risk allele (C) of rs72759609 was associated with higher gene expression when compared with reference allele (T). Electrophoretic mobility shift assay (EMSA) and mass spectrometry analysis further indicated that the T-to-C mutation of rs72759609 in the 25-bp probe altered the binding affinity of the DNA probe to the NT2 nuclear proteins, validating the regulatory function of rs727559609.

For in vivo analysis, a Pdzd2 conditional knockout (cKO) mouse model was generated by crossing Pdzd2+/fl mice with β-actin-cre mice to suppress PDZD2 expression in the eye. As shown in immunohistochemical analysis, PDZD2 was expressed in multiple layers in mouse retina and Pdzd2-deficient mice displayed reduced RGC number at the peripheral retina. In addition to RGC loss, the viable RGCs exhibited impaired function in terms of declined amplitudes of positive scotopic threshold response (pSTR) and photopic negative response (PhNR) in electroretinogram (ERG). Enhanced vertical cup-disc ratio (VCDR) was also demonstrated in Pdzd2-deficient mice when compared to wild-type (WT) littermates.

Taken together, the findings suggested an increase in PDZD2 expression due to the identified polymorphism in the intronic silencer-like region positively modulates Hh signaling and increases the risk of developing POAG, whereas deficiency of PDZD2/sPDZD2 in Pdzd2 mutant mice also leads to abnormal eye phenotypes, indicating that both increased and suppressed PDZD2/sPDZD2 levels might contribute to glaucoma, especially POAG. As a result, genotyping of PDZD2 SNPs and monitoring of sPDZD2 levels in human may be of high diagnostic potential for POAG risk assessment.