Dysfunction of heat shock factor 4 impairs retinal structure and visual function in mice and zebrafish

Abstract

Purpose Loss of function of heat shock factor 4(HSF4) causes microphthalmia with lens opacification. The objective of this study is to uncover the regulation of HSF4 on retinal homeostasis. Methods Hsf4^del mutant mice and Hsf4^null zebrafish models were recruited in this study. H&E was used to determine retinal structure. The immunoblot, qRT-PCR and immunofluorescence staining were used to measure the expression of mRNA and protein. AAV2-Hsf4-Flag virus were used to the reconstitution assay. Results The retinal structure of Hsf4^del mice and Hsf4^null zebrafish, which is comparable to wild-type at P10 days old, undergoes atrophy at 7 and 13 months old. Dysfunction of Hsf4 downregulates the expression of visual cycle enzymes (e.g., RPE65, RLBP1 and RDH5 ) and heat shock proteins (e.g., HSP90 and HSP25), and simultaneously activates retinal gliosis (e.g., upregulating the expression of GFAP, GS, CRYAB, inflammatory interleukins, and VEGFA) and the expression of senescent P16^INK4a and P21^cip1 in the retina of postnatal P1- P10 mice and embryonic zebrafish, and those changes are enhanced in 7 and 13 months old mice and zebrafish. Subretinal administration of AAV2-Hsf4b to the retina of one-month Hsf4^del mice partially rescued the expression of changed proteins. ERG results showed that the downregulation of amplitude of a- and b- waves at scotopic response was detected at P15. Overexpression of Flag-Hsf4b in the in vitro cultured primary Hsf4^del RPE cells restores the expression of visual cycle enzymes and heat shock proteins. TUNEL assay shows that there are more apoptotic cells in the ONL and the RPE of 7-and 13-month-Hsf4^del retina than in P10 retina. Conclusion In addition to causing cataracts, the loss of function of HSF4 impairs the visual cycles and activates the gliosis in early postnatal age, which are associated with the retinal atrophy.