A deletion mutation in HSF4 (Hsf4 del ) causes congenital cataracts and retinal degeneration in mice and zebrafish
The missense mutations in HSF4’s DNA binding domain and N-terminal hydrophobic region are associated with the inherited autosomal dominant lamellar and all white cataracts[10]. To determine whether Hsf4 participates in regulating retinal development, the Hsf4del mice was recruited [20]. As expected, Hsf4del mice developed lens opacification after 17 days old (p17) (Fig. S1A), and the cataractous lens underwent vacuoles, shrink, and fibrosis at 7 and 13 months old (Fig. S1B). Interestingly, we found that the retinal structure exhibited the disorganized in 7- and 13-month-old Hsf4del mice (Fig. S1B). Immunoblot results showed that HSF4 is expressed in the retinal tissues and its expression level decreased with age increases (Fig. S1C). The immunofluorescent results showed that Hsf4 is predominantly expressed in the cells of ONL and RPE (Fig. S1D ). The expression of HSF4 protein in Hsf4del retina was smaller than HSF4 wild-type (Fig. S1E). These results implied that HSF4 exerts a regulation on retinal homeostasis.
To characterize the regulation of Hsf4del on the retina, we analyzed the retinal structure of Hsf4del vs. wild-type mice at P10, 7and 13 months old by using H&E, PAS staining and oil-red staining. The results showed that the retinal structure exhibited disordered in 7 and 13 months old Hsf4del mice as compared to wild-type mice, characterized by disorganization of inner nuclear layer (INL) and inner-outer plexiform layers (IPL and OPL), retinal pigment epithelium (RPE) disconnected (Fig. 1A and B), lipofuscin deposit underlying RPE (Fig. 1C), the outer nuclear layer (ONL) atrophy (Fig. 1A, D-F), and the increase of neovascular vessels (Fig. 1B). However, this disordered change did not observe in P10 Hsf4del mice (Fig. 1A-D). Those results indicated that dysfunction of Hsf4 results in retinal disorder in mice in an age-related manner. To verify this, we recruited Hsf4null zebrafish model, in which Hsf4 gene was knocked out [17]. Interestingly the structure of Hsf4null retina looked normal at 10 dpf except the ONL of Hsf4null retina became thinner at 13 mpf as compared to that in wild-type (Fig. 1G). Those results demonstrated that Hsf4 is essential for retinal homeostasis. To determine whether HSF4-mutant affects the visual function, we performed the ERG to Hsf4del vs. wild-type mice at P15, 3 and 7 months old. The results showed that Hsf4del mice exhibited low amplitude of a- and b-wave at dark adapted 0.01 cd-s/m2 or 3.0 cd-s/m2 as compared to wild-type mice regardless of mouse ages (Fig. 1H and I). Since no cataract was observed in P15 Hsf4del mice, we proposed that the changed ERG in Hsf4del mice was due to the dysfunction of retina but not the cataracts. Taken together, these results demonstrated that the Hsf4del mutant impaired the vision at postnatal ages, and triggered the retina to undergo age-related atrophy.
Hsf4 del mice and Hsf4null zebrafish attenuate photoreceptor cells in an age-related manner
Since Hsf4del mice exhibited the abnormal ERG at postnatal age, we further studied the regulation of Hsf4 on the photoreceptor cells. we compared the photoreceptor cells of Hsf4del mice to wild-type mice in postnatal age P10 and adult mice of 7 and 13 months old. The biomarkers of Rhodopsin (RHOD) for rod cells or OPN1/SW and OPN1/MW for cone cells were measured by immunofluorescent staining. The results indicated that the expression of RHOD, OPN1/SW, and OPN1/MW, which exhibited no difference between Hsf4del and WT retina in P10 old mice, was significantly downregulated in 7 and 13 months Hsf4del retina as compared to that in the wild-type (Fig. 2A), and these changes were confirmed again at both protein and mRNA levels by using immunoblotting (Fig. 2B-E) and qRT-PCR (Fig. 2F-H). In zebrafish models, Hsf4null retina downregulated the expression of RHOD, GNB1 and GNAT2 at 7 mpf, and 13mpf as compared to wild-types at both protein (Fig. 2I-K) and mRNA levels (Fig. 2L and M). These results are consisted with the results of H&E in Fig. 1G. Taken together, these results confirmed again that dysfunction of HSF4 results in photoreceptor cells atrophy with age increase.
Hsf4 del activates glial cells and inflammation in postnatal retina, and this regulatory effect is enforced with mice ageing
Glial cells play an important role in modulating retinal homeostasis. Abnormal activation of the glial cells is associated with the retinal injury by upregulating the inflammation[25]. To determine whether gliosis is associated with disordered retina of Hsf4del, we measured the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) in the retina of HSF4del mice vs wild-types at P1, P10, 7 and 13 months old. The immunofluorescent results showed that both GS and GFAP proteins were slightly upregulated in P10 HSF4del retina and this upregulation was enforced in 7- and 13- months HSF4del retina as compared to that in wild-type (Fig. 3A). The results of immunoblot and qRT-PCR showed that the expression of GS, which was downregulated in P1 HSF4del retina at both protein and mRNA levels as compared to wild-types (Fig. 3B, D and F), was upregulated at protein and mRNA levels in P10 HSF4del retina, and the upregulation was enforced in 7- and 13-months Hsf4del retina. (Fig. 3B, D and F). It is unclear the mechanism underlying the downregulation of GS in P1 Hsf4del retina. GFAP, which is no difference between HSF4del and wild-type retina at P1 day, were upregulated at both mRNA and protein levels in HSF4del retina as compared to wild-types at P10, 7 and 13 months old (Fig. 3B, C and E). These results suggested that HSF4del mutation activates gliosis in the postnatal retina, and the activation was enforced with age increase. Since activation of glial cells is associated with non-infectious inflammation, which is the pathological cause of retinopathy and neuronal diseases, we then measured the expression of inflammatory factors using qRT-PCR. The results showed that the expression of IL-1β, IL-6, IL-8 and VEGFA were significantly upregulated in P10, 7 and 13 months HSF4del retinal tissues as compared to that in wild-types (Fig. 3G and H). These results suggested that Hsf4del mice upregulated the expression of inflammatory interleukins in the retina at early postnatal age. In addition to glial cells, the RPE cells were also the resource of inflammatory interleukins in the retina. we then separated the RPE from neuronal tissues of P10 Hsf4del vs. wild-type mice. The qRT-PCR results showed that the expression of IL-6 and VEGFA were upregulated in both RPE and neuronal tissues of the Hsf4del retina as compared to that in wild-type counterparts (Fig. 3I and J). These results suggested that both RPE and glial cells were the resource of inflammatory factors in Hsf4del mice. To confirm the activation of gliosis is relying on HSF4, we performed a subretinal injection of AAV-Hsf4-Flag to 0.5 month Hsf4del mice followed by recovery for 2 weeks. The expression of glial proteins and inflammatory cytokines were measured by using immunoblot and qRT-PCR. The results showed that ectopic expression of HSF4b partially downregulated the high expression of GS, GFAP, IL-1β, IL-6 and IL-8 at both protein and mRNA levels (Fig. 3K-N). To determine whether AAV2-hsf4b-Flag exerts any regulation on retinal structure, we performed a subretinal injection of AAV-Hsf4-Flag to one month Hsf4del mice followed by recovery for 2 months. The H&E staining results showed that administrated AAV2-Hsf4-Flag could partially reduced the disordered retinal structure of Hsf4del mice (Fig. S2B). Those results indicated that the activation of gliosis in Hsf4del retina is due to dysfunction of Hsf4b.
In zebrafish models, Hsf4null mutation slightly upregulated the expression of glial GFAP, GS, and inflammatory interleukins (including IL-1, Il-6 and IL-8) in 10 dpf and 7mpf, and this upregulation was enforced at 13 mpf zebrafish retina as compared to wild-type counterparts (Fig. 3, O-S). Taken together, these results suggested that dysfunction of HSF4 activates the gliosis in the retina at early postnatal age.
In the results of Fig. 1B, The PAS staining results showed that there were more small blood vessels in the aged Hsf4del retina than in wild-types. This upregulation was then confirmed by using immunohistochemistry staining with antibodies against CD31 and IB4 (Fig.S2A). Accordingly, we proposed that the upregulation of angiogenesis in the aged Hsf4del retina is associated with the high expression of VEGFA (Fig. 3H and I).
HSF4 del mice downregulate the expression of visual cycle enzymes in RPE
The retina of Hsf4del mice exhibits lipofuscin deposite in RPE and the abnormal upregulation of cytokines and VEGF expression in RPE cells (Fig. 3), implying that HSF4 participates in regulating RPE cell homeostasis. Since RPE’ s predominant function is to modulate visual cycle, we then tested the potential regulatory effect of Hsf4 on the visual cycle by measuring the expression of visual cycles proteins, such as RPE65, RLBP1 and RDH5 in Hsf4del vs wild-type retina at P1, P10, 7 and 13 months old [26]. Using the immunofluorescent and RPE whole mount staining, we found that the expression of RPE65, RDH5 and RLBP1 in RPE were downregulated in P1 and P10 HSF4del retina as compared to that in wild-type, and the downregulation was enforced with age increase (Fig. 4A and Fig. S3A). This downregulation was also confirmed by the immunoblot results followed by densitometry quantitation (Fig. 4B-E), and the qRT-PCR (Fig. 4F-H). To confirm the regulatory role of HSF4, we tested the expression of these visual-cycle proteins in the AAV2-Hsf4b-Flag-reconstituted Hsf4del retina. AAV2-Hsf4b-Flag were subretinally injected to 0.5 month Hsf4del retina followed by two weeks recovery. The expression of RPE65, RLBP1 and RDH5 was tested by immunofluorescence, immunoblot and qRT-PCR. The results showed that ectopic expression of HSF4b-Flag rescued the low expression of RPE65, RDH5 and RLBP1 in both protein (Fig. 4I-K) and mRNA levels (Fig. 4L) in Hsf4del retina. the immunofluorescent results showed that the subretinal injected AAV2-Hsf4-Flag was predominantly expressed in RPE cells (Fig. S3B). These results suggested that HSF4 participates in regulating the expression of visual cycle enzymes in RPE. To further confirm the regulatory effect of Hsf4 on the visual cycle-associates enzymes, we isolated and in vitro cultured the primary RPE cells from 2-months-Hsf4del retina or wild-type. After that, the plasmid pCMV-3xFlag-Hsf4b was transiently transfected into the primary RPE/Hsf4del cells (Fig. 4M and N). The immunoblot results showed that the expression of RPE65, RDH5 and RLBP1 was downregulated in RPE/Hsf4del cells as compared to that in RPE/wt. The overexpression of Hsf4b-Flag restored the expression of those enzymes (Fig. 4M and N). These results confirmed again that HSF4 participates in regulating the expression of visual cycle proteins in RPE cells.
Furthermore, we verified this regulation in Hsf4null zebrafish. The Hsf4null lens exhibited transparency at 4 and 10 dpf, and opacification in 7 and 10 mpf (Fig. S3C). Consistent with the results in Hsf4del mice, the expression of visual cycle proteins RPE65, RDH5 and RLBP1 was downregulated at both mRNA(Fig. S3D) and protein levels (Fig. S3E-H) in 4 dpf, 10dpf, 7mpf and 13 mpf Hsf4null retina as compared to wild-type. Taken together, those results demonstrated that dysfunction of HSF4 impairs the expression of visual cycle enzymes at very early postnatal age.
HSF4 del mice triggers cellular senescence or apoptosis in the retina.
The previous data showed that HSF4del mutation upregulated P21cip1 expression, which triggered lens epithelial cells to undergo senescence[13]. In the results of Fig. 3, Hsf4del retina expressed a high level of the inflammatory interleukins at the postnatal age, implying that HSF4del might cause cells to undergo senescence or apoptosis in the retina. To prove this, we immunoblotted the expression of P21cip1 and P16INK4a in the retina of Hsf4del vs. wild-types. P16INK4a was upregulated at both protein and mRNA in Hsf4del retina of P10, 7 and 13 months old mice but not in P1 mice (Fig. 5A, B and D). P21cip1 is slightly upregulate in P1 Hsf4del retina as compared to that in wild-type, and aging underpinned the upregulation (Fig. 5A, C and E). Reconstitution of AAV2-HSF4b-Flag to one-month HSF4del retina partially downregulated P21cip1mRNA expression (Fig. 5F). In addition, the upregulation of P16INK4a and P21cip1 mRNA was also observed in zHsf4null retina at 4dpf, 10dpf, 7mpf and 13mpf as compared to that in wild-type counterparts (Fig. 5G and H). These results indicated that dysfunction of Hsf4 upregulated CKIs expression in retinal tissues. To determine whether Hsf4del retina undergo apoptosis, we then performed TUNEL assay. The TUNEL-positive cells were much more in 7 -and 13- months retina than that in P10 Hsf4del retina (Fig. 5I). Few TUNEL- positive cells were observed in the wild-type retina (Fig. 5I). Those results indicated that dysfunction of HSF4 elevated the cells’ senescence or apoptosis in ONL and RPE.
HSF4 del mice deregulate the expression of heat shock proteins in the retina
The fundamental function of Hsf4 is to regulate the expression of heat shock proteins. In lens tissue, HSF4 exerts a dual regulation on heat shock protein expression. It upregulates the expression of small heat shock proteins, such as Hsp25, Cryab and γ-crystallins, and simultaneously downregulates the expression of Hsp70, FGF4/7[9, 11, 16]. However, its regulatory effects on the expression of heat shock proteins in the retina remain unclear. The immunoblot results showed that HSF4del mutation downregulated the expression of Hsp90 and Hsp25, but upregulated CRYAB expression at both protein (Fig. 6A-D) and mRNA levels (Fig. 6E-G) in the retina of P1, P10, 7 M and 13 M mice as compared to that in wild-type counterparts. The results of immunofluorescent and RPE whole mount staining assays indicated that CRYAB expression, was significantly upregulated in Hsf4del retina as compared to wild-type (Fig. S4, A and B). The recapitulation of AAV2-Hsf4b-Flag into one-month Hsf4del retina restored the changed expression of heat shock proteins at both protein and mRNA levels (Fig. 6H-J). Since Hsf4b-Flag is predominantly expressed in RPE cells (Fig S3B), we further tested the regulation of Hsf4 on those heat shock proteins’ expression in the primary cultured RPE cells in vitro. As the results indicated in Fig. 6K, the expression of Hsp90 and Hsp25 was downregulated and CRYAB expression was upregulated in RPE/Hsf4del cells as compared to that in RPE/wt cells (Fig. 6K, lanes 1–6 and L). Overexpression of Hsf4b-Flag partially restored the expression change of Hsp90, Hsp25 and CRYAB in the RPE/Hsf4del cells (Fig. 6K, lanes 4–9 and L). These results confirmed that Hsf4b exhibits a distinctive regulation on different heat shock proteins’ expression in RPE cells. In addition, we also tested the expression of heat shock proteins in the retina of Hsf4null vs. wild-type zebrafish. Deficiency of Hsf4 upregulated CRYAB and downregulated Hsp25 in the retina of 4dpf, 10dpf, 7mpf, and 13mpf zebrafish compared to wild-type (Fig. 6M-O). In contrast, Hsf4null downregulated Hsp90 only in 7 and 13mpf zebrafish retina, but not in embryonic zebrafish (Fig. 6M). These results suggested that dysfunction of HSF4 deregulated the expression pattern of heat shock proteins during retinal development.