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Divergent Effects of HSP70 Overexpression in Photoreceptors During Inherited Retinal Degeneration

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Divergent Effects of HSP70 Overexpression in Photoreceptors During Inherited Retinal Degeneration Biochemistry and Molecular Biology Divergent Effects of HSP70 Overexpression in Photoreceptors During Inherited Retinal Degeneration Ke Jiang, Elizabeth Fairless, Atsuhiro Kanda, Norimoto Gotoh, Tiziana Cogliati, Tiansen Li, and Anand Swaroop Neurobiology, Neurodegeneration, and Repair Laboratory (NNRL), National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States Correspondence: Anand Swaroop, PURPOSE. Disruption of proteostasis is a key event in many neurodegenerative diseases. NNRL, National Eye Institute, Heat shock proteins (HSPs) participate in multiple functions associated with intracellular National Institutes of Health, transport and proteostasis. We evaluated the effect of augmented HSP70 expression in MSC0610, 6 Center Drive, Bethesda, mutant photoreceptors of mouse retinal degeneration models to test the hypothesis that MD, USA; failure to sustain HSP70 expression contributes to photoreceptor cell death. [email protected]. Ke Jiang, NNRL, National Eye METHODS. We examined HSP70 expression in retinas of wild-type and mutant mice by Institute, National Institutes of RNA and protein analysis. A transgenic mouse line, TgCrx-Hspa1a-Flag, was generated Health, MSC0610, 6 Center Drive, to express FLAG-tagged full-length HSP70 protein under control of a 2.3 kb mouse Crx Bethesda, MD, USA; promoter. This line was crossed to three distinct retinal degeneration mouse models. [email protected]. Retinal structure and function were evaluated by histology, immunohistochemistry, and Current affiliations: electroretinography. EF: Yale School of Medicine, Yale University, New Haven, Connecticut, RESULTS. In seven different mouse models of retinal degeneration, we detected tran- United States; sient elevation of endogenous HSP70 expression at early stages, followed by a dramatic AK: Laboratory of Ocular Cell reduction as cell death ensues, suggesting an initial adaptive response to cellular stress. Biology and Visual Science, Augmented expression of HSP70 in RHOT17M mice, in which mutant rhodopsin is Department of Ophthalmology, misfolded, marginally improved photoreceptor survival, whereas elevated HSP70 led to Faculty of Medicine and Graduate more severe retinal degeneration in rd10 mutants that produce a partially functional School of Medicine, Hokkaido PDE6B. In Rpgrip1−/− mice that display a ciliary defect, higher HSP70 had no impact on University, Sapporo, Hokkaido, photoreceptor survival or function. Japan; NG: Center for Genomic Medicine, CONCLUSIONS. HSP70 overexpression has divergent effects in photoreceptors determined, Kyoto University Graduate School of at least in part, by the nature of the mutant protein each model carries. Additional inves- Medicine, Kyoto, Japan; tigations on HSP pathways and associated chaperone networks in photoreceptors are TC: Ophthalmic Genetics and Visual needed before designing therapeutic strategies targeting proteostasis. Function Branch, National Eye Institute, National Institutes of Keywords: photoreceptors, retinal degeneration, heat shock protein, proteostasis, chap- Health, Bethesda, Maryland, United erone States. Received: June 13, 2020 Accepted: October 2, 2020 Published: October 27, 2020 Citation: Jiang K, Fairless E, Kanda A, et al. Divergent effects of HSP70 overexpression in photoreceptors during inherited retinal degeneration. Invest Ophthalmol Vis Sci. 2020;61(12):25. https://doi.org/10.1167/iovs.61.12.25 nherited retinal degenerative diseases (IRDs), charac- essential for cell’s health and survival. Given the highly I terized by progressive dysfunction or loss of photore- active phototransduction cascade and continuous renewal of ceptors, constitute a major cause of vision impairment, outer segments (OS), maintenance of proteostasis is critical which severely impacts patients’ quality of life. Although for healthy photoreceptors and for the survival of stressed much progress has been achieved with mutations identi- photoreceptors in IRDs. Disrupted cellular proteostasis and fied in over 200 genes,1 we lack a precise understand- resulting apoptosis driven by the unfolded protein response ing of pathogenic mechanisms and effective treatments for (UPR) are among several hypotheses to explain the mech- most IRDs. Proteostasis, defined as the appropriate produc- anism underlying photoreceptor death in degenerating tion, folding, trafficking, and degradation of proteins, is retina.2–5 Copyright 2020 The Authors iovs.arvojournals.org | ISSN: 1552-5783 1 This work is licensed under a Creative Commons Attribution 4.0 International License. Downloaded from iovs.arvojournals.org on 09/28/2021 HSP70 Augmentation in Degenerating Photoreceptors IOVS |October2020|Vol.61|No.12|Article25|2 Molecular chaperones, especially the heat shock protein photoreceptors35 compared with whole retina36 and then (HSP) family, act to support cellular proteostasis by restoring examined HSP70 protein levels in several retinal degen- protein function under physiological conditions or by trig- eration mouse models. We augmented HSP70 expression gering programed cell death under stress.6,7 The HSP super- in the photoreceptors of RHOT17M, Rpgrip1−/−,andrd10 family is classified by apparent molecular mass into subfam- mice by crossing them with a transgenic mouse line, TgCrx- ilies. Eight HSP70 proteins identified in humans, belong- Hspa1a-Flag, that overexpresses HSP70. Our studies demon- ing to the 70-kDa subfamily, share 52% to 99% amino acid strate divergent effects of HSP70 expression in degenerating sequence homology and exhibit similar functions yet some- photoreceptors carrying different mutations and highlight what distinct characteristics.8 Hereafter we use the term the importance of distinct approaches when targeting cellu- “HSP70” to collectively refer to two proteins, HSP70-1a and lar proteostasis for therapy. HSP70-1b, which only differ from each other by two amino acid residues and are encoded in mice by Hspa1a and ETHODS Hspa1b genes, respectively.8,9 HSP70 is one of the most stud- M ied chaperones, especially in the stressed retina. In addi- Mice tion to the stress-inducible HSP70, a cognate 70-kDa HSP HSC70 (Hspa8)10 is constitutively expressed. Under phys- Mice were reared under ∼200 lux white, fluorescent cyclic iological conditions, HSPs protect and improve folding of (12 hr/12 hr light/dark cycle) light conditions following newly synthesized proteins, participate in quality control, recommendations of the Guide for the Care and Use of guide their translocation into distinct organelle destina- Laboratory Animals, the Institute of Laboratory Animal tions, and assist in the assembly/disassembly of multipro- Resources, and the Public Health Service Policy on Humane tein complexes.11–13 When under stress, transcription regu- Care and Use of Laboratory Animals. All protocols were lator heat shock factor 1 (HSF1) activates expression of vari- approved by the Animal Care and Use Committee of the ous HSPs, including HSP70.14 HSP70 binds to misfolded National Eye Institute (NEI ASP# 650). Animals were fed proteins and facilitates their recovery with assistance of NIH-31 Open Formula Mouse Diet (7017) (ENVIGO, Fred- other HSPs, such as HSP40 and HSP90, and co-chaperones erick, MD, USA). The following retinal degeneration mouse such as Hip and Hop.15,16 Alternatively, when the damage lines were used: TgRHOT17M37 (referred to as RHOT17M), − − is not repairable, HSP70 facilitates the degradation of aggre- Rpgrip1tm1Tili38 (Rpgrip1 / ), and Pde6brd10/rd10 (rd10)from gated proteins by delivering them to the ubiquitin protea- Jackson Laboratory (Bar Harbor, ME, USA). Other mouse some system with assistance of co-chaperone CHIP.17,18 models include: C57BL/6 (wild-type), Pde6brd1/rd1 (rd1), The nexus between disruption in cellular proteostasis Cep290rd16/rd16 (rd16),39 Prph2rd2/rd2 (rds) (Jackson Labora- − − − − and progression of neurodegenerative disease has uncov- tory), and Nrl / ;Nrl-GFP (Nrl / ).40 Both male and female ered an important role for chaperone proteins in regulat- mice were used in this study. ing the survival or death of stressed neuronal cells.19–21 Disease-causing proteins, such as β-amyloid and tau in Generation of TgCrxp-Hspa1a-Flag Mice Alzheimer disease, huntingtin in Huntington disease, α- A 2.3 kb mouse cone-rod homeobox (Crx) promoter synuclein in Parkinson disease, and mutant rhodopsin in (from −2286 to +72) and the Hspa1a-coding region with retinitis pigmentosa, all interact with the HSP chaperone an additional Kozak sequence and FLAG-tag were cloned machinery.20–22 Therefore HSPs are potential therapeutic into a modified promoter-less pCl vector (Promega, Madison, targets for neurodegenerative diseases. WI, USA). The Crx:Hspa1a-Flag fragment was purified and Although HSP70 deficient mice were generated over a injected into fertilized FVB/N mouse oocytes. Transgenic decade ago, the role of HSP70 in retinal development has founder mice and their progeny were identified by PCR. The not been investigated.23 In the adult retina, HSP70 maintains founders were backcrossed to C57BL/6J mice to establish a proteostasis and plays an essential role in the maturation of stable colony. TgCrxp-Hspa1a-Flag mice are referred to as phototransduction proteins.24,25 In cooperation with HSP90 TgHspa1a+, whereas littermate controls without the trans- and a photoreceptor specific co-chaperone AIPL1, HSP70 is gene are called TgHspa1a-. implicated in facilitating correct folding and assembly of retinal cGMP phosphodiesterase (PDE6) holoenzyme.26–28 Elevated HSP70 expression
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