Alpha-crystallins protect photoreceptors during retinal detachment through FAIM2 regulation

doi:10.21203/rs.3.rs-1997941/v1

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Alpha-crystallins protect photoreceptors during retinal detachment through FAIM2 regulation

https://doi.org/10.21203/rs.3.rs-1997941/v1

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We evaluated the role of α-crystallin in photoreceptor survival in an experimental model of retinal detachment, and its association with Fas apoptosis inhibitory molecule 2 (FAIM2). Retina/RPE separation was induced in rat and mouse eyes by the subretinal injection of hyaluronic acid, 1%. Retinal tissues were analyzed for the presence αA-crystallin and αB-crystallin proteins using immunohistochemistry and immunoblot. Indices of photoreceptor death were analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and cell counts. The 661W cells subjected to FasL were used as a cell model of photoreceptor degeneration to assess the mechanisms of the protective effect of αA-crystallin and its phosphorylation on T148. In addition to the associated cell-death, FAIM2 and αA-crystallin interaction were evaluated using co-immunoprecipitation assay. α-Crystallin protein levels were rapidly induced in response to retinal detachment, with αA-crystallin playing a particularly important role in protecting photoreceptors during retinal detachment. Our data also shows that FAIM2, an intrinsic neuroprotective protein in photoreceptors, interacts with α-crystallins following retinal detachment. Mechanistically, our work also demonstrated that the phosphorylation of αA-crystallin is essential for the interaction of αA-crystallin with FAIM2 and their neuroprotective effect. Thus, αA-crystallin is involved in the regulation of photoreceptor survival during retinal detachment, playing a key role in the stabilization of FAIM2, an important modulator of photoreceptor cell survival under chronic stress condition.

α-crystallin

Fas-apoptotic inhibitory molecule 2

FAS-mediated apoptosis

Retinal detachment

Photoreceptors

Retinal detachment (RD) is defined as separation of the neurosensory retina from the underlying retinal pigment epithelium (RPE). RD is a common form of injury and a component of many retinal diseases ¹. Retina-RPE separation results in the activation of death pathways within the photoreceptor cells, the light sensing elements within the retina, with the Fas pro-apoptotic pathway being a primary regulator of this death ²^,³. The effect of these pro-death pathways is attenuated, though, by the activation of pro-survival pathways including autophagy ⁴, interleukin-6 and hypoxia-inducible factors ⁵^,⁶.

Our previous studies have shown that RD increases photoreceptor levels of Fas-apoptotic inhibitory molecule 2 (FAIM2) in vivo, or after exogenous activation of Fas signaling in photoreceptor-like 661W cells in vitro ²^,⁷. FAIM2 is an evolutionarily conserved, 35 kDa membrane protein that is predominantly expressed in neuronal cells. FAIM2 prevents apoptosis by direct interaction with Fas upstream of Fas-associated death domain containing protein (FADD). Faim2 expression in neuronal cells, including cerebellar granule neurons and Purkinje cells, increases resistance to Fas-mediated apoptosis, oxygen-glucose deprivation, and cerebral ischemia in several experimental models of neuronal injury ^8-10.

Studies have reported a neuroprotective function of α-crystallin in the retina in diseases such as diabetes ¹¹ and in autoimmune uveitis ¹². Its role in RD, however, is less explored. A recent study reported that high levels of αB-crystallin were found in the vitreous of patients with rhegmatogenous retinal detachment ¹³. Crystallins have been shown to be highly upregulated in a rodent model of retinal tear, consistent with a potential protective response in the context of retinal injury ¹⁴. Additionally, a study by Hamadmad et al., 2019 reported that αΑ-crystallin is significantly upregulated following RD in mouse and humans ¹⁵.

We previously reported that several members of the crystallin family were induced at the mRNA level in the rat model of experimental retinal detachment after 7 days ¹⁶. However, the precise time-course of this induction and the exact function of crystallins during retinal detachment remain unknown. In this study we sought to evaluate the role of α-crystallin on photoreceptor survival and its association with FAIM2 in an experimental model of RD.

Animals. Long Evans rats and the various mice used in this study were housed under a 12h light/dark cycle with free access to standard chow and water. áA- and áB-crystallin knockout mice were previously characterized and described ¹⁷^,¹⁸ and a generous gift from Dr. Wawrousek of the National Eye Institute (NEI). All animals were checked as negative for the rd8 mutation and genotyped as previously described ¹⁷^,¹⁸. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research while the animal protocol was previously verified and approved by the University Committee on Use and Care of Animals of the University of Michigan.

Experimental model of retinal detachment. Retinal detachments were created in rats and mice per the protocol previously used and described in ². The animals were anesthetized with a mix of ketamine (100 mg/ml) and xylazine (20 mg/ml) prior to dilation of the pupils with topical phenylephrine (2.5%) and tropicamide (1%). A sclerotomy was made with a 25-gauge needle approximately 1–2 mm posterior to the limbus paying particular attention to avoid damaging the lens. Subsequently, a subretinal needle was introduced through the sclerotomy into the vitreous cavity and then through a peripheral retinotomy into the subretinal space. The neurosensory retina was detached from the RPE by slowly injecting Sodium hyaluronate (10 mg/ml, Abbott Medical Optics, Abbott Park, IL, USA, Healon OVD). The injection was performed until approximately one-third to one-half of the neurosensory retina was detached in the left eye. Except for introduction of the subretinal injector and injection of the sodium hyaluronate, the right eye was subjected to the same procedure and served as the internal control.

Immunoblot. Retinas were dissected from the RPE-choroid at various time points and prepared by homogenization in RIPA lysis buffer as previously described ¹⁹. Protein concentrations were assessed with the Pierce BCA reagent in order to adjust all samples for equal protein amount. Retinal lysates were analyzed using the following antibodies: αA-crystallin (Santa Cruz, Cat No: sc-28306) and αB-crystallin (Enzo life Sciences, Cat No: ENZ-ABS706 ) and FAIM2 (Origene, Cat No: TA-317786 ) in 1:1000 in TBST. Immunoblots were performed as previously described ²⁰ but using NuPage gels 4-12% and MES buffer following the manufacturer’s instructions. Results were normalized by probing the same membrane with an antibody against ß-actin (Millipore) as the loading control.

Immunohistochemistry. Immunohistochemical labeling was carried out using the indirect immunofluorescence method as previously described ². For this procedure, whole eyes were recovered at various time points after retinal detachment and fixed in 4% paraformaldehyde overnight at 4°C. The whole globes were then placed in a tissue processor (Tissue-Tek II, Sakura, Tokyo, Japan) for standard paraffin embedding. A standard paraffin microtome was then used to obtain 10 μm thick sections. Immunohistochemistry was then carried out starting wtith epitope unmasking using Proteinase K Antigen Retrieval. The sections were stained with the same rabbit polyclonal antibodies for FAIM2, αA-crystallin or αB-crystallin listed for immunoblot followed by secondary goat anti-rabbit IgG antibody coupled to Alexa fluorophore 488 and 594 (Jackson ImmunoResearch, West Grove, PA). Cell nuclei were counterstained using Hoescht. No specific staining could be detected in the negative controls prepared by omitting the primary antibody during the incubation (data not shown).

Cell death quantification. Apoptosis was measured by DNA Fragmentation ELISA (Roche Diagnostics) as previously described ²¹. In brief, the retinal tissue or cells were mechanically homogenized in lysis buffer prior to being incubated rocking for 30 min, and then centrifuged at 1,000g for 10 min at room temperature. The supernatant, as well as of the positive and negative controls (20µl each) were then moved into the ELISA plate before adding the immunoreagent complex. After the manufacturer’s recommended incubation and washes, the colorimetric solution was added. Following development of the colorimetric reaction, the stop solution was added and the colorimetric signal was measured with excitation at 405 and normalized to the reference wavelength of 490nm using a 96-well plate reader (SpectraMax Gemini EM, Molecular Devices).

Alternatively, cell death was measured by terminal transferase dUTP nick end labeling (TUNEL) with horseradish peroxidase detection as described previously ²². TUNEL-positive cell counts were expressed per surface area relative to the extent of detachment.

Cell culture. All cell experiments were repeated 3 times, each time including 3 individual technical replicates. The 661W photoreceptor cell line is a generous gift by Dr. Muayyad al-Ubaidi (Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA). The 661 W cell line was maintained as previously described ² in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 300 mg/l glutamine, 32 mg/l putrescine, 40 μl/l of β-mercaptoethanol, 40 μg/l of both hydrocortisone 21-hemisuccinate and progesterone, penicillin (90 units/ml) and streptomycin (0.09 mg/ml). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

Transfection. Cells were transfected with either empty vector, wild type, phosphomimetic (T148D) or non-phosphorylatable (T148A) form of αA-crystallin, using the Neon Transfection System (Invitrogen) as previously described ¹¹. After being trypsinized and washed in PBS, the cells were resuspended in suspension buffer and electroporated with 2.5μg of the appropriate plasmid. Cells were then plated in 100mm dishes before proceeding to further experimental procedures.

Fas receptor activation. The cells were treated with Fas ligand (FasL) as previously described ². Specifically, the cells were incubated with 500 ng/ml FasL (Recombinant Mouse Fas Ligand/TNFSF6 Protein Cat#: 6128-SA-025R&D Systems Inc., Minneapolis, MN, USA) and 250 ng/ml HA (Hemagglutinin/HA Peptide Antibody Cat. #: MAB060 R&D Systems Inc., Minneapolis, MN, USA) for 8 hours. At the end of incubation, cells were harvested and prepared as described below for immunoprecipitation analysis.

Immunoprecipitation. 50 μL of Dynabeads protein G suspension (Dynabeads® Protein G Immunoprecipitation Kit, 10007D, Life Technologies Corporation) previously preincubated with FAIM2 antibody (OriGene) for 1 hr at room temperature was added to 500μg of retinal or cell culture lysates and incubated at 4°C overnight. Following incubation, the beads were collected with a magnetic device andthe bead-antibody-antigen complex was washed with immunoprecipitation buffer with as previously described ². After the washes, the antibody-antigen complex was eluted with 30 μL of premixed Nupage LDS sample buffer and elution buffer and analyzed by immunoblot as described above.

Statistical Analysis and ARRIVE guidelines. Per the editorial policy, the described study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Power analysis were performed to calculate the number of animals necessary based on previous studies with the retinal detachment model and other studies with alpha-crystallin knockout animals suggesting that groups of 6 were expected to be sufficient for expression and 8 for cell death effects. Exclusion criteria were established a priori and only included ocular infection and retinal tear post-retinal detachment procedure. Investigators could not be blinded to the mouse strain at the time of the retinal detachment induction due to the lens aspect, however at the time of post-processing, samples were coded and the technician performing the analysis was blinded as to the genotype or condition. Data were normalized to the corresponding ß-actin signal for all immunoblot experiments before statistical analysis. ANOVA models with heterogeneous variances, adjusted for the replication of the experiment, were fit to the data to assess differences between detached and attached retinas from the different genotypes using Prism software. Reported in the figures are the means ± SEM and statistically significant differences. Analyses were performed using non-repeated measures ANOVA followed by the SNK test for multiple comparisons or t-test for a single comparison.

α-Crystallins are induced in the detached retina. Retinal detachment was created in Long Evans rats and its impact on α-crystallin expression was assessed to obtain a time course of expression at both the messenger RNA and protein levels. αA-crystallin messenger RNA levels showed a delayed and transient induction, peaking at 3 days post-detachment and almost back to normal after 7 days (Figure 1A), while αB-crystallin messenger demonstrated a rapid induction, detectable as early as 1-day post-detachment and consistent all the way to 7 days, the latest time point we analyzed (Figure 1B). We further analyzed the protein expression levels of α-crystallins in response to retinal detachment. As previously shown ¹¹, αA- and αB-crystallins are basally expressed in the retina at very low levels, however their levels increased significantly starting 1 day after retinal detachment (Figure 1C). The rapid increase in αA-crystallin levels could be detected as early as 2 hours after detachment (data not shown). While less pronounced, this increase in protein level remained significant up to 7 days post detachment, the latest time-point analyzed in our study. αB-crystallin protein levels also increased in detached retinas. Similarly, to αA-crystallin, this increase could be detected after a few hours, with a peak after 1 day and started to resolve after 7 days of retinal detachment.

To test the implications of this upregulation of α-crystallins during retinal detachment, we created detachments in wild-type, αA- and αB-crystallin knockout mice. We first analyzed the impact of retinal detachment on the expression of αA- and αB-crystallin in mice and showed that like the rat model, αA- and αB-crystallin are rapidly elevated in response to retinal detachment, the increase being maximal 3 days post-detachment (Figure 2A). As expected, no αA-crystallin could be detected in the αA-crystallin knockout (Figure 2B), only a faint signal being detected at Day 3 and corresponding to the known cross-reactivity of the αA-crystallin antibody with αB-crystallin. Similarly, no αB-crystallin could be detected in the αB-crystallin knockout (Figure 2C), the only signal being due to some slight cross-reactivity with αA-crystallin (double band). Interestingly, αB-crystallin induction was reduced in intensity and only detectable 3 days post-detachment in αA-crystallin deficient mice when compared to wild-type animals (Figure 2B). Conversely, the αB-crystallin knockout showed a sustained increased expression of αA-crystallin in response to retinal detachment, in addition to the previously described basal induction in this strain, which is visible in the attached retina (figure 2C) ¹¹.

Loss of αA-, but not αB-crystallins leads to increased photoreceptor death after retinal detachment. We further analyzed the impact of retinal detachment on photoreceptor cell survival by measuring the density of TUNEL positive cells at 3 days post-detachment when cell death is maximal, or by measuring the thickness of the outer nuclear layer 2 months post RD. In both cases, our data strongly support a protective role of αA-crystallin, as demonstrated by increased number of TUNEL positive cells (Figure 3A) and further reduction of ONL thickness (Figure 3B) in αA-crystallin deficient mice when compared to age-matched wild-type animals. Consistent with previous findings in another model of retinal degeneration ¹¹, lack of αB-crystallin, which is associated with basal increase of αA-crystallin, did not significantly change the rate of retinal detachment-induced photoreceptor degeneration, consistent with a protective role for αA-crystallin. The αA- and αB-crystallin double-knockout mice demonstrated increased photoreceptor loss after retinal detachment compared to WT controls, similar to the phenotype seen αA-crystallin knockout mice (Figure 3).

α-Crystallins are induced and accumulate in photoreceptor inner and outer segments in the detached retina. We further analyzed the cellular localization of αA- and αB-crystallin by immunofluorescence analysis on retinal cross-sections. As previously reported ¹¹, both αA- and αB-crystallins are mainly expressed at low levels in the ganglion cell layer in the attached retina (Figure 4A). Analysis of the retina 24 hours post-detachment showed that αA-crystallin staining was predominantly increased in the outer retina (Figure 4B), more specifically in the inner and outer segments of photoreceptors in the detached area as shown in the higher magnification images of attached (Figure 4C) and detached regions (Figure 4D). αB-crystallin was also induced in the outer retina, but throughout the photoreceptor cells from the outer segments, all the way to the outer plexiform layer (Figure 4E-F).

FAIM2 expression in stressed photoreceptors requires the presence of α-Crystallins. We recently showed that FAIM2 is an important neuroprotective factor in photoreceptors during periods of retina-RPE separation ². To determine the potential role of crystallins in the regulation of FAIM2 expression in the retina, we assessed cellular localization of FAIM2 in WT and α-crystallin knockout mice. This analysis confirmed an increased immunoreactivity (Figure 5A-B) in the photoreceptor outer segments following retinal detachment in WT animals and is represented with higher magnification images of attached (Figure 5I) and detached regions (Figure 5J). In contrast, no change in FAIM2 levels were detected in single αA-crystallin (Figure 5C-D) or αB-crystallin (Figure 5E-F) or dual αA/αB-crystallin knockout (Figure 5G-H) retinas following retinal detachment (Figure 5 D, F and H respectively). These findings strongly suggest that increased FAIM2 protein in photoreceptors after retinal detachment requires the presence of αA- and αB-crystallins.

Increased interaction of FAIM2 with αA-crystallin after retinal detachment. To confirm the role of αA- and αB-crystallins in the regulation of neuroprotective factors, we immunoprecipitated FAIM2 and assessed the pull-down of αA- or αB-crystallin shortly after experimental retinal detachment. We first demonstrated that both αA- and αB-crystallin showed some interaction with FAIM2 in the attached retina (Figure 6 – A lanes). However, while a clear increased association of FAIM2 with αA-crystallin was detected (Figure 6 – left panel), there was a decrease in FAIM2 association with αB-crystallin in response to experimental retinal detachment (Figure 6 – right panel).

FAIM2 interaction with αA-crystallin in photoreceptors during Fas activation is T148 phosphorylation dependent. Photoreceptor death following retina-RPE separation is mediated by Fas receptor signaling and activation of apoptosis ³. Fas-ligand induced apoptosis of 661W cone-like cells mirrors this in vivo finding closely and has been used as a robust in vitro model ²^,⁴. Consistent with the protective role of αA-crystallin and its phosphorylation on T148, overexpression of either wild-type or the phosphomimetic form (T148D) of αA-crystallin reduced FasL-induced cell death by more than 40% when compared to empty vector transfected cells (Figure 7A). Counting the number of pyknotic cells showed that 8 hours of FasL lead to an average of 110 cells in EV transfected cells compared to 58, 63 and 106 in WT, T148D and T148A transfected 661W cells respectively. To further assess the role of phosphorylation of αA-crystallin on residue 148 with FAIM2 interaction during Fas activation in photoreceptors, we immunoprecipitated FAIM2 and assessed its interaction with wild type, phosphomimetic (T148D) or non-phosphorylatable (T148A) form of αA-crystallin in 661W in vitro photoreceptor cell model. The data also demonstrated that phosphorylation of αA-crystallin on residue 148 led to the increased interaction with FAIM2 in Fas activated photoreceptors, whereas pull-down of the non-phosphorylatable form was significantly reduced (Figure 7B). The observation suggests that phosphorylation on residue 148 of αA-crystallin is essential for its interaction with FAIM2 and thus plays a key role during Fas-mediated cell death in photoreceptors.

In the current study, we directly tested the role of αA- and αB-crystallin in photoreceptor survival in the context of experimental retinal detachment as a model of acute retinal injury. We demonstrate that both proteins are rapidly induced in response to retinal detachment in both rat and mouse models, and αA-crystallin’s absence is associated with increased photoreceptor cell death, while loss of αB-crystallin isn’t, most likely due to compensation by αA-crystallin. Our data shows that FAIM2, an intrinsic neuroprotective protein in photoreceptors, interacts with αA and αB crystallins following retinal detachment. This interaction may be important for stabilizing FAIM2 and increasing total protein levels in photoreceptors, facilitating their ability to resist apoptotic stress after retinal detachment. We further analyzed the regulation of αA-crystallin protective function and demonstrated that the newly described phosphorylation on S/T148 is key for its protective effect during Fas activation in photoreceptors, and that this in part may be driven by its interaction and regulation of FAIM2 function.

Our study has demonstrated the marked increase in the protein level of α-crystallin in the experimental retinal detachment model. The finding is consistent with previous reports of up-regulation of α-crystallins in retinal degeneration mouse model ²³ and in the retina of human donors with age-related macular degeneration ²⁴^,²⁵. Consistent with the key role of αA-crystallin in the retina, a recent study by Hamadmad et al has reported that post retinal detachment αA-crystallin displayed the highest level of increase among all the other crystallins in the mouse. They also reported a significant increase in protein levels of αΑ-crystallin in the vitreous of retinal detachment patients compared to their control counterparts ¹⁵.

Following experimental retinal detachment, α-crystallins were induced and accumulated in photoreceptor inner and outer segments. This localization is consistent with another study at later stages of retinal detachment that reported that αΑ-crystallin expression is highly increased compared to control and that its immunoreactivity is found primarily in the inner and outer segments of the photoreceptors, with slightly more diffuse staining in other retinal layers ¹⁵. This is also consistent with another disease mostly impacting the outer retina, experimentally-induced uveitis, in which αA-Crystallin is also highly upregulated in the retina and is primarily localized in the photoreceptor inner segments ¹².

The protective role of αA-crystallin reported in our study is consistent with multiple previous studies reporting that αA-crystallin up-regulation plays a protective role in the suppression of photoreceptor apoptosis associated with intraocular inflammation ²⁶, experimental autoimmune uveitis ²⁷ and Rpe65⁻/⁻ mouse model of Leber's congenital amaurosis ²⁸. On a mechanistic level, we previously reported that photoreceptor apoptosis is accelerated in Faim2 knockout mice following experimental retinal detachment. We also reported that FAIM2 is primarily involved in reducing stress-induced photoreceptor cell death, but that this effect was transient ²^,⁷. The transient nature of the effect of FAIM2 is similar to the one we recently reported relative to αA-crystallin during another chronic condition: diabetes ¹¹. Thus, we decided to assess in the current study the functional relationship between FAIM2 and αA-crystallin in photoreceptors after retinal detachment. We report here that while basal level of interaction between FAIM2 and αA- and αB-crystallin was observed in the attached retina, a significant increase in FAIM2 and αA-crystallin association was detected following retinal detachment.

α-Crystallins are known to undergo numerous post-translation modifications (PTMs), which further affect their chaperone activity ^29-32. We recently reported that the serine/threonine 148 residue of αA-crystallin is highly phosphorylated in the retina under normal conditions. Further, we uncovered that phosphorylation on this specific residue is significantly decreased in the retina of diabetic donors with retinopathy and that this serine/threonine 148 phosphorylation essentially controls the protective role of αA-crystallin ¹¹. Consistent with our previous study, we show that this phosphorylation is also essential for the interaction of αA-crystallin with FAIM2 in 661W cells.

In conclusion, our current study highlights a new mechanism by which αA-crystallin is involved in the regulation of photoreceptor survival during retinal detachment, playing a key role in the stabilization of FAIM2, an important modulator of photoreceptor cell survival under chronic stress conditions such as unresolved retinal detachment.

Author Contributions

M.N., J.Y., M.P., and A.M.M. collected and analyzed data and edited the manuscript. C.B., D.Z. and P.E.F. obtained funding, designed the experiments, analyzed data and wrote the manuscript.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Acknowledgements

This project and its authors were supported by grants from the NEI 5K08EY023982 and R01EY029675 (C.B.), Research to Prevent Blindness (C.B, D.Z. and P.E.F.), and NIH EY020895 (P.E.F.). This work utilized the Core Center for Vision Research funded by P30 EY007003 from the National Eye Institute. The authors thank Dr. Eric Wawrousek for providing the knockout mice.

Conflict of Interest

Dr. Besirli and Dr. Zacks have intellectual property interests and are listed as inventors on a patent for the use of FAIM2 as a retinal neuroprotective treatment. Dr. Fort has intellectual property interests and is listed as inventors on a patent for the use of α-crystallins as a retinal neuroprotective treatment.

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Competing interest reported. Dr. Besirli and Dr. Zacks have intellectual property interests and are listed as inventors on a patent for the use of FAIM2 as a retinal neuroprotective treatment. Dr. Fort has intellectual property interests and is listed as inventors on a patent for the use of α-crystallins as a retinal neuroprotective treatment.

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Alpha-crystallins protect photoreceptors during retinal detachment through FAIM2 regulation

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