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. Author manuscript; available in PMC: 2016 Jun 23.
Published in final edited form as: Cell Rep. 2015 Jun 11;11(11):1686–1693. doi: 10.1016/j.celrep.2015.05.023

Iron toxicity in the retina requires Alu RNA and the NLRP3 inflammasome

Bradley D Gelfand 1,2,3,*, Charles B Wright 1, Younghee Kim 1, Tetsuhiro Yasuma 1, Reo Yasuma 1, Shengjian Li 1, Benjamin J Fowler 1,4, Ana Bastos-Carvalho 1, Nagaraj Kerur 1, Annette Uittenbogaard 1, Youn Seon Han 1, Dingyuan Lou 1, Mark E Kleinman 1, W Hayes McDonald 5, Gabriel Núñez 6, Philippe Georgel 7, Joshua L Dunaief 8, Jayakrishna Ambati 1,4
PMCID: PMC4481133  NIHMSID: NIHMS692654  PMID: 26074074

Abstract

Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fenton's reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components Caspase-1/11 or Nlrp3 or by inhibition of Caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron.

Introduction

Iron is a critical component of dozens of enzymatic processes, and in excess can induce oxidative damage via Fenton's reaction, in which iron catalyzes the formation of highly reactive hydroxyl radicals. Accordingly, iron overload is implicated in the pathogenesis of numerous diseases including neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (Urrutia et al., 2014). Retinal iron overload is also implicated in the pathogenesis of age-related macular degeneration (AMD). For example, human eyes with AMD display increased iron deposition in the retina and aqueous humor (Flinn et al., 2014; Hahn et al., 2003; Junemann et al., 2013), and animal models of iron overload reproduce AMD-like phenotypes of the retinal pigmented epithelium (RPE) and outer retina (Flinn et al., 2014; Gnana-Prakasam et al., 2012; Hadziahmetovic et al., 2008; Hahn et al., 2004). Although the damaging effects of iron overload are often attributed to oxidative damage, the precise mechanisms driving iron-induced retinal toxicity have not been defined.

Here, we report a mechanism of iron-induced retinal degeneration unique among several Fenton-capable metals that requires the specific activation of the NLRP3 inflammasome, an innate immune signaling complex recently implicated in AMD pathogenesis. We also report that iron induces NLRP3 inflammasome signaling via induction of Alu RNAs derived from short interspersed nuclear elements (SINEs), which are endogenous inflammasome activators abundant in human AMD. These findings suggest that the intrinsic toxicity of excess iron in the retina depends upon the activation of SINE RNA-mediated innate immune signaling.

Results

Iron overload activates the NLRP3 inflammasome

Given the apparent role of iron overload in retinal degeneration, we sought to determine the effect of administering iron via subretinal injection on the RPE cell layer, which provides essential support to photoreceptors and the degeneration of which demarcates atrophic AMD. The NLRP3 inflammasome is an immune signaling complex implicated in the pathogenesis of AMD (Anderson et al., 2013; Kauppinen et al., 2012; Liu et al., 2013; Marneros, 2013; Tarallo et al., 2012; Tseng et al., 2013). In models of atrophic AMD, NLRP3 inflammasome activation induces cellular death via the inflammasome effector Caspase-1 (Tarallo et al., 2012). Therefore we sought to determine whether iron overload activated the NLRP3 inflammasome. We examined mice doubly deficient in genes encoding the cellular iron exporters Ceruloplasmin and Hephaestin (Cp−/−Heph−/−) that exhibit age-dependent retinal iron deposition and AMD-like pathologies (Hadziahmetovic et al., 2008; Hahn et al., 2004). Similar to previous reports of human geographic atrophy tissues (Tarallo et al., 2012; Tseng et al., 2013), we detected robust expression of the inflammasome-related gene Nlrp3 in the RPE layer of Cp−/−Heph−/− mice compared to age-matched wild-type controls (Figure 1A). We sought to determine whether acute administration of Fe(III) induced RPE degeneration and inflammasome activation. Seven days after subretinal injection of Fe(III), we detected degeneration of the RPE (Figure 1B) reminiscent of both human atrophic AMD and mouse models of NLRP3 inflammasome-induced RPE degeneration (Fowler et al., 2014; Kaneko et al., 2011; Tarallo et al., 2012). Both subretinal delivery of iron in wild-type mice and iron loading of human RPE cells induced Caspase-1 maturation (Figures 1C and 1D). To further investigate the anatomic location of inflammasome activation, we assessed in situ Caspase-1 activity in unfixed retinal cryo-sections by administering a Caspase-1 peptide substrate that becomes fluorescent upon cleavage. We observed elevated Caspase-1 proteolytic activity in the RPE cell layer of wild-type mice following iron subretinal injection (Figure S1A). We confirmed the signal was not due to accumulation of auto-fluorescent material in iron-treated wild-type mice without substrate and in iron-treated Caspase-1/11 knockout mice (Figure S1B). Together, these results implicate iron overload as an inflammasome agonist in the RPE. We next sought to determine whether iron induced inflammasome activation was due to Fenton catalytic activity. Other multivalent metal ions such as Cr(VI), Cu(I) and Zn(I) are also efficient Fenton catalysts. Loading RPE cells with metal ions induced free radical formation (Figure S1C). However, unlike Fe(III), loading human RPE cells with Cr(VI), Cu(I) or Zn(I) did not induce Caspase-1 maturation (Figure S1D). These findings indicate that overload of Fenton catalytic metals alone are not sufficient to drive inflammasome activation.

Figure 1. Iron toxicity depends on the NLRP3 inflammasome.

Figure 1

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(A) Immuno-labeling of Nlrp3 (yellow signal) and nuclei (DAPI, blue signal) in the RPE/choroid of 6-month old wild-type (top) or Cp−/−Heph−/− mice (middle). Bottom row depicts the same area from a serial section of Cp−/−Heph−/− immunolabeled with isotype goat IgG. Representative of N=3 mice. Scale bar denotes 20 μm. (B) Fundus (top) and ZO-1 staining of RPE flat mount preparations (bottom) of wild-type mice 7 days after injection of 1μL Fe(III) at indicated concentrations. Blue arrows denote injection site. (C and D) Western blotting of pro- and active forms of Caspase-1 in ARPE-19 cells and wild-type mouse retina and RPE/choroid (pooled from 4 eyes) treated with indicated doses of Fe(III). Fundus and ZO-1 staining of RPE flat mount preparations following delivery of Fe(III) into the subretinal space of mice lacking inflammasome components Caspase-1/11 (E) and Nlrp3 (F). (G) Fundus and ZO-1 staining of RPE flat mount preparations of wild-type mice that received Caspase-1 peptide inhibitor preceding delivery of Fe(III) into the subretinal space. (H, I) Fundus and ZO-1 staining of RPE flat mount preparations following administration of Cr(VI), Cu(I) or Zn(I) into the subretinal space of wild-type mice (H) and in mice lacking inflammasome components Caspase-1/11 (I). Indicated doses represent the minimum concentration of metals required to consistently induce RPE degeneration. (B-I) Scale bars denote 50 μm. Fundus images representative of at least 4 replicates.

Inflammasome is required for iron overload toxicity

We next sought to determine whether inflammasome signaling contributed to iron-induced RPE degeneration. Mice lacking the inflammasome components Caspase-1/11 or Nlrp3 were protected from RPE degeneration upon administration of iron compared to wild-type mice (Figures 1E and 1F). Interestingly, Nlrp3 ablation reduced RPE degeneration due to a high dose of iron injection, albeit less effectively than Caspase-1/11-deficiency, implying that in response to large doses of iron, compensatory Nlrp3-independent pathways that also require Caspase-1/11 contribute to RPE degeneration. Additionally, blockade of Caspase-1 activity via delivery of a cell-permeable peptide inhibitor prevented RPE degeneration due to Fe(III) administration in wild-type mice (Figure 1G). Together, these data implicate the NLRP3 inflammasome in mediating iron-induced retinal toxicity.

We next tested whether other metal ions capable of participating in Fenton's reaction induced retinal toxicity. As with Fe(III) each of Cr(VI), Cu(I) and Zn(I) were toxic to the RPE cell layer (Figure 1H). However, unlike Fe(III), the toxicity of Cr(VI), Cu(I) and Zn(I) overload persisted in Caspase-1/11 deficient mice (Figure 1I), suggesting that iron toxicity in this system is uniquely inflammasome-dependent.

Iron overload causes accumulation of Alu, B1 and B2 SINE RNAs

RNAs transcribed from ubiquitous Alu repetitive elements are inflammasome activators implicated in atrophic AMD (Dridi et al., 2012; Kaneko et al., 2011; Tarallo et al., 2012). We sought to determine whether iron overload induced Alu RNA accumulation. Iron overload induced elevated Alu RNA levels in human RPE cells (Figure 2A). Rodents also carry SINE repeats such as B1 and B2 elements that, like Alu repeats are derived from non-LTR, non-autonomous retrotransposons (Jurka et al., 2005). B1 and B2 RNAs can also induce inflammasome signaling and retinal degeneration (Kaneko et al., 2011; Tarallo et al., 2012). Subretinal delivery of iron induced a dose-dependent increase in B1 and B2 RNA levels in the RPE/choroid of wild-type mice but left Dicer1 RNA levels unchanged (Figure 2B). Fluorescent in situ hybridization of B1 and B2 RNAs in retina of 6-month-old Cp−/−Heph−/− mice revealed robust enrichment of these RNAs in multiple retinal layers including the RPE, choroid and neural retina of double knockout mice compared to age-matched wild-type controls (Figure 2C).

Figure 2. Iron toxicity depends on SINE RNA induction.

Figure 2

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(A) Densitometry of Alu RNA northern blotting and real-time qPCR of DICER1 mRNA in human ARPE-19 cells exposed to iron overload for 4 days at indicated doses. (B) Densitometry of B1 and B2 RNAs northern blotting and real-time qPCR of Dicer1 mRNA RPE/choroid lysates from wild-type C57BL6/J mice 7 days after subretinal injection with iron. (C) Fluorescent in situ hybridization of B1 and B2 RNAs in retinal cross-sections of 6-month old Cp−/−Heph−/− (top row) or wild-type mice (bottom row). Scale bar denotes 50 μm. (D) Western blotting of pro- and active forms of Caspase-1 in ARPE-19 cells exposed to Fe(III) after treatment with either scrambled or Alu RNA-targeted antisense oligonucleotides. (E) Representative fundus photographs and ZO-1 immunostained RPE flat mounts of wild-type mice treated with a cell-permeable antisense oligonucleotide targeting B2 RNA (and scrambled control) 1 day prior to subretinal injection of 10 nM Fe(III). Images were acquired 6 days after Fe(III) administration. Blue arrows denote injection site. Scale bar denotes 50 μm. For all panels, N=3-6, *P < 0.05, Error bars denote SEM.

We hypothesized that iron toxicity could be due to its ability to induce Alu RNAs. Administration of an Alu RNA-targeted antisense oligonucleotide in iron-treated human RPE cells abolished Caspase-1 maturation (Figure 2D), implicating Alu RNA as an intermediate in this process. We previously developed an antisense oligonucleotide targeted against B2 RNA that is chemically modified for in vivo cell entry and that prevents RPE degeneration due to SINE RNA accumulation in mice (Kaneko et al., 2011; Tarallo et al., 2012). Antagonizing B2 RNA in wild-type mice prevented iron-induced RPE degeneration in 4/6 eyes treated with B2 antisense (compared to 0/6 eyes treated with a scrambled antisense, Fisher's exact test P < 0.03) (Figure 2E). Collectively, these data support the concept that iron overload induces Alu RNA accretion and resulting inflammasome-mediated retinal toxicity.

Iron overload enhances DICER1-dependent Alu RNA stability

Alu RNA levels are controlled in part by the RNase DICER1 (Hu et al., 2012; Kaneko et al., 2011; Ren et al., 2012; Ren et al., 2013; Tarallo et al., 2012; Yan et al., 2013). We previously reported that DICER1-mediated Alu RNA clearance is essential for maintaining retinal health (Kerur et al., 2013; Tarallo et al., 2012). We confirmed that as with Alu RNAs, both B1 and B2 RNAs are substrates for DICER1 enzymatic cleavage (Figure S2). Because free iron generates reactive oxygen species and the abundance of DICER1 is reduced by oxidative stress (Kaneko et al., 2011; Mori et al., 2012; Wiesen and Tomasi, 2009), we predicted that iron-induced Alu RNA accumulation was due to transcriptional repression of DICER1 levels. Surprisingly, iron overload did not affect DICER1 levels in RPE cells or in wild-type mice (Figure 2A, B). Dicer1 mRNA was also unchanged in the RPE of 6-month-old Cp−/−Heph−/− compared to age-matched wild-type mice (Kaneko et al., 2011), suggesting that induction of Alu, or B1 and B2 RNAs by iron overload was not due to suppression of DICER1 abundance.

We next sought to determine whether iron affected the stability of Alu RNA transcripts. Iron overload delayed RPE cell processing of a transfected biotin-labeled Alu RNA (Figure 3A). Further, whereas excess iron did not affect the transcription rate of endogenous Alu RNAs, degradation of native Alu RNAs was delayed as assessed by a run-on assay (Figure 3B). To determine whether iron overload-induced Alu RNA accumulation involved DICER1 enzymatic activity, we examined this activity in cells treated with an antisense oligonucleotide targeting DICER1 that itself induces Alu RNA accumulation (Kaneko et al., 2011; Tarallo et al., 2012). In DICER1 antisense-treated cells iron overload did not further enhance Alu RNA levels (Figure 3C), suggesting iron may work by suppressing DICER1 enzymatic activity. Conversely, iron did not affect recombinant human DICER1-mediated Alu RNA processing in vitro (Figure 3D), implicating an indirect, iron-sensitive mediator of DICER1 activity.

Figure 3. Iron overload enhances stability of Alu RNA.

Figure 3

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(A) Processing of a synthetic biotin-labeled Alu RNA transiently transfected in ARPE-19 exposed to 1 mM Fe(III). X-axis denotes time following 2 h RNA loading period. (B) Quantity of endogenous Alu RNA accumulated during a 4 hour modified nucleotide doping pulse (4 h) or after washout (20 h) in ARPE-19 cells exposed to 1 mM Fe(III). Alu RNAs were quantified by q-PCR of size-separated RNA samples, in which RNAs 100-800 nt were isolated after poly-acrylamide gel electrophoresis. (C) Northern blotting of Alu RNA of human ARPE-19 cells treated with control or DICER1-targeted antisense oligonucleotides, and exposed to indicated doses of Fe(III). (D) Polyacrylamide gel separated Alu RNA after incubation with recombinant DICER1 in the presence of indicated quantities of Fe(III).

Poly(C)-binding protein 2 (PCBP2) binds to and enhances DICER1 processing of Alu RNA

We investigated potential iron-sensitive Alu RNA binding partners by performing 2-D liquid chromatography tandem mass spectrometry on proteins from primary human RPE cell lysates captured by pull-down of synthetic biotin-labeled Alu RNA. We identified Poly(C)-binding protein 2 (PCBP2; Figure S3A), an RNA binding protein reported to be both iron-sensitive and capable of modulating DICER1 enzymatic activity for micro-RNA processing (Li et al., 2012). It is reported that free cellular iron impairs DICER1 micro-RNA processing by decreasing its affinity for PCBP2 (Li et al., 2012). Because, like micro-RNAs, Alu RNAs are enzymatic targets of DICER1, we investigated whether a similar mechanism was responsible for iron-induced Alu RNA accumulation. We confirmed the interaction of PCBP2 and Alu RNA by streptavidin-mediated pull-down of a biotin-labeled Alu RNA followed by immunoblotting for PCBP2 (Figure 4A) and conversely via antibody-mediated pull-down of endogenous PCBP2 followed by northern blotting for native Alu RNA (Figure 4B). Whereas iron treatment neither impaired Alu RNA-DICER1 binding nor decreased PCBP2 expression, PCBP2-Alu RNA binding was decreased by iron overload (Figure 4A, B). The affinity of DICER1 for Alu RNA was unchanged by iron overload (Figure 4A), as is the case for DICER1 pre-micro-RNA processing activity inhibition by iron (Li et al., 2012). Alu RNA/PCBP2 binding specificity was further confirmed by performing a competition assay with 5-fold excess of unlabeled in vitro transcribed human pre-Let-7a micro-RNA, which binds to PCBP2 and DICER1 (Li et al., 2012) or unlabeled tRNA (with no known binding affinity for PCBP2) (Figure S3B). To examine the effect of PCBP2 on DICER1-mediated processing of Alu RNAs, we measured the efficiency of Alu RNA processing by DICER1 in vitro in the presence of recombinant human PCBP2 and iron. Whereas iron or PCBP2 alone did not affect DICER1-mediated Alu RNA processing, recombinant PCBP2 enhanced DICER1-mediated Alu RNA cleavage, which was significantly impaired in the presence of iron (Figure 4C). Collectively, these data identify PCBP2 as an iron-sensitive co-factor for DICER1-mediated Alu RNA enzymatic processing.

Figure 4. Iron sensitivity of DICER1 processing of Alu RNAs is mediated by PCBP2.

Figure 4

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(A) Western blotting of streptavidin-mediated pull-down and whole cell lysates from biotin-Alu or biotin-tRNA transfected human ARPE-19 cells that were exposed to 1 mM Fe(III). (B) Northern blotting of native Alu and 5S RNAs in human ARPE-19 exposed to 1 mM Fe(III) following immuno-precipitation with anti-PCBP2 antibody or in whole cell lysates. (C) DICER1-mediated Alu RNA cleavage quantified from in vitro dicing reactions containing recombinant human DICER1, recombinant human PCBP2, and 100 μM Fe(III) where indicated. For all panels, N=3-6, *P < 0.05, Error bars denote SEM. (D) Proposed model of iron overload-induced retinal toxicity, involving the sequestration of PCBP2 from Alu RNA/DICER1 complexes, leading to an accumulation of Alu RNAs, NLRP3 inflammasome activation and retinal degeneration.

Discussion

Our data establish an alternative paradigm for iron overload-induced retinal toxicity – namely that iron overload enhances Alu RNA stability and thereby promotes RPE degeneration via the NLRP3 inflammasome (Figure 4D). Iron toxicity in the retina and other tissues is widely attributed to catalysis of hydroxyl radicals via Fenton's reaction. However, our findings reveal that absent SINE RNA accumulation or Caspase-1 signaling, iron overload is not sufficient to drive degeneration of the retina. We further report that iron overload-induced Alu RNA/inflammasome-mediated retinal toxicity is not a generic response to excess of Fenton catalysts, as this pathway was not common to chromium, copper or zinc-induced retinal toxicity.

Although our data suggest that hydroxyl radical formation is not sufficient to drive inflammasome-mediated retinal toxicity, the necessity of iron-mediated hydroxyl radical formation on Alu RNA-mediated iron toxicity is not known. We previously reported that Alu RNAs induce mitochondrial reactive oxygen species generation, which is an essential component of its inflammasome signaling and toxicity (Tarallo et al., 2012). That Alu RNA signaling involves reactive oxygen species generation, and iron's ability to catalyze free radical formation suggests that Fenton activity of iron could augment Alu RNA toxicity.

Iron antagonism of DICER1 in the RPE likely impacts not only Alu RNA metabolism, but micro-RNA biogenesis as well. Although we previously found no anatomical disruption of micro-RNA-deficient RPE of adult mice (RPE-specific knockouts of Drosha, Dgcr8 and Argonaute-2 appeared normal upon fundus and flat mount examination (Tarallo et al., 2012)), we cannot exclude the possibility that global micro-RNA perturbation due to DICER1 inhibition also contribute to or modulate iron-overload induced retinal toxicity.

These findings also provide support for pathogenic roles for both inflammasome activation and iron overload in human AMD. Inflammasome signaling in human AMD tissues has now been observed by multiple laboratories (Chan et al., 2013; Tarallo et al., 2012; Tseng et al., 2013). Experimentally, multiple AMD-related stimuli including Alu RNA (Kerur et al., 2013; Tseng et al., 2013), A2E (Anderson et al., 2013), complement cascade components (Doyle et al., 2012; Triantafilou et al., 2013), amyloid-β (Liu et al., 2013), and excess VEGF-A (Marneros, 2013) have been reported to act via inflammasome signaling. Our results suggest that targeting inflammasome signaling components as a ‘next-generation’ therapeutic for AMD may provide additional, unanticipated benefits with respect to phenotypes arising from iron overload. Notably, our findings reveal that NLRP3 alone does not account for the entirety of iron-induced RPE degeneration. Therefore it will be important in future studies to determine the extent to which NLRP3-independent inflammasome activation contributes to the AMD and pathological effects of AMD-related inflammasome agonists. Further, our studies utilized a mouse line lacking both Caspase-1 and the non-canonical inflammasome component Caspase-11, which were protected from treatment with a relatively high dose of iron. Whether non-canonical inflammasome activation is required for iron-induced toxicity is an important future direction for study.

PCBP2 is an RNA binding protein and iron chaperone, with diverse cellular functions such as RNA stability (Czyzyk-Krzeska and Bendixen, 1999; Paulding and Czyzyk-Krzeska, 1999; Xin et al., 2011), protein stability (You et al., 2009), immunity (Blyn et al., 1996; Blyn et al., 1997; You et al., 2009), micro-RNA processing (Li et al., 2012), iron homeostasis (Lane and Richardson, 2014), preventing stress-induced apoptosis (Ghosh et al., 2008), cell growth (Han et al., 2013; Waggoner et al., 2009), and tumor growth (Han et al., 2013; Hu et al., 2014). The extent to which these diverse functions of PCBP2, as well as its SINE RNA processing activity, contribute to inflammasome activation and iron-induced RPE degeneration in geographic atrophy warrants further investigation

Iron overload has been observed in numerous neurodegenerative disorders including in senile plaques and neurofibrillary tangles of human cadaver brains with Alzheimer's disease (Quintana et al., 2006; Sayre et al., 2000; Smith et al., 1997) and in the substantia nigra of humans with Parkinson's disease (Ayton and Lei, 2014; Dexter et al., 1987). Interestingly, the involvement of the NLRP3 inflammasome has been independently implicated in the pathogenesis of these diseases (Codolo et al., 2013; Freeman et al., 2013; Heneka et al., 2013). This study raises the intriguing possibility that the damaging effects of Alu RNA accretion due to iron overload could extend beyond AMD and contribute to neurodegenerative diseases with recognized etiological similarities. These findings also raise the interesting possibility that systemic iron overload promotes NLRP3 inflammasome activation outside of the eye, such as in hepatic macrophages, where inflammatory phenotypes such NF-κB activation and cytokine expression are noted effects of iron overload in liver injury (Lin et al., 1997; Xiong et al., 2008)

Experimental Procedures

Intraocular injections and imaging

Subretinal injections and intravitreous injections (1 μL each) were performed with a 35-gauge Exmire microsyringe (Ito Corporation). Fundus imaging was performed on a TRC-50 IX camera (Topcon) linked to a digital imaging system (Sony). RPE flat mounts were immunolabeled using antibodies against Zonula Occludens-1 (Invitrogen)and visualized by confocal microscopy (Leica). Cryosections of 4% PFA fixed retinal cross sections were immuno-labeled with anti-Nlrp3 (Abcam) and visualized by fluorescent microscopy (Nikon).

RNA and protein abundance analysis

In situ hybridization was performed on frozen, fixed tissue sections from Ceph−/−/Heph−/− and wild-type (C57BL/6J) age-matched mice were processed using custom designed probes for B1 and B2 RNAs (Advanced Cell Diagnostics) according to the manufacturer's instructions. Probes were visualized by confocal microscopy (Leica). Quantities of B1, B2 U6 and 5S RNAs were determined by northern blotting. Protein abundance was measured by western blotting with the following primary antibodies: mouse-anti-PCBP2 (Abnova, 1:500), rabbit-anti-DICER1 (Bethyl, 1:1,000), mouse-anti-α-Tubulin (Sigma, 1:1,000), rabbit-anti-Caspase-1 (Invitrogen, 1:1,000 or Abcam, 1:1,000), mouse-anti-NLRP3 (Enzo, 1:1,000), anti-β-actin (Sigma-Aldrich, 1:1,000), and rabbit-anti-Vinculin (Sigma-Aldrich, 1:1,000).

Statistical analysis

Results are expressed as mean ± SEM, with p values < 0.05 considered statistically significant. Differences between groups were compared by Mann–Whitney U test or Student t-test, as appropriate.

Supplementary Material

supp 1
supp 2

Acknowledgments

Vector to express recombinant human PCBP2 was provided by Dr. Peng Jin (Emory University). L. Toll, G. R. Pattison, R. King, L. Xu, M. McConnell, C. Payne, D. Robertson, G. Botzet provided technical assistance. B.D.G. was supported by American Heart Association and International Retinal Research Foundation (IRRF); T.Y. was supported by Fight for Sight postdoctoral award; J.A. was supported by NIH grants (DP1GM114862, R01EY018350, R01EY018836, R01EY020672, R01EY022238, and R01EY024068), Doris Duke Distinguished Clinical Scientist Award, Burroughs Wellcome Fund Clinical Scientist Award in Translational Research, Ellison Medical Foundation Senior Scholar in Aging Award, Foundation Fighting Blindness Individual Investigator Research Award, Harrington Discovery Institute Scholar-Innovator Award, Dr. E. Vernon Smith and Eloise C. Smith Macular Degeneration Endowed Chair, and Research to Prevent Blindness departmental unrestricted grant; B.J.F. was supported by NIH T32HL091812 and UL1RR033173; A.B.C. was supported by the Programme for Advanced Medical Education (sponsored by Fundação Calouste Gulbenkian, Fundação Champalimaud, Ministério da Saúde and Fundação para a Ciência e Tecnologia, Portugal) and Bayer Global Ophthalmology Research Award; Y.H. was supported by Alcon Japan Research award; N.K. was supported by Beckman Initiative for Macular Research and NIH K99EY024336; and C.B.W. was supported by The Loris and David Rich Postdoctoral Scholar Award (IRRF). J.A. and B.J.F. are named as inventors on patent applications relating to the treatment of macular degeneration filed by their employer, the University of Kentucky. J.A. is a co-founder of iVeena (Holdings, Pharma, Delivery) LLCs, which has licensed some of this technology from the University of Kentucky. J.A. has received honoraria from Allergan, Inc.

Footnotes

Author Contributions: B.D.G, C.W., Y.K., T.Y., R.Y., S.L., B.J.F., A.B.C., N.K., A.U., Y.S.H., D.L., M.E.K., and W.H.M. performed experiments. G.N., P.G. and J.L.D. provided reagents. B.D.G. conceived and directed the project and wrote the paper with assistance from J.A. All authors had the opportunity to discuss the results and comment on the manuscript.

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