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1 Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment 2 epithelia utilizes microtubule-associated protein 1 light chain 3B 3 4 Lauren L. Daniele1, Jennifer Caughey1, Stefanie Volland2, Rachel C. Sharp1, Anuradha 5 Dhingra1, David S. Williams2, Nancy J. Philp3 and Kathleen Boesze-Battaglia1 6 7 1. Dept. of Biochemistry, SDM, University of Pennsylvania, Philadelphia, PA 19104, 8 2, Departments of Ophthalmology and Neurobiology, Jules Stein Eye Institute, David Geffen 9 School of Medicine, University of California, Los Angeles, Los Angeles, CA 10 3 Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 11 Philadelphia, PA. 12 13 14 15 16 17 18 19 20 Running Title: Role of LC3B in RPE peroxisome regulation 21 22 23 24 * Corresponding Author 25 Kathleen Boesze-Battaglia, PhD 26 Department of Biochemistry 27 SDM 28 University of Pennsylvania 29 Philadelphia, PA 19104 30 Ph: 215-898-9167 31 e-mail: [email protected] 32 33 34 bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

35 Abstract (244/250 words allowed) 36 The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the 37 retinoid the visual cycle, nutrient supply, ion exchange and waste removal. Each day the RPE 38 removes the oldest ~10% of photoreceptor outer segments through phagocytic uptake, which 39 peaks in a synchronous burst following light onset. Impaired degradation of phagocytosed OS 40 material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, 41 inflammation and cell death. OSs are rich in very long chain fatty acids which are preferentially 42 catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the 43 regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively 44 unexplored. Using immunohistochemistry, immunoblotting and catalase activity assays, we 45 investigated peroxisome abundance and activity at 6 am, 7 am (at lights on), 8 am, and 3 pm, in 46 WT mice and mice lacking microtubule-associated protein 1 light chain 3B (LC3B), that have 47 impaired degradation of phagosomes. We found that catalase activity, but not protein 48 expression, is 50% higher in the morning compared with 3 pm, in RPE of WT but not LC3B-/- 49 mice. Surprisingly, we found that peroxisome abundance was stable during the day, however 50 numbers are elevated overall in LC3B-/- mice, implicating LC3B in autophagic organelle turnover 51 in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with 52 phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE 53 peroxisomes for daily surges in ingested lipid-rich OS. 54 bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

55 Introduction 56 57 The retinal pigment epithelium (RPE) supports photoreceptor activity and outer-segment (OS) 58 renewal through the retinoid visual cycle and ingestion of spent photoreceptor outer 59 segments (36, 41, 42, 66, 73). On a daily basis, the RPE ingests 10% of the lipid and protein- 60 rich OS, for recycling and degradation, while new OS material is added basally (69, 74). 61 Incomplete phagosome degradation in the highly oxidative environment of the RPE predisposes 62 undigested lipids to oxidative damage and the formation of lipid peroxidation adducts 63 contributing over time to an immune response and tissue damage (3, 19, 27, 35, 70). 64 65 The ingested OS are a complex mix of saturated and highly unsaturated fatty acids, which 66 make up 50% of OSs (by weight) and provide the RPE with substrate for mitochondrial and 67 peroxisomal fatty acid oxidation (FAO) (1, 23, 61, 64, 68). Within the lipid pool, OS membranes 68 are estimated to contain ~ 30% very long chain fatty acids VLCFAs (23). VLCFAs (Cƚ20) are 69 preferentially catabolized in peroxisomes via β-oxidation (64, 68). Therefore, efficient lipid 70 catabolism in the RPE requires coordination of β-oxidation pathways within both mitochondria 71 and peroxisomes (1, 23, 61). Once VLCFA are shortened, they are substrates for mitochondrial 72 β-oxidation (1, 61). Some of the resultant acetyl-CoA is funneled into the mitochondrial 73 ketogenesis pathway, producing beta hydroxybutyrate (βHB). βHB is subsequently secreted into 74 the subretinal space and taken up by photoreceptors as a metabolic substrate (1). Given their 75 intimate metabolic coordination it is not surprising that numerous micro-peroxisomes can be 76 found in RPE cells where they are contiguous with ER tubules and proximal to mitochondria (45, 77 47, 62). 78 79 Peroxisomes are ubiquitous organelles highly enriched in liver, intestine, adipose tissue and 80 brain. In addition to their functions in α and β oxidation-mediated catabolism of fatty acids (48) 81 peroxisomes play essential roles in ether lipid synthesis, docosahexaenoic acid (DHA) 82 synthesis, and bile acid synthesis. Peroxisomes are also sites for both the generation and 83 reduction of reactive oxygen and nitrogen species (ROS and RNS) which serve as mediators for 84 cellular signaling (56, 72). The most abundant of these is hydrogen peroxide, produced as a 85 byproduct of the initial desaturation of Acyl-CoA esters by Acyl CoA Oxidases (ACOXs) in the 86 committed step of peroxisome fatty acid oxidation (64, 68). Hydrogen peroxide generated in the 87 ACOX reaction is rapidly degraded by the antioxidant enzyme, catalase. Peroxisome antioxidant 88 activity declines with cellular age (44). Dysregulation of peroxisome antioxidant capacity with bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

89 age may have broad cellular impact as inhibition of catalase activity in fibroblasts resulted in 90 increased mitochondrial ROS levels and disrupted activity (44, 67, 71). Catalase activity is also 91 lower in peroxisomes of human primary RPE from aged donors, and those with age-related 92 macular degeneration (AMD) (46), and paradoxically, this is accompanied by an overabundance 93 of peroxisomes (6, 7, 21), suggesting a role for impaired peroxisome turnover in declining 94 peroxisome function.

95 Although countless studies have focused on understanding peroxisome proliferator-activated 96 receptor (PPAR) regulation, and correlation with disease (50, 60, 63, 76), to our knowledge little 97 information is available regarding the activity and numbers of peroxisomes in the RPE 98 over a 12hr light/dark cycle. In this study, we investigate peroxisome function and dynamics in 99 the context of phagocytic degradative processes in RPE cells. We find that catalase activity is 100 increased by ~50% during the time of peak phagocytosis, and this diurnal regulation of 101 antioxidant activity is absent in mice lacking LC3B. We also find that while LC3B-dependent 102 processes contribute to peroxisome turnover, peroxisome number does not depend on time of 103 day. Our data suggest peroxisome function in the RPE is regulated dynamically in coordination 104 with phagocytosis, likely through direct regulation of enzyme activity. Catalase activity can be 105 regulated through diverse post-translational modifications (9, 10, 16, 26, 39, 59) which are often 106 sensitive to the redox state of the cell. Elevated catalase activity during the early morning 107 suggests the possibility of a homeostatic mechanism to ready RPE peroxisomes for the daily 108 synchronous surge in ingested lipid-rich outer segments. bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

109 Results 110 111 Mouse RPE contain abundant peroxisomes. 112 Fatty acids comprised of 20 carbons and longer are catabolized in peroxisomes (64, 68), where 113 the rate limiting step is desaturation of a fatty acyl-CoA by the enzyme ACOX1, producing the

114 byproduct hydrogen peroxide (H2O2) (Fig.1A). Catalase, the most abundant peroxisomal

115 antioxidant, reduces H2O2 produced in peroxisomes during this reaction, preventing its toxic 116 accumulation. Using a publicly available microarray data set (GSE10246; Fig 1B) we analyzed 117 gene expression profiles of peroxisome lipid-associated proteins in mouse tissues. RPE express 118 fatty acid transporters and peroxisome β-oxidation enzymes. Three members of the ABC lipid- 119 transporter superfamily reside in peroxisomes, these include, Abcd1 (ALDP), Abcd2 (ALDR) 120 and Abcd3 (PMP70). Of these, Abcd1 is expressed in RPE (mouse) in relative abundance with 121 levels of Abcd3 in RPE similar to that in liver (Fig. 1B). Transcripts for Acox1, and catalase as 122 well as 3-ketoacyl-CoA thiolase (Acaa1/thiolase) are highly expressed in liver but are also 123 enriched in the RPE relative to retina, cornea and neural tissues (Fig 1A, B). Two thiolase genes 124 are expressed in mouse, Acaa1a, and Acaa1b, with the expression of thiolase b restricted to 125 liver, with lower expression in kidney, intestine and adipose tissue (13). The complete β- 126 oxidation of polyunsaturated fatty acids (FA) within peroxisomes requires additional enzymes, 127 due to the presence of cis- and trans- double bonds (68), including peroxisome 2,4, Di-enoyl 128 CoA reductase (Decr2). We find that transcripts for these enzymes are also enriched in RPE 129 relative to retina and brain (Supplemental Fig. S1). 130 131 We next examined peroxisome localization and abundance in the RPE, using 3,3'- 132 diaminobenzidine (DAB) reactivity by transmission EM complemented with immuno-electron

133 microscopy analysis. Catalase reacts with DAB in the presence of excess H2O2 to form a dense 134 precipitate localized to catalase-positive peroxisomes (15). DAB was localized to small oval 135 shaped organelles with ~100 nm diameter in ultrathin sections of RPE (Fig. 1C, D). When 136 Immuno-electron microscopy (EM) analysis was used to detect catalase, gold particles labeled 137 small rounded structures adjacent to mitochondria (Fig 1E), morphologically consistent with our 138 DAB labeling. In RPE whole mounts labeled with PMP70 and Pex14, we observe numerous 139 peroxisomes throughout the cytoplasm, some with a tubular morphology (Supplemental Fig. S2) 140 as was recently observed for peroxisomes of mouse fibroblasts using stimulated emission 141 depletion (STED) microscopy (65). Catalase immunolabeling appeared most intense in the 142 RPE relative to other retina layers in cryosections of mouse eye (Fig 2 A). In RPE, catalase- bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

143 positive puncta were enriched in the peroxisome-specific membrane protein, PMP70, a fatty 144 acid ABC transporter used as a marker of peroxisome number (22, 51) (Fig 2B,C). Linear 145 intensity profiles show that peaks in catalase signal are spatially correlated with that of PMP70 146 (Fig 2C) and co-distribution analysis of catalase and PMP70 have a Pearson’s correlation 147 coefficient of 0.89 (± 0.006 n=38). We also examined catalase expression in RPE of 148 microtubule-associated protein 1 light chain 3B-/- (LC3B-/-) mouse, a model of defective 149 phagosome maturation and lipid dysregulation (18, 19). LC3B-/-, like WT also show a relative 150 enrichment of catalase in RPE relative to retina (Fig 2 D), with linear intensity profiles of PMP70 151 and catalase well correlated (Fig 2 E,F). Our data shows that relative to other cells within the 152 eye, peroxisomes are abundant in RPE, with catalase co-distributing with PMP70-positive 153 peroxisomes in both LC3B-/- and WT mice. 154 155 Peroxisome numbers do not depend on time of day 156 Peroxisomes respond to changes in the cellular environment by adapting their number, 157 morphology and metabolic functions. In RPE, the level of VLCFA substrate for peroxisome 158 oxidation is regulated in a synchronous circadian manner through phagocytosis of OSs (28, 41, 159 42). Phagocytic ingestion of rod photoreceptor OS peaks following light onset (42). Because 160 peroxisome number can be dynamically regulated by cellular conditions, we wanted to 161 determine if OS disc phagocytosis influences peroxisome abundance and function in the RPE. 162 We analyzed peroxisome abundance in WT mouse RPE at various times of day and compared 163 this to peroxisome abundance in the RPE of the LC3B-/- mouse, having defective phagosome 164 maturation (18, 19). RPE/choroid was isolated at: 6 am - one hour before lights on, 7 am – at 165 lights on, 8 am - an hour after lights on, and 3 pm (8h after light onset). We compared the levels 166 of two membrane proteins widely used as markers for peroxisomes; Pex14 is an essential 167 transmembrane component of the peroxisome translocation machinery that imports cytosol- 168 translated enzymes into the peroxisome lumen (77) and PMP70, one of 3 ATP binding cassette 169 family members that transport FAs into peroxisomes (22, 51). Pex14 (Fig 3 A, C) and PMP70 170 levels (Fig 3 B, D) remained relatively stable throughout the day in RPE/choroid isolated from 171 WT mice. When compared to WT mice, the levels of Pex14 in the LC3B-/- mouse RPE were 172 elevated by 20% of WT at 7 am and 12% at 8 am, whereas PMP70 expression was elevated 173 above WT by an average of 32% at all time points (Fig 3 E, F). Peroxisome abundance was 174 next assessed in LC3B-/- and WT mouse RPE by confocal imaging (Fig.4 A, B). Pex14 175 immunoreactivity increased by at least 50% in LC3B-/- vs WT in 2 data sets (Fig 4C). PMP70 176 immunoreactivity was increased by 50% in one data set with another data set showing a trend bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

177 towards higher PMP70 immunoreactivity that was not statistically significant. A third data set 178 showed no difference between LC3B-/- and WT immunoreactivities. Collectively, these studies 179 suggest that peroxisome degradation is reduced in RPE of LC3B-/- mice. 180 181 LC3B contributes to early morning peak in catalase activity in RPE. 182 Although peroxisome numbers are relatively constant over the time period tested, because RPE 183 ingests a lipid rich OS at light onset, we sought to determine if peroxisome enzymatic activity 184 depends on substrate availability. We compared ACOX1 activity as coupled catalase function in 185 WT and LC3B-/- mice. Therefore, we compared peroxisomal catalase activity at 6 am, 7 am (at 186 lights on) and 8 am – during which the OS-derived LCFA and VLCFA β-oxidation substrates are 187 maximal- and at 3 pm, 8 hours later. At the 8hr time point virtually no phagosomes were 188 observed in WT mice while over 50% of the phagosomes remain undegraded in the LC3B-/- 189 mouse RPE (18, 19). Catalase activity was elevated ~1.5 fold during the early morning 190 compared with the afternoon in WT mice (Fig 5 A), but not in LC3B-/- mice, suggesting that an 191 LC3B dependent process influenced peroxisome catalase activity. To determine if elevated 192 catalase activity was due to increased enzyme levels, we investigated catalase protein amounts 193 from the same mice at 6 am, 7 am (at lights on), 8 and at 3 pm, by immunoblot analysis. 194 Catalase protein levels remained relatively constant over the time periods studied (Fig. 5B, C). 195 In contrast to our catalase activity data, we observed an apparent increase in relative catalase 196 protein levels between 8 am and 3 pm in WT, however catalase protein levels at 6 am and 7 am 197 were not significantly different from those at 3 pm. We quantified catalase immunoreactivity from 198 our immune-EM analysis of WT mouse eyes removed at 1 hour and 6 hours after light onset. At 199 1 hour after light onset, we found 467 gold particles / µm2 (± 30 sem, n=41), which was 200 comparable to our finding of 432 particles/ µm2 (± 20 sem, n=39) at 6 hours after light onset. 201 Taken together, our data suggest that changes in catalase protein levels do not account for the 202 elevated activity during the morning. We also tested whether diurnal modulation of metabolic 203 activity was unique to peroxisomes, and so we investigated whether mitochondrial activity was 204 also regulated diurnally by assessing activity of citrate synthase, the first enzyme of the 205 tricarboxylic acid (TCA) cycle (Fig 5D), and found no difference in citrate synthase activity as a 206 function of time of day. We directly tested whether catalase is activated by increased VLCFA

207 and LCFA β-oxidation substrate after uptake of OSs or by elevated H2O2, by measuring

208 catalase activity in RPE/choroid explants treated with OSs or H2O2. Catalase activity was 209 increased by ~50% above Ringers control (p<0.05) in RPE explants incubated with 0.5 mM bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

210 H2O2 but not OSs (Fig 5E). We found no statistically significant differences in catalase activity in 211 RPE explants from LC3B-/- for either treatment. bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

212 Discussion 213 RPE peroxisomes are enriched in β-oxidation genes. 214 Peroxisomal β-oxidation preferentially breaks down very long chain fatty acids abundant in POS 215 membranes. The chain- shortened FAs can then be utilized by mitochondria for further rounds 216 of β-oxidation. In this study we show that genes encoding key enzymes of fatty acid β-oxidation 217 within peroxisomes, such as Acox1, and catalase are highly expressed in mouse RPE relative 218 to other eye tissues. Peroxisome β-oxidation genes are most highly expressed in liver, 219 consistent with the liver’s principal role in fatty acid metabolism and regulation of systemic fatty 220 acid availability (48, 53). Peroxisomes in the liver, unlike adipocytes, brain and intestinal 221 mucosa, are large and contain a matrix enriched in luminal enzymes, including catalase (48). β- 222 oxidation pathways of the liver play multifaceted roles in fatty acid metabolism, including the 223 breakdown of dicarboxylic acids and dietary pristanoyl-CoA. Single rounds of β-oxidation in liver 224 are also critical for synthesis of bile acids from cholesterol and DHA from essential fatty acids 225 (5, 68). Previous studies have shown that RPE like the liver, is ketogenic, with the ability to 226 catabolize fatty acids (FAs) derived from phagocytosed photoreceptor outer segments (POS) 227 through mitochondrial β-oxidation pathways that produce acetyl-CoA and the secreted ketone 228 metabolite β-hydroxybutyrate (1, 61). Peroxisomes of the RPE are found adjacent to 229 mitochondria and their proximity supports their coordination of metabolic pathways. In addition 230 to metabolic coordination, mitochondria and peroxisomes overlap in redox sensing and 231 ROS/RNS signaling and regulation. Mitochondria and peroxisomes both produce significant 232 amounts of ROS through their different metabolic pathways. Each round of β-oxidation in

233 peroxisomes produces H2O2. We show that the antioxidant catalase, the primary enzyme

234 responsible for the removal of H2O2 is enriched in peroxisomes within RPE. Peroxisomal 235 antioxidant activity, plays a key role in redox homeostasis, and disrupted catalase activity has 236 serious consequences for mitochondrial health (44, 67, 71). 237 238 Catalase activity may be regulated post-translationally 239 We show that catalase, the heme-containing antioxidant enzyme enriched in RPE peroxisomes, 240 has highest activity during the early morning when OS derived VLCFA’s are expected to reach 241 their maximum in RPE cells and β-oxidation reaches its peak (61). The elevated catalase 242 activity is not due to increased expression of catalase protein, suggesting that diurnal 243 modulation of catalase activity occurs at the post-translational level. Catalase activity is 244 regulated by a diverse array of post-translational modifications depending on cell context, 245 including phosphorylation, S-nitrosylation, oxidation and ubiquitination (9, 10, 16, 26, 39, 59), as bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

246 well as regulation of its multimerization (59). Catalase is thought to exist mainly in a tetrameric, 247 enzymatically active form, but higher-order oligomer and dimer forms have been encountered

248 inside cells (2, 58). The enzymatic activities of catalase in H2O2 removal as well as its 249 peroxidase activity, requires its heme group and heme binding is required for tetramerization

250 and maturation (11). During small elevations in cellular H2O2 in cultured mouse fibroblasts, the 251 redox sensitive kinases, c-Abl and Arg, increase catalase activity by phosphorylation of key 252 tyrosines and directly binding, protecting catalase from de-phosphorylation (9, 10). (59) 253 Catalase is an important antioxidant whose activity declines with age and in AMD-afflicted RPE 254 (46) and the regulation of its activity in RPE is largely unexplored. 255 256 Diurnal modulation of Catalase activity depends on LC3B 257 In contrast to WT RPE, RPE of LC3B-/- mice lack a boost in catalase activity during the morning. 258 RPE of LC3B-/- mice have impaired degradation of phagocytosed OS – derived material, 259 depressed fatty acid oxidation, eventual accumulation of lipid deposits, and low levels of 260 esterified and free DHA, due in part to the role of LC3B in LC3-associated phagocytosis (19). 261 Lower catalase activity in RPE of LC3B-/- mice during the morning is consistent with impaired β- 262 oxidation of POS-derived FAs in LC3B-/- (19). Elevated catalase activity in RPE may reflect 263 higher peroxisomal β-oxidation activity during the morning when VLCFA availability may be 264 maximal. The synchronous burst in POS uptake by phagocytosis in mammalian RPE occurring 265 ~1 hour after light onset is necessary for the maintenance of photoreceptor health (52). We

266 hypothesized that catalase activity is modulated in step with this burst, through increased H2O2 267 produced when availability of OS – derived LCFA and VLCFA substrate reaches its peak. If this 268 were true, RPE explants made from WT mice fed OS may respond with a boost in catalase -/- 269 activity, and this may be impaired in RPE explants from LC3B mice. However, increasing H2O2 270 independently of substrate availability may override the impairment and act more directly on a

271 modifier of catalase activity (e.g. an H2O2-sensitive kinase) resulting in increased catalase

272 activity in both genotypes. Our results show that H2O2 resulted in ~50% increase in catalase -/- 273 activity for both WT and LC3B RPE explants, making H2O2 - sensitive kinase mediated 274 phosphorylation a plausible mechanism in RPE cells. However, elevated catalase activity was 275 not observed after treatment of RPE explants of either WT or LC3B-/- mouse with OS, leaving 276 open the possibility of catalase activity modulation through another pathway which is impaired in 277 the absence of LC3B. Future studies are needed to decipher the role of LC3B in modulating 278 catalase activity. 279 bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

280 RPE peroxisome number does not vary with time of day 281 Peroxisome number is dynamically regulated to adapt to the changing cellular milieu. The 282 number of peroxisomes at any given time emerges from the dynamic balance of biogenesis, 283 replication, and degradation (14, 30). In this study we explored the possibility that peroxisome 284 number is dynamically regulated after the synchronous peak in outer segment phagocytosis, 285 when ingested very-long chain fatty acids derived from photoreceptor outer segments, is 286 maximal. However, our immunoblot evidence suggests RPE peroxisome mass is relatively 287 stable over time. This was a surprise as previous studies in mouse show 2 brief peaks in RPE 288 peroxisome number 3 hours before and 3 hours after light onset (47), suggesting rapid 289 expansion followed by rapid removal of peroxisomes in RPE. The chronology of the observed 290 peak in peroxisome numbers by EM roughly corresponds to our observations of peak catalase 291 activity in the morning spanning an hour before and after light onset. However, we saw no 292 evidence for rapid removal of peroxisome material. It is likely that peroxisome removal and 293 replacement is a relatively slow process, as pulse-chase studies of fluorescently-tagged or 294 isotope-labeled peroxisomal proteins indicated a half-life of 1-3 days for liver peroxisomes (31, 295 57). The observations of a steep decline in peroxisome numbers 1-2 hours after light onset track 296 a peak in numbers of phagosomes in this study, suggesting the possibility of false negatives or 297 misclassification of structures, when phagosomes accumulate and traffic more basally. 298 299 Peroxisome degradation depends on LC3B; implications for RPE homeostasis 300 Turnover is important in maintaining healthy functional organelles. Degradation of dysfunctional 301 and damaged peroxisomes and other organelles is principally achieved through regulated 302 macroautophagy (or pexophagy) a lysosomal degradative process whereby a double membrane 303 autophagosome engulfs the entire organelle prior to fusion with lysosomes (4, 34). The signals 304 which select old dysfunctional peroxisomes for degradation are beginning to be elucidated and 305 selective ubiquitination plays an important role (37). Prolonged residency of monubuqitinated 306 Pex5 at the peroxisomal membrane is a signal for binding of pexophagy adapters P62 and 307 NBR1 (17, 55) . P62 contains an LC3 interaction motif and ubiquitin binding domains allowing its 308 co-recruitment of peroxisome cargo and autophagosome machinery. In a distinct pexophagy 309 pathway, under conditions of elevated ROS, ataxia-telangiectasia mutated kinase 310 phosphorylates Pex5, allowing monoubiquitination at a lysine residue, leading to recruitment of 311 P62 and autophagosomal membranes (75). Our results show that peroxisomes are more 312 numerous in RPE of LC3B-/- mice, which likely reflects sluggish degradation and turnover of 313 peroxisomes, given the well-established role of LC3B in autophagosome formation and bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

314 autophagosome- lysosome fusion (54). LC3B is one of 6 different members of the mammalian 315 Atg8 family (LC3/GABARAP proteins) involved in autophagy (43). Of the LC3 subfamily, only 316 LC3A and LC3B are expressed in mouse RPE (18). Our data suggests that the LC3B isoform is 317 necessary for efficient turnover of peroxisomes within the RPE. This is supported by evidence of 318 elevated peroxisome membrane proteins (Pex14 and PMP70) in RPE of LC3B-/- mice from 319 western blot and IHC. Our data is consistent with recent finding that LC3B was necessary for 320 autophagic degradation of p62, normally degraded along with its cargo, in HEK293T cells (49). 321 322 In this study we found that RPE of mice lacking LC3B expression have elevated peroxisome 323 numbers but lack the ability to increase activity of peroxisome antioxidant catalase during the 324 early morning. Elevated peroxisome numbers with lower antioxidant function is reminiscent of 325 aged and diseased RPE (6, 21). Reduced autophagic flux, accompanied by accumulation of 326 autophagosomes and lysosomes, in RPE derived from AMD patients was proposed to 327 contribute to poor mitophagy and mitochondrial dysfunction (29). The role for autophagic 328 degradation in mitochondrial quality control in AMD has been the subject of more intense focus 329 (32). Mitochondria and peroxisomes intimately cooperate in metabolic pathways and regulation 330 of ROS. Impairments in autophagy in ageing and diseased RPE may have more far reaching 331 consequences when the health and function of peroxisomes is also considered. Peroxisome 332 turnover defects may impact the resilience of RPE to oxidative stress in ageing and contribute to 333 disease pathogenesis. bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

334 Materials and Methods 335 336 Animals: 337 C57BL6/J (WT) mice and the LC3B-/- mouse line (strain name: B6;129P2-Map1Lc3btm1Mrab/J; 338 stock # 009336, (8) were purchased from Jackson Laboratory (Bar Harbor, ME). The LC3B-/- 339 mice were backcrossed for at least 5 generations onto a C57BL6/J background. The LC3B-/- 340 and the WT mice were confirmed to be free of the rd8 mutation by genomic DNA PCR using 341 primers as described in (12). Maintenance of mouse colonies and all experiments involving 342 animals were as described previously (24, 25). Mice were housed under standard cyclic light 343 conditions: 12-h light/12-h dark and fed ad libitum, with both female and male mice used in 344 these studies. All procedures involving animals were approved by the Institutional Animal Care 345 and Use Committees of the University of Pennsylvania and the University of California, Los 346 Angeles, and were performed in accordance with the Association for Research in Vision and 347 Ophthalmology guidelines for use of animals in research. 348 349 Microarray analysis for heat map 350 The heat map was generated using the publicly available high throughput Mus musculus gene 351 expression microarray data set GSE10246 (40) available through NCBI GEO (GNF Mouse 352 GeneAtlas V3). The probe IDs for each gene of interest were obtained from an up to date 353 microarray annotation file (Mouse430_2na36annot) obtained from the Affymetrix website and 354 used to search GSE10246. The heat map was made in MultiExperiment Viewer version 4.9.0 355 using Log2 scale for values averaged from 2 replicates for each tissue. 356 357 Immunohistochemistry (IHC) 358 Eyes were removed from euthanized mice and immediately placed in 4% paraformaldehyde 359 (PFA). For cryosections, the anterior segment and lens were removed during fixation, leaving an 360 intact posterior eye (eyecup). Eyecups were fixed overnight at 4oC. Following cyroprotection in 361 30% sucrose, 1x PBS at 4oC overnight, eyecups were embedded in OCT compound (Sakura 362 Finetek, Torrance, CA), frozen, and sectioned with a cryostat (Microm HM 550) at 10-20 µm 363 thickness. Cryosections were rehydrated and washed in 1x PBS followed by incubation with 364 antibodies diluted in blocking buffer (5% BSA, 0.1% triton X-100, 1X PBS). Sections were 365 washed 3x in 1x PBS and bound primary antibodies were detected by incubation with Alexa- 366 fluor conjugated secondary antibodies diluted in blocking buffer (Thermo Fisher/Invitrogen, 367 Eugene, OR) for 1 hr at 37oC. For flat mounts of RPE/choroid/sclera, melanin and other pigment bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

368 was bleached after fixation, using the method of Kim and Assawachananont (38). Briefly, each 369 eyecup was incubated at 55o C in 10% peroxide/1x PBS for 2.5 hr and immunolabeled as above 370 after 3-4 washes in PBS. Immunolabeled tissue was imaged with a Nikon A1 confocal 371 microscope (Nikon Instruments, Melville, NY). We used the following primary antibodies for IHC 372 (with company and dilution): rabbit anti-catalase (Abcam ab1877, 1/500), rabbit anti-Pex14 373 (ProteinTech 10594-1-AP, 1/500), mouse anti-PMP70 (Sigma SAB4200181, 1/300). 374 375 Electron microscopy with DAB labeling of peroxisomes 376 Peroxisomes were labeled with alkaline 3, 3′-diaminobenzidine (DAB) following established 377 methods (20). Briefly, mouse eyes were enucleated, and anterior segment removed as above, 378 and immersed in fixative (2.5% glutaraldehyde, 2% paraformaldehyde, in 0.1M cacodylate 379 buffer) overnight at 4oC. After fixation, small pieces of eyecup were washed 2x 30 min in 0.1M 380 Cacodylate buffer, then washed 20 min in Tris HCl, pH 8.5, then in 0.2% DAB for 0.5 hr in 381 darkness in a water bath at 37oC. Control tissue was incubated at 37oC with Tris HCl buffer 382 alone. DAB solution contained 0.2 DAB (from a 4% stock; EMS 13080), 0.1 M Tris HCl, 0.15%

383 H2O2. Following DAB incubation, tissue was washed 15 min 0.1M Tris HCl in darkness, then 15 384 min 0.1M Cacodylate in darkness. The labeled tissue was embedded in Epon. All post-fixation, 385 embedding and ultrathin sectioning was performed by the University of Pennsylvania EM 386 Resource Laboratory. Ultrathin sections were imaged with a Jeol-1010 transmission electron 387 microscope. 388 389 Immuno EM 390 Mouse eyes were enucleated and fixed at 4oC in 4% formaldehyde and 0.2% glutaraldehyde in 391 0.1 M sodium cacodylate buffer. The anterior segment was removed, and eyecups were 392 dissected along the dorsal ventral axis. After washing in 0.1 M sodium cacodylate buffer, 393 samples were dehydrated with an ethanol series, and embedded in LR-white resin. Ultrathin 394 sections were collected on formvar coated nickel mesh grids, quenched with 0.1% glycine in 395 0.1M phosphate buffer for 15 min, blocked in 2% BSA in 0.1M phosphate buffer for 30 min, and 396 incubated at 4oC overnight, with catalase ab15834 (Abcam, Cambridge UK) primary antibody, 397 diluted by 1:500. Sections were then washed in 0.1M phosphate buffer, incubated with anti- 398 rabbit IgG conjugated to 18-nm gold particles (Jackson Immuno Research Labs, West Grove, 399 PA), washed again, and stained with 5% uranyl acetate in ethanol for 5 min. All samples were 400 imaged with a JEM 1200-EX (JEOL, Peabody, MA) at 80kV at magnifications of 30,000 to 401 60,000x. Labeling density of the peroxisomes was determined by counting the number of gold bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

402 particles per μm2 of sectioned peroxisome. Measurements were carried out using Fiji (ImageJ 403 Version2.0.0; image processing software package; available at https://fiji.sc/). Data were 404 collected from animals fixed at 1 and 6 hours after light onset. 405 406 RPE/Choroid explants, OS and peroxide treatment 407 RPE explants were incubated in bicarbonate buffered Ringers with L-carnitine, or Ringers and 408 either bovine OS particles (100,000 per chamber) or peroxide (0.5 mM) using published 409 methods (61). Bovine outer segments were obtained from InVision BioResources (Seattle, WA). 410 Stocks of 2 x 107 particles/ml were made and stored in 15% sucrose. OS or peroxide were 411 diluted to their final concentrations in Ringer’s and explants were incubated for 1-2 hrs at 37C

412 with 5% CO2/O2. 413 414 Western blotting 415 RPE explant lysates were prepared as described (Reyes-Reveles, 2017). RPE proteins were 416 loaded into precast 4-12% NuPage Bis-Tris gels (Thermo Fisher/Life Tech, Carlsbad, CA) and 417 resolved with PAGE. Proteins were transferred to PVDF membranes and the membrane 418 blocked for 1 hr at RT in 5% dry milk, TBST (137 mM NaCl, 2.7 mM KCl, 19 mM Tris base with 419 0.1% Tween-20). The membranes were probed overnight at 4oC with antibodies dissolved in 420 blocking buffer. We used the following antibodies for Western blotting: rabbit anti- catalase 421 (Abcam ab1877, 1/5000), rabbit anti-Pex14 (ProteinTech 10594-1-AP, 1/1000), mouse anti- 422 PMP70 (Sigma SAB4200181, 1/1000), rabbit anti- β-catenin (Cell Signaling, D10AB; 8480, 423 1/1000), mouse anti- β-actin (Sigma; A2228, 1/2500), and mouse anti-cyclophilin A (Cell 424 Signaling; 2175S, 1/1000). Following washes, membranes were probed for 1 hr at RT with 425 Horseradish peroxidase (HRP) - conjugated secondary antibodies were used to detect mouse 426 (Thermo Fisher Scientific; G-21040, 1/2500) and rabbit IgG (ThermoFisher Scientific; 31460, 427 1/2500). Chemiluminescence signals were developed using SuperSignal® West Dura extended 428 duration substrate (ThermoFisher Scientific; 34075). 429 430 Catalase assays 431 Catalase activity was measured as amount of peroxide depleted in samples by detection with a

432 probe reacting with H2O2 in the presence of HRP to produce the fluorescent resorufin (Amplex 433 Red Catalase Assay Kit, Eugene, OR). Fluorescence at 590 nm was measured with a 434 microplate reader (Fluoroskan Ascent microplate fluorometer, Thermo Systems). Catalase 435 activity in experimental samples was determined as change in fluorescence compared to known bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

436 standards. Lysates for measuring catalase activity were prepared similarly to Western blotting, 437 except that solubilization was performed with manual homogenization in PBS. Triton X-100 was 438 added to a final concentration of 0.5% followed by vigorous vortexing to enhance the extraction 439 of catalase from peroxisomal membranes. 440 441 Citrate synthase activity assays 442 Citrate synthase activity was measured using Mitocheck citrate synthase activity assay (Cat. # 443 701040, Cayman Chemical, Ann Arbor, MI.) as described (33). This assay is based on the 444 change in absorbance measured with the release of SH-CoA due to the citrate synthase- 445 catalyzed condensation of oxaloacetate and acetyl-CoA to form citrate. The reaction was 446 initiated with the addition of oxaloacetate to reaction wells containing RPE lysates diluted ~50- 447 fold. Absorbance at 412 nm as a function of time was measured for lysate samples, positive 448 controls (using a known amount of enzyme), as well as negative controls (with either no enzyme 449 or no enzyme and no oxaloacetate). Specific activity was determined by dividing the reaction 450 rate by lysate protein concentration. bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

451 Figure Legends 452 Figure 1. Enrichment of β-oxidation genes in peroxisomes of mouse RPE and their 453 identification with TEM 454 A. Schematic depicting key enzymes involved in fatty acid β-oxidation within peroxisomes. The

455 initial committed step, desaturation of acyl - CoA esters by ACOX1, produces H2O2 as a 456 byproduct, which is then neutralized by catalase. FFA – free fatty acid, OS – photoreceptor 457 outer segments. B. Microarray expression analysis from GSE10246 showing relative enrichment 458 of peroxisomal β oxidation enzymes, including catalase, in mouse RPE. C. Transmission EM 459 image of WT RPE labeled with DAB. BM - RPE basement membrane, M-mitochondria, MG – 460 melanin granule, MV - microvilli. D. Boxed field (in panel C) at higher magnification. Yellow 461 arrowheads in C and D indicate peroxisomes. E. Example images of Immuno-electron 462 microscopy in WT mouse RPE showing gold particle labeling of catalase. 463 464 Figure 2. Mouse RPE peroxisomes are enriched in catalase 465 A and D. Single plane confocal micrographs of cryosections of WT and LC3B-/- mouse eyes 466 harvested and fixed at 10 am and immunolabeled with antibodies to PMP70 (green) and 467 catalase (red). GCL - ganglion cell layer, IPL-inner plexiform layer, INL – inner nuclear layer, 468 OPL-outer plexiform layer, IS – photoreceptor inner segments, OS -photoreceptor outer 469 segments, RPE - retinal pigment epithelium. B, E. Higher magnification views of the boxed 470 regions in A and D respectively. C, F. Intensity profiles for PMP70 (green), Catalase (red) and 471 Hoechst nuclear dye (blue) across the lines depicted in B, and E, respectively. The degree of 472 overlap of catalase and PMP70 was estimated with a Pearson’s correlation coefficient of 0.89 (± 473 0.006 n=58) for WT and 0.89 (± 0.005 n=55) for LC3B-/- measured from circular ROIs (5 μm 474 diam). 475 476 Figure 3. Expression of Pex14 and Pmp70 is elevated in LC3B-/- during the day but does 477 not vary diurnally 478 A, B. Western blots of RPE lysates made at indicated times of day. Immunoblotting was 479 performed with antibodies to peroxisomal membrane proteins Pex14 (A) and PMP70 (B) and 480 loading control actin. C, D. Quantification of intensities of Pex14 (C) or PMP70 (D) relative to 481 loading control band intensities as a function of time of day for WT (left plots) and LC3B-/- lysates 482 (right plots each panel). Values were not significantly different in C and D. Ratio of 483 immunoreactivity of Pex14 (E) or PMP70 (F) in LC3B-/- vs. WT estimated from the same data in 484 C and D, respectively. Values were not significantly different across each time point in E and F. bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

485 486 Figure 4. Peroxisomes are more numerous in LC3B-/- mice 487 A, B. Cryosections of eyes from WT and LC3B-/- mice harvested at 10 am showing the RPE 488 layer, and immunolabeled with antibodies to peroxisome specific markers Pex14 (red) and 489 PMP70 (green) Hoechst nuclear dye (cyan). C. Example ratios of fluorescence intensities for 490 Pex14 or PMP70 in LC3B-/- vs. WT images, asterisks denote statistical significance between WT 491 and LC3B-/- intensities: ** p < 0.01, * p < 0.02. 492 493 Figure 5. Elevated catalase activity during early morning in RPE of WT but not LC3B-/- 494 A. Catalase activity measured from RPE/choroid lysates made at different times relative to light 495 onset (7 am). Activity is expressed relative to mg protein. 8 data sets from 3 mice of each 496 genotype were averaged. *p<0.02, **p<0.0006, **p<0.0001. Values were not significantly 497 different across time points for LC3B-/- mice (one-way ANOVA). Differences in catalase activities 498 between WT and LC3B-/- were statistically significant (p<0.009 two-way ANOVA), but at 3 pm, 499 WT and LC3B-/- catalase activity differences did not reach statistical significance (p<0.1). B. 500 RPE choroid lysates from WT and LC3B-/- mice made at the indicated times of day were 501 immunoblotted with antibodies to catalase, and cyclophilin A (as a loading control). C. Relative 502 intensities of catalase vs. cyclophilin A in immunolabeled bands from Western blots of 503 RPE/choroid lysates made at different times of day. D. Citrate synthase activity in WT and 504 LC3B-/- lysates of posterior eyecup after removal of neural retina (RPE/choroid). Values were 505 not significantly different. E. Catalase activity in lysates of RPE/choroid explants incubated with

506 Ringers, or with added bovine outer segments and 5 mM glucose, or 0.5 mM H2O2. Catalase

507 activity was increased by 55% in WT after H2O2 treatment compared with Ringers control 508 (*p<0.05). 509 510 511 Acknowledgments This work was supported in whole or in part, by the National Institutes of 512 Health Grants EY010420-21 (to KBB), EY026525-02 (to KBB and NJP), R01EY027442 and 513 P30EY000331 (to DSW), and PENN Core grant. The authors thank the PDM- live cell imaging 514 core and the UPENN EM core for analyses. 515 516 bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

517 Abbreviations used 518 ABCD - ATP-binding cassette transporter subfamily D 519 ACOX - Acyl CoA Oxidase 520 AMD - age-related macular degeneration 521 βHB - β-hydroxybutyrate 522 DAB - diaminobenzidine 523 DHA - docosahexaenoic acid 524 FAO - fatty acid oxidation 525 HRP - Horseradish peroxidase 526 LC3B – microtubule-associated protein 1 light chain 3B 527 NBR1 - neighbor of BRCA1 gene 1 528 OS - photoreceptor outer segment 529 P62 - sequestosome 1; p62 protein 530 Pex14 - peroxisomal biogenesis factor 14 531 PMP70 - The ATP-binding cassette transporter subfamily D member 3 532 PPAR - peroxisome proliferator-activated receptor 533 RPE - retinal pigment epithelium 534 VLCFA - very long chain fatty acids bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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Figure 1. A B

FA OS

ABCDs

FFA

Abcd1 (ALDP) Abcd2 (ALDR) Abcd3 (PMP70) Acox1 Acox2 Catalase Acaa1a/b Acaa1a (thiolase/ Acaa1b PTHIO) Hsd17b4 (bi-functional protein)

C D

BM

M

MG 0.5 µm

E Catalase-Au

1 µm MV bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2.

A D WT catalase, PMP70 LC3B-/- catalase, PMP70

RPE RPE

OS OS

IS

IS ONL

ONL OPL

OPL INL

INL

IPL

IPL GCL

25 µm 25 µm GCL B E catalase catalase

PMP70 PMP70

merge merge

5 µm 5 µm C F bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3.

A B

light light Time: 6 am 7 am 8 am 3 pm Time: 6 am 7 am 8 am 3 pm LC3B +/+ -/- +/+ -/- +/+ -/- +/+ -/- LC3B +/+ -/- +/+ -/- +/+ -/- +/+ -/- kDa 62 kDa 62 PMP70 49 Pex14 49 actin actin 38 38 C D

WT LC3B-/- WT LC3B-/-

6 am 7 am 8 am 3 pm 6 am 7 am 8 am 3 pm 6 am 7 am 8 am 3 pm 6 am 7 am 8 am 3 pm

Time of RPE/choroid isolation Time of RPE/choroid isolation

E F

6 am 7 am 8 am 3 pm 6 am 7 am 8 am 3 pm bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4. A WT

PMP70

PEX14

5 μm merge

B LC3B-/-

PMP70

PEX14

merge 5 μm

C ** * bioRxiv preprint doi: https://doi.org/10.1101/687533; this version posted July 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5.

A * ** *** WT LC3B-/-

6 am 7 am 8 am 3 pm 6 am 7 am 8 am 3 pm light light Time of RPE/choroid isolation B time 6 am 7 am 8 am 3 pm LC3B +/+ -/- +/+ -/- +/+ -/- +/+ -/- kDa 62 catalase 49

28 cycl. A

C p<0.02 WT LC3B-/-

light D Citrate Synthase activity WT LC3B-/-

6 am 7 am 8 am 3 pm 6 am 7 am 8 am 3 pm light light Time of RPE/choroid isolation E Catalase activity p<0.05 WT LC3B-/-

Ringers H O OS 2 2 + glucose