Photoreceptor Cell–Derived CAPN5 Regulates Retinal Pigment Epithelium Cell Proliferation Through Direct Regulation of SLIT2 Cleavage

Retinal Cell Biology Photoreceptor Cell–Derived CAPN5 Regulates Retinal Pigment Epithelium Cell Proliferation Through Direct Regulation of SLIT2 Cleavage

Yan Wang,1,2 Heming Li,1,2 Shihu Zang,3 Fanfan Li,4 Yingying Chen,4 Xiao Zhang,1,2 Zongming Song,1,2,5 Qing Peng,6 and Feng Gu1,2 1School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang Province, China 2State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang Province, China 3Fuyang City Suburban Middle School, Fuyang, Anhui, China 4The Second Afﬁliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China 5Henan Eye Institute, Henan Eye Hospital, Henan Provincial People’s Hospital and People’s Hospital of Zhengzhou University, Zhengzhou, Henan, China 6Shanghai Tenth People’s Hospital, Tongji University, School of Medicine, Shanghai, China

Correspondence: Feng Gu, Eye Hos- PURPOSE. To identify the causative gene and investigate the corresponding mechanisms for an pital, Wenzhou Medical University, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) family. 270 Xueyuan West Road, Wenzhou, 325027, Zhejiang, China; METHODS. Clinical examination and genetic analysis were performed in a Chinese ADNIV [email protected]. family. To dissect the molecular consequence, we used gene targeting to knock-in a patient’s specific mutation in the mouse genome. Immunostaining and immunoprecipitation were Submitted: July 25, 2017 Accepted: March 7, 2018 harnessed to analyze the colocalization and interaction of CAPN5 with SLIT2 in photoreceptors. The purified SLIT2-N, SLIT2-C fragments, and the conditioned medium from Citation: Wang Y, Li H, Zang S, et al. 661W cells with the overexpression of CAPN5 were treated on ARPE-19 cells. The viability of Photoreceptor cell–derived CAPN5 ARPE-19 cells was determined by MTT assays. The activation of protein kinase A (PKA) was regulates retinal pigment epithelium cell proliferation through direct regu- analyzed by immunofluorescence and Western blotting in 661W and ARPE-19 cells as well as lation of SLIT2 cleavage. Invest Oph- in frozen retina tissue from wildtype (WT) and knock-in mice. thalmol Vis Sci. 2018;59:1810–1821. RESULTS. We identified a novel CAPN5 mutation (p.R289W) in a Chinese family and generated https://doi.org/10.1167/iovs.17-22689 the knock-in CAPN5R289W mouse. This mutation caused abnormal proliferative RPE in both humans and mice. CAPN5 directly cleaved WT SLIT2 in vitro, but not the mutant SLIT2 (p.R1113I). CAPN5 interacted with the SLIT2 in mouse retinal photoreceptors (661W cells) and increased cleavage and secretion of the SLIT2 fragments (SLIT2-N and SLIT2-C). Conditioned medium induced higher levels of secreted SLIT2 fragments, which promoted PKA activation and promoted proliferation of ARPE-19 cells.

CONCLUSIONS. The novel CAPN5 mutation (p.R289W) is responsible for the present ADNIV family. The mutant CAPN5 stimulated secretion and cleavage of SLIT2 fragments that may act as a bystander to regulate abnormal RPE cell proliferation for ADNIV. Keywords: ADNIV, CAPN5, SLIT2, PKA, cleavage, retinal pigment epithelium, cell proliferation

alpainsareproteasesthatregulatemanybiological photoreceptor degeneration, reactive retinal pigment epitheli- C functions through their proteolytic activity.1 Active cal- um (RPE) cells, retinal neovascularization, autosomal inflam- pains have been linked to neurodegenerative diseases such as mation, and blindness.12,13 Unlike the classical calpains CAPN1 Huntington’s disease, Alzheimer’s disease, and several neuro- and CAPN2, CAPN5 is expressed mainly in the outer segments traumas.1–5 CAPN1 and CAPN2 are well-known classical of the retina and also in synapses of photoreceptors, as well as calpains and activated in neurodegenerative conditions and in the inner plexiform layer and retinal ganglion cells.14 cell death. They also have been identified as important Although the CAPN5 protein has no calcium-binding domains components of several eye pathologies, including retinal unlike CAPN1/2, its activity is related to calcium ion levels in degeneration, retinal hypoxia, retinal detachment, and glauco- the retina in a fashion similar to CAPN1 and CAPN2.11 ma.6–9 The catalytic domain of the calpain protease family is highly CAPN5 is a member of the calcium active-related protease conserved in different isoforms and across species.15 Many calpain family.10,11 Mutations in CAPN5 cause inherited substrates of classical calpains are known that are related to cell autosomal dominant neovascular inflammatory vitreoretinopa- death, the cytoskeleton, and cell metabolism.16,17 A single thy (ADNIV).12,13 CAPN5 is hyperactive in ADNIV and is also amino acid mutant locating at the catalytic domain of CAPN5 related to many additional retinal pathologies, including increases its activation and is expressed in a very cell-specific

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manner.18,19 The molecular mechanisms underlying several 1.6 3 1.6-mm field of view by rotating the cassette that holds pathologies, such as photoreceptor degeneration and eye the animal. inflammation, are not clear in ADNIV. It has been shown that secreted proteins regulated the development, differentiation, Antibodies and Reagents and proliferation of the RPE layer as well as the vitreous space in a paracrine manner.20 Whether factors secreted by Antibodies to the following antigens were used: SLIT2 (sc- photoreceptor cells can also influence the function of RPE 16619, E-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cells or other tissues in the eye has not been well studied. CAPN5 (sc-271271, A-5; Santa Cruz Biotechnology), phosphor- Therefore, here we identified a novel mutation in CAPN5 in an PKAa/b/c (Thr198) (sc-136461; Santa Cruz Biotechnology), ADNIV family and investigated the molecular consequence of mouse PKAa/b/c (sc-98951,H-56; Santa Cruz Biotechnology), CAPN5 mutation. We found that it is physically associated with Ki67 (Ruiying Biotechnology, Hangzhou, China), glyceralde- the secreted neural repulsion molecule SLIT2 in retinal hyde-3-phosphate dehydrogenase (GAPDH) (Bioworld, Wuhan, photoreceptors. China). Secondary antibodies coupled to horseradish peroxi- dase or fluorescein were rabbit anti-mouse IgG (Boshide, Wuhan, China), goat anti-mouse IgG (Boshide), goat anti-rabbit MATERIALS AND METHODS IgG (Boshide), Alexa Fluor 488-conjugated donkey anti-mouse IgG and Alexa Fluor 555-conjugated donkey anti-rabbit IgG Genetic Analysis (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The family was recruited and ocular and hearing examinations were performed in the Eye Hospital of Wenzhou Medical Cell Culture University. Whole exome sequencing was utilized, and Four different cell lines (HEK-293T cells, human neuroblasto- candidate disease-causing mutation in CAPN5 was evaluated ma SHSY5Y cells or mouse photoreceptor-like 661W cells, in the ADNIV family using Sanger sequencing. human RPE ARPE-19 cells) were cultured in the present study. Transfection of SHSY5Y and 661W cells was performed using a Generation of CAPN5 Knock-In Mice reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 72 hours of Knock-in mice were created on a B6 (C57BL/6J/N) back- R289W transfections of CAPN5 vectors in 661W cells, the fresh ground. Before we generated B6 knock-in (CAPN5 ) mice, medium (400 lL DMEM with 0.1% FBS) was added in a 24-well we confirmed the strain background is wildtype (WT) B6, RD8/RD8 plate, which is used as conditioned medium for the culture of which ruled out C57BL/6J/N with Crb1 linked to human RPE ARPE-19 cells. retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). CAPN5R289W mice were maintained in the Wenzhou Medical University Animal Care Services Facility. All experi- Immunostaining mental procedures were approved by the Institutional Cells (5 3 104) were transfected with the plasmids on Committee of Wenzhou Medical University in accordance with coverslips 60 hours post transfection, and cells were fixed the Declaration of Helsinki and the ARVO Statement for the Use with 4% paraformaldehyde (PFA) for 10 minutes at 258C. Cells of Animals in Ophthalmic and Vision Research, and were were incubated with 10% bovine serum albumin (BSA) diluted conducted in compliance with the Animal Research: Reporting in PBS, then incubated with anti-Flag antibody and anti-SLIT2 of In Vivo Experiments guidelines. antibody for 18 hours at 48C. After three washes in PBS for 5 The gene-targeting vector was sequenced to confirm that it minutes each, slips were incubated with secondary antibodies, had only the desired point mutation. Gene targeting was Alexa 555 goat anti-rabbit and Alexa 488 goat anti-mouse performed in mouse embryonic stem cells, and the knock-in (1:700 dilution each, prepared in blocking solution). The mice were identified by analyzing genomic DNA isolated from coverslips were attached on glass sides with glycerol diluted in tail tips. The CAPN5 knock-in mice contained FRT-neo-FRT PBS (1:1). Images were captured in a digital format using a (intron 5) and p.R289W point mutation (exon 6); CAPN5 Zeiss microscope (Carl Zeiss, Chester, VA, USA). For retina knock-in mice were mated with FLP mice to remove the analysis, mouse eyes were enucleated and embedded in antibiotics gene. The final CAPN5 knock-in mice without the optimal cutting temperature compound at 808C. Sections (8 R289W antibiotics gene were labeled with CAPN5 . lm) were cut on a cryostat, placed on glass slides, and allowed to dry for 2 hours. Sections were fixed in cold acetone on ice Electroretinography and Optical Coherence for 10 minutes, followed by three washes with PBS, 5 minutes Tomography each. Sections were incubated in blocker (1% BSA, 0.2% skim milk, and 0.3% Triton X-100 in PBS) for 15 minutes at room ERG was performed with a standard protocol. Differences temperature. Sections were then incubated with rabbit anti- between WT (n ¼ 15) and CAPN5R289W (n ¼ 12) mice were CAPN5 (1:500; Santa Cruz Biotechnology) and anti-SLIT2 assessed for six ERG parameters in three age-matched groups (1:300; Santa Cruz Biotechnology) overnight at 48C in humid by means of t-tests corrected for multiple comparisons (a ¼ environment. After three 5-minute washes in PBS, sections 0.95). In a subset of the mice (n ¼ 7forCAPN5R289W; n ¼ 6for were incubated with secondary antibodies. WT), S- and M-opsin–mediated cone function was compared. Both stimuli were presented in a ganzfeld lined with aluminum Western Blotting Assay and Immunoprecipitation foil. Retinal cross-sections of WT and CAPN5R289W mice were acquired with a 3.2-lm resolution spectral-domain optical Western blotting analysis and immunoprecipitation was coherence tomography (SD-OCT) system (Bioptigen, Inc., performed as described.21 In brief, cell lysates were incubated Durham, NC, USA). Corneas were lubricated with ophthalmic overnight at 48C with anti-CAPN5 antibody or anti-SLIT2 lubricant frequently during the imaging session (Systane Ultra; antibody or with nonimmune rabbit or mouse IgG controls. Alcon Ltd., Fort Worth, TX, USA). Using the fast fundus mode Immunoprecipitations were then captured with protein G- (200 raster scans of 200 longitudinal reflectivity profiles each), Sepharose beads for 2 hours at 48C and eluted with 10 mM we first centered the location of the optic nerve head within a glutathione according to manufacturer’s instructions (GE

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Healthcare, Shanghai, China). The cells were transfected with black arrow in Fig. 1A). She has cataracts (Fig. 1B), retinitis pSin CAPN5-Flag or pSin CAPN5 R289W vector for 60 hours, pigmentosa (RP; Fig. 1C), and mild dysaudia (Fig. 1D). Other and cell lysates were incubated with anti-Flag M2 antibody affected individuals have a similar clinical manifestation, with conjugated with magnetic beads (Sigma-Aldrich Corp., St. the exception of the mild dysaudia, which is close to the Louis, MO, USA) for 2 hours at 258C. Immunoprecipitates were phenotypes from reported ADNIV families.12 We identified a then separated with a magnetic shell and eluted according to novel mutation (c. 865 C>T, p. Arg [R] 289 Trp [W]) (Fig. 1E) the manufacturer’s instructions (Sigma-Aldrich Corp.). in this family. The Arg289 residue located in exon 6 is highly conserved in different vertebrate species. Results from direct Semiquantitative RT-PCR sequencing confirmed this mutation cosegregated with all affected individuals in the family and was not present in any of RT-PCR was performed using primers for mouse Capn5: sense, the unaffected family members or normal controls (from in- 50-GATCCCCGGCTCTTCGTAGAT-30, anti-sense 50-GACGATTA house exome databases with 1402 samples from a Chinese CGTCCACCCACTC-30; for mouse SLIT2: sense, 50-GGCAGACA population). Thus, the p. R289W mutation in CAPN5 is a CTGTCCCTATCG-30, anti-sense, 50-ATCTATCTTCGTGAT disease-causing mutation in this family. CCTCGTGA-30; for loading control mouse Gapdh: sense, 50-AG CTTCGGCACATATTTCATCTG-30, anti-sense, 50-CGTTCACTCC 0 Elevated Ki67 Staining in the RPE Layer in CATGACAAACA-3 . Experiments were performed in triplicate R289W and the density of bands was measured by ImageJ software CAPN5 Mice (http://imagej.nih.gov/ij/; provided in the public domain by To investigate molecular mechanism of CAPN5 mutation, we the National Institutes of Health, Bethesda, MD, USA) and generated CAPN5R289W knock-in mice. We did not observe any normalized to Gapdh. obvious differences in RPE layer thickness between 3- and 8- month-old knock-in and WT mice by OCT (Fig. 2A). Also no Plasmid Construct and Protein Purification functional differences of photoreceptors by ERG examination between 8-month-old CAPN5R289W mice and their WT The Flag-tagged full-length human CAPN5 isoform, CAPN5 counterparts have been observed (Figs. 2B, 2C). However, R289W mutant, His-tagged single-chain variable fragment when we examined levels of the proliferative antigen Ki67 by (scFv) against CAPN5 was inserted into the pSin-EGFP vector. immunofluorescence, we observed a significant increase in Site-directed mutagenesis of the SLIT2 was performed using both 3- and 8-month-old CAPN5R289W mice (n ¼ 4) when high-fidelity enzyme Pfu (TIANGEN Biotech Co., Ltd., Beijing, compared with WT mice (n ¼ 6) in RPE layers (Fig. 2D, 2E). China). The pET28a vector was used as backbone for the This suggests that abnormal proliferative activation exists in expression of CAPN5, SLIT2, SLIT2-N, and SLIT2-C. The CAPN5R289W mouse RPE layers. plasmids were transformed into Escherichia coli BL21 cells, þ CAPN5 Interacts With SLIT2 in Mouse Photoreceptor- and His-tagged proteins were purified using Ni affinity Like Cells and Neural-Like Cells. It has been reported that columns. CAPN5 is specifically expressed in the nucleus and cytoplasm of neural-like cells and photoreceptor cells and that CAPN5 Determination of Cell Viability mutations commonly affect the subcellular location of CAPN5.12,21 SLIT2 is expressed in the cytoplasm, at the plasma ARPE19 (5 103) cells were suspended in DMEM/F-12 medium 3 membrane, and can also be secreted by neurons or retinal with 0.1% FBS or conditioned medium from 661W transfected 22,23 neural cells. Previous studies using a yeast two-hybrid with CAPN5 vectors to a concentration of 5 104 cells/mL, and 3 system have shown that the large subunit 1 associates with 100 L was added to each well of a 96-well plate. After 24 l SLIT2/3. To identify the subcellular location and interaction treatment with recombinant SLIT2, SLIT2-N, and SLIT2-C between CAPN5 and SLIT2, we transfected CAPN5 and CAPN5 proteins for 48 hours, cells were incubated separately with 10 R289W mutant plasmids into 661W cells, and immunostaining lLMTT(500lg/mL) for 4 hours. The culture medium was then was performed. It showed that WT CAPN5 was expressed removed, and 100 lL dimethylsulfoxide was added to each well, mainly in the nucleus and partly colocalized with SLIT2. followed by a 30-minute incubation period at 258C. Optical CAPN5 R298W was expressed and translocated mainly into the density was measured spectrophotometrically at 540 nm. cytoplasm, and most of the signals colocalized with SLIT2 in the cytoplasm (Fig. 3A). These results suggested that the Statistics mutant increased translocation of CAPN5 from the nucleus to Statistical analyses were performed with statistical software the cytoplasm and promoted CAPN5 colocalization with SLIT2 (SPSS 13.0; IBM Corporation, Armonk, NY, USA). All data are in the cytoplasm. To further investigate whether CAPN5 presented as mean 6 SEM unless otherwise specified. Student’s interacted with SLIT2 in 661W cells, we used anti-Flag beads to precipitate overexpressed CAPN5 and CAPN5 mutant t-test was used for comparison in experiments with only two groups. In experiments with more than two groups, ANOVA protein in 661W and SHSY5Y cells, respectively. We found was performed, followed by Tukey’s post hoc test for pairwise that CAPN5 coprecipitated with SLIT2 using a specific anti- comparisons among three and more than three groups. For SLIT2 primary antibody in both 661W and human neuroblas- analysis of more than two groups of nonparametric data, the toma SHSY5Y cells (Fig. 3B). Taken together, these results Kruskal-Wallis test was used. demonstrated that CAPN5 colocalized and interacted with SLIT2 at cytoplasm in photoreceptor-like 661W cells and neural-like SHSY5Y cells, and mutation leads to the mislocal- ization of the CAPN5. RESULTS CAPN5 Associates With SLIT2 and Affects its Proteo- Identification of a Novel CAPN5 Mutation (p. lytic Cleavage in Mouse Retinal Photoreceptors. To R289W) in an ADNIV Family confirm the localization of tissue expression of CAPN5 and SLIT2 in the retina, we performed immunofluorescence and The family included nine affected individuals in a four- Western blotting in adult WT mice (n ¼ 10) and knock-in generation family (Fig. 1A) originating from Anhui Province, CAPN5R289W mice (n ¼ 8) using specific primary antibodies China. The proband was a 9-year-old female (indicated with against CAPN5 and SLIT2. It has been reported that CAPN5 is

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FIGURE 1. ADNIV pedigree, clinical manifestations, and genetic analysis. (A) Pedigree of an ADNIV family. Black symbols represent affected family members. The slashes indicate the deceased in this family. The dots denote females, and squares denote males. The arrow points to the proband. (B) Cataracts have been observed. (C) RP of the proband and normal control fundus image (right panel). The arrows indicate pigmentation. (D) Mild dysaudia of the proband. (E) Sequences of proband and control.

expressed on the photoreceptor layer, inner layer, and retinal the complex captured by CAPN5 antibody by Western blotting. ganglion cells (RGCs) in mouse retina.14 SLIT2 expression has SLIT2 was not present in mouse IgG control groups. IgG heavy- been observed in retina neuronal cells.25,26 Here, we detected chain bands were present in input, IgG, and WT mouse retina CAPN5 expression mainly in the outer segment of the retina lysates (Fig. 4D). The protein levels of CAPN5 were not altered and partly in the inner layer and ganglion cell layer. SLIT2 was in WT mice and CAPN5R289W mice. The full-length SLIT2-N and expressed in the outer segment of photoreceptor cells and the SLIT2-C fragments were all detected in CAPN5R289W mice (n ¼ inner layer (Fig. 4A). We also found that CAPN5 and SLIT2 were 4) retinas, while we could not detect the SLIT2-C fragment in mainly coexpressed in the outer segments of photoreceptors WT mice retinas (n ¼ 4) (Fig. 2E, P < 0.001). We also did not in WT mice and CAPN5R289W mice (Fig. 4B). Both CAPN5 and find the differently expressed levels of SLIT2 and CAPN5 in SLIT2 proteins were also detected in the mouse retina (n ¼ 3), adult CAPN5R289Wþ/ heterozygous mice and CAPN5R289Wþ/þ but we did not detect a specific 75-kDa CAPN5 band in HEK- homozygous mice (Fig. 4E, 4F, not significant; P > 0.05). These 293T cells (Fig. 4C); it has been shown that SLIT2 is expressed results indicate that CAPN5 associated with SLIT2 and by HEK-293T cells.25 regulated proteolysis of SLIT2 in retina photoreceptors, as To further clarify the possible association between CAPN5 levels of the cleaved SLIT2-N and SLIT2-C fragments were and SLIT2 in vivo, we used a CAPN5 antibody to immunopre- increased in CAPN5R289W mouse retinas in vivo. cipitate CAPN5 protein complexes from whole retina lysates in CAPN5 Promotes Proteolytic Cleavage of SLIT2 in adult WT mice (n ¼ 4). Both full-length SLIT2 and 140-kDa SLIT- Neuronal-Like Cell Lines. To demonstrate that CAPN5 N forms were detected using a specific anti-SLIT2 antibody in regulates the cleavage of SLIT2 in vitro, we transfected CAPN5

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R289W FIGURE 2. Retinal examination in CAPN5 mice. (A) Fundus photo. The green line indicates the section of OCT shown. A1, A2, 8-month B6, and WT controls; B1, B2, 3-month CAPN5R289W mice; C1, C2, 8-month CAPN5R289W mice. (B) Amplitude of a-wave. (C) Amplitude of b-wave. (D) Ki67 immunostaining in CAPN5R289W mice. 1, 2 represent retina per group. Scale bar: 150 lm. (E) High magniﬁcation of Ki67 immunostaining in CAPN5R289W RPE layers. Scale bar:20lm, 403. OS, outer segment; IS, inner segment.

and CAPN5 R289W mutant vectors into 661W cells and the SLIT2 protein. Also, recombinant CAPN5 was incubated with human neuroblastoma SHSY5Y cells. After 72 hours post His-tagged mouse SLIT2 protein and the noncleavable R1113I transfection, the levels of SLIT2 isoforms were measured by SLIT2 mutant protein in physiological buffers. After 1-hour Western blotting. We found that an increase in both 140-kDa incubation, proteins were analyzed by Western blotting using an SLIT2-N and 55-kDa SLIT-C fragments was observed in 661W anti-SLIT2 specific antibody. We found that WT recombinant cells after CAPN5 transfection (Fig. 5A). The levels of cleaved SLIT2 was proteolytically cleaved into SLIT2-N and SLIT2-C SLIT2-N plus SLIT2-C fragments were increased with the fragments by human CAPN5. However, SLIT2 R1113I mutant overexpression of CAPN5 R289W compared to WT CAPN5 was resistant to cleavage as expected (Fig. 5G). These results (Fig. 5B, P < 0.05). We also detected higher levels of SLIT2-C suggest that recombinant CAPN5 protein proteolytically cleaves fragment compared with SLIT2-N fragment after CAPN5 SLIT2intoSLIT2-NandSLIT2-Cfragmentsbutdidnot R289W transfection (Fig. 5C, P < 0.05).These increased levels proteolytically cleave the mutant SLIT2 R1113I protein. of secretion were also detected in SHSY5Y cells with the To further explain how CAPN5 activity drives the cleavage transfection of CAPN5 R289W. The levels of SLIT2-C versus of SLIT2, we used an intracellularly expressed antibody SLIT2-N were unchanged after WT CAPN5 transfections (Fig. fragment plasmid, pSinscFv, to target and inhibit CAPN5. We 5D, 5E, P < 0.01; Fig. 5F, P < 0.05).These data suggested that cotransfected CAPN5 R289W and pSinscFv vectors into 661W CAPN5 R289W mutant promoted cleavage of SLIT2 fragments. cells and found that the total levels of SLIT2-N and SLIT2-C CAPN5 R289W overexpression induced higher levels of were significantly decreased 60 hours post transfection cleaved SLIT2-C than SLIT2-N in cells. compared with CAPN5 R289W and the vector control group It has been shown that native secreted mouse SLIT2 is (Fig. 5H). Taken together, these results suggested that usually proteolytically cleaved at R1113 to form the long SLIT2-N overexpression of CAPN5 induced increased proteolytic and short SLIT2-C fragments. This cleavage site is highly cleavage of SLIT2 in 661W and SHSY5Y neuronal-like cell conserved in different species.26 To further examine whether lines. CAPN5 R289W showed a much stronger proteolytic CAPN5 directly cleaves SLIT2, we incubated purified His-tagged activity on SLIT2 as a substrate compared to WT CAPN5. Also, recombinant human CAPN5 with purified recombinant mouse recombinant WT SLIT2 protein was degraded by CAPN5, while

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FIGURE 3. CAPN5 interacts with SLIT2 in vitro. Here, 661W cells were transfected with vectors and were then ﬁxed and stained using an anti-Flag antibody (green) and an anti-SLIT2 antibody (red). (A) CAPN5 colocalizes with SLIT2 in 661W cells. Scale bar:20lm. (B) Sixty hours posttransfection with CAPN5 vectors, 661W cells and SHSY5Y cells were lysed and immunoprecipitated with M2 anti-Flag beads. The protein complex was then separated by SDS-PAGE and detected using an anti-SLIT2 antibody. CAPN5 was associated with SLIT2 in 661W and SHSY5Y cells.

CAPN5 did not proteolytically cleave the mutant SLIT2 CAPN5 Drives Secretion of SLIT2 Fragments and (p.R1113I) protein in vitro. Moreover, a speciﬁc inhibitory Promotes Proliferation of Human RPE Cells via Activa- intracellular antibody blocked the activity of CAPN5 and tion of Protein Kinase A (PKA). We prepared SLIT2 inhibited cleavage of SLIT2, which may have therapeutic recombinant protein (Fig. 6A). We found overexpression of effects for this disease. CAPN5 leads to the increased expression of SLIT2-N and SLIT2-

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R289W FIGURE 4. CAPN5 associates with SLIT2 and cleaves SLIT2 in vivo. Retina sections from 3-month-old WT and CAPN5 mice. (A) Low- magnification images of CAPN5 (red) and SLIT2 (green) in retina sections. ONL, outer nuclear layer; INL, inner nuclear layer; OS/IS, outer segment/ inner segment of photoreceptor cells. Scale bar:50lm. (B) Higher-magnification images of OS/IS. Scale bar:20lm. (C) SLIT2 and CAPN5 were expressed in mouse retina by Western blotting. The cell lysate from HEK-293T cells was set as a positive control for SLIT2 expression. The lysate from 3-month-old WT mouse retinas was precipitated with the anti-CAPN5 antibody. The protein complex was separated by SDS-PAGE and detected by Western blotting with an anti-SLIT2 antibody. (D) CAPN5 associates with SLIT2 in the mouse retina. IP,immunoprecipitation. The loading control was anti-mouse IgG heavy chain, and positive control is shown as detected CAPN5. The protein and mRNA levels of CAPN5 and SLIT2 in WT mice (n ¼ 3) and CAPN5R289W mice (n ¼ 3) were measured by Western blotting. (E)(i) The expression levels of CAPN5 and SLIT2 in the mouse retina. The density of SLIT-C bands was measured and calculated compared to the values of GAPDH loading control; the mean 6 SEM values from three independent experiments (WT, n ¼ 5; CAPN5R289W) are shown compared with WT mice. (ii) The mRNA levels of CAPN5 and SLIT2 in the mouse retina. The density of SLIT2 bands was measured and calculated compared to the values of a GAPDH loading control; the mean 6 SEM values from the CAPN5R289Wþ/ heterozygous mice (n ¼ 4) and CAPN5R289Wþ/þ homozygous (n ¼ 4) mice in three independent experiments were compared to WT mice and are shown on the lower bands. ***P < 0.001, Student’s t-test. n.s., no significance.

C fragments compared to controls (Fig. 6B, 6C; P < 0.05). The CAPN5 R289W Mutant Decreased PKA Phosphoryla- level of the SLIT2-C fragment was higher than the level of tion in Retinal Photoreceptor Cells. It has been shown that SLIT2-N (Fig. 6D; P < 0.05). It has been reported that secreted the inhibition of PKA may be of beneﬁt in several neurodegen- SLIT2-C stimulates robust activation of PKA in adipocytes.27 erative diseases.28 We therefore examined the level of phosphor- We used the recombinant SLIT2, SLIT2-N, and SLIT2-C ylation and activation of PKA in retinas from WT and fragments to stimulate human RPE cells and found that by CAPN5R289W mice by immunostaining. The level of phosphor- activating PKA, phosphorylation levels were increased. The ylated PKA was decreased in the outer segments of photorecep- phosphorylation of PKA was also enhanced by conditioned tors in transgenic CAPN5R289W mice compared to WT (Fig. 7A). medium (from 661W cells after transfection with CAPN5 These results were consistent with the levels of phosphor-PKA in vectors; Fig. 6E). To investigate whether secretion of SLIT2-C 661W photoreceptor cells after CAPN5 R289W overexpression from photoreceptors inﬂuences the viability of human ARPE- (Fig. 7B). We found that the levels of phosphor-PKA were 19 cells, we cultured ARPE-19 cells with conditioned medium reduced in CAPN5R289W mouse retinas (Fig. 7C, 7D). The levels or treated human RPE cells with recombinant SLIT2, SLIT2-N, of phosphor-PKA were decreased in 661W cells with overex- and SLIT2-C fragments. The viability of retinal epithelium cells pression of CAPN5 R289W (Fig. 7E, 7F). Taken together, these was higher in groups grown in conditioned medium or with results demonstrate that R289W mutation inhibits phosphoryla- SLIT2 and fragments treatment (Fig. 6F; P < 0.001, P < 0.01). tion of PKA in photoreceptor cells both in vivo and in vitro. These results revealed that photoreceptor-like 661W cells CAPN5R289W Mice Show Abnormalities in the Prolif- secreted cleaved SLIT2-N and SLIT2-C fragments that both erative RPE Layer. To investigate whether CAPN5 R289W promoted ARPE-19 cell proliferation by activation of PKA. promoted proliferation in the RPE layer in vivo, we performed

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FIGURE 5. CAPN5 promotes proteolytic cleavage of SLIT2 in neuronal cells. Cells (661W and SHSY5Y) were transfected with vectors. Sixty hours post transfection, SLIT2 expression was measured. (A) Isoforms of SLIT2 expressed in 661W cells. (B) Relative levels of SLIT2-N plus (þ) SLIT2-C fragments in 661W cells. (C) The normalized levels of SLIT2, SLIT2-N, SLIT2-C are shown in 661W cells. (D) Forms of SLIT2 expressed in SHSY5Y cells. (E) Levels of SLIT2-N plus SLIT2-C fragments in SHSY5Y cells. (F) The normalized levels of SLIT2, SLIT2-N, SLIT2-C are shown in SHSY5Y cells. GAPDH was used as the loading control. Expression levels of CAPN5 are shown in the lower panels as the positive control. The density of SLIT2-N plus (þ) SLIT2-C fragment bands, SLIT2, SLIT2-N, SLIT2-C bands compared and normalized to GAPDH bands were measured and calculated using Image J software. The mean 6 SEM values from three independent experiments were compared between CAPN5 WT and CAPN5 R289W groups. *P < 0.05, **P < 0.01; 1-way ANOVA, with Tukey’s test was used. Human recombinant CAPN5/His protein was incubated with mouse recombinant SLIT2/His and SLIT2 mutant R1113I protein at the indicated concentrations. After 1-hour incubation, proteins were detected by Western blotting with an anti-SLIT2 antibody. (G) The native recombinant CAPN5 cleaved SLIT2. , untreated control. (H) Inhibitory intracellular antibody pSinscFv decreased CAPN5-mediated cleavage of SLIT2. *P < 0.05, **P < 0.01, Student’s t-test. , cells transfected with pSin empty vector control; þ, pSinscFv/CAPN5 R289W vector transfections.

hematoxylin and eosin (H&E) staining and immunoﬂuores- We observed that phosphorylation of PKA was increased in cence analysis. We found an abnormal proliferative RPE layer in CAPN5R289W mouse RPE layers but reduced in the photore- CAPN5R289W mice, while no notable degeneration of the ceptor layer (Fig. 8B). These results indicated that the CAPN5 photoreceptor layer or other layers were observed (Fig. 8A). R289W mutant led to abnormal proliferation and increased

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FIGURE 6. Overexpression of CAPN5 promotes secretion of SLIT2 fragments from 661W cells as well as increasing cell proliferation and phosphorylation of PKA in RPE cells. (A) Puriﬁed SLIT2-N, SLIT2-C, and SLIT2 proteins were detected by anti-His tag antibody. Cells (661W) were transfected with vectors; 72 hours post transfection, the cultured media were collected, and detected by Western blotting with an anti-SLIT2 antibody. (B) Western blotting of SLIT2 isoforms in 100 lL 661W conditioned medium (CM). (C) Relative levels of SLIT2-N plus SLIT2-C fragments in CM. (D) Normalized levels of SLIT2, SLIT2-N, SLIT2-C in 100 lL 661W cell CM. The density of SLIT2-N plus (þ) SLIT2-C, SLIT2, SLIT2-N, SLIT2-C bands were normalized to vector control groups. The mean 6 SEM values from three independent experiments were compared between CAPN5 WT and CAPN5 R289W groups. *P < 0.05, **P < 0.01. (E) SLIT2 and its fragments increased phosphorylation of PKA in human RPE cells. CM, the conditioned medium from CAPN5 vector-transfected 661W cells. Medium with 0.1% FBS was used as a control. The images are representative of triplicate experiments. The density of p-PKA bands compared and normalized to GAPDH bands were measured and calculated using ImageJ software, respectively. The mean 6 SEM values from three independent experiments were compared to control medium or between CAPN5 WT and CAPN5 R289W groups. (F) ARPE-19 cells (5 3 103) were treated with recombinant SLIT2-C fragment at 1 lg, the CM from CAPN5 vector- transfected 661W cells, and control medium with 0.1% FBS. *P < 0.05, **P < 0.01.

phosphorylation of PKA in RPE cells (Fig. 8C), but reduced conformation. The proband shows features of cataracts, RP, phosphorylation of PKA in the photoreceptor layer when and mild dysaudia. To dissect its molecular consequence, we compared with WT mice in vivo. generated a knock-in mouse. While, to our surprise, no obvious abnormal features (cataracts or retinitis pigmentosa) have been observed in mice, we did find that CAPN5R289W mice have an DISCUSSION abnormally proliferative RPE layer. Thus, the different phenotypes between the ADNIV patients and CAPN5R289W mice Mutations in CAPN5 cause human ADNIV, the process of could be caused by activated CAPN5-mediated multipathway multiple pathologies including photoreceptor degeneration, proteolytic regulation of pathways in different species. autoinflammatory responses, and proliferative vitreoretinopa- SLIT2 classically exists in one of three different forms: full 12,13 thy. The molecular mechanisms underlying many of these length, a long fragment (SLIT2-N), and a short fragment SLIT-C, pathologies have not been well clarified. We identified a novel which are generated by proteolytic cleavage. The full-length mutation in CAPN5 (p.R289W) in a Chinese ADNIV family. We SLIT2 and SLIT2-N have been well studied as activators of speculated that this mutant stabilized catalytic core domain II roundabout axon guidance receptor (ROBO) signaling path- and enhanced CAPN5 catalytic activation in the protein ways in retinal axonal guidance.22,23,29 However, the

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R289W FIGURE 7. Phosphorylation of PKA was decreased in photoreceptors in CAPN5 mice. (A) Immunofluorescence activity of phosphorylated PKA in the retina of mice. Double-stained CAPN5 (red) and phosphor-PKA (p-PKA, green) in mouse retina sections. The left panels show the lower magnification (203), scale bar: 100 lm, and the right panels show higher magnification (403), scale bar:25lm. Sections were 8 lm in thickness. WT (n ¼ 5), mice were used as the control group. (B) Double-staining of p-PKA and CAPN5 in 661W cells. 661W cells transfected with WT CAPN5 vector and CAPN5 R289W vector. Sixty hours post transfection, cells were fixed and incubated with anti-CAPN5 (green) and anti-phosphor-PKA (red) followed by the appropriate secondary antibodies. Merged channels are shown. Scale bar:5lm. (C) Decreased levels of phosphor-PKA in CAPN5R289W mouse retinas. The retinal lysate from 3-month-old WT (n ¼ 5) and CAPN5R289W mice (n ¼ 7) were collected and separated by SDS- PAGE. (D) The levels of p-PKA, GAPDH, and PKA were detected by Western blotting. The bands were measured, calculated, and normalized to B6 WT group; the mean 6 SEM values were from three independent experiments and are compared with WT control groups. **P < 0.01; Student’s t- test was used. (E) Decreased levels of p-PKA in 661W cells, which were transfected with empty vector, CAPN5 WT vector, and CAPN5 R289W vector. Cells were lysed 60 hours post transfection, and Western blotting was performed to measure the levels of p-PKA, GAPDH, and PKA. Images are representative of experiments performed in triplicate. (F) The mean 6 SEM values were from three independent experiments and are compared with vector control groups. *P < 0.05; 1-way ANOVA, with Tukey’s test was used.

functional consequences of secreted SLIT2 fragments in the cells,31,32 as well as adipose-secreted SLIT2-C fragments that retina are still unknown. SLIT2 plays an important role as a regulate thermogenesis and metabolic function of brown and secreted factor in neural guidance and migration and beige fat.24 The secreted SLIT2 and SLIT2-N fragments development of the retinal optical chiasma via the SLIT2- promoted retinal neovascularization in the developing retina ROBO pathway.29,30 Recently, it has been shown that SLIT2- and abnormal pathologies in diabetic retinopathy. Indeed, ROBO, especially SLIT2-N/ROBO signaling, enhances retinal these features of SLIT2-related diabetic retinopathy overlap neovascularization by regulating VEGF expression and increas- with diabetic retinopathy-like features in CAPN5 mutation– ing RPE cell proliferation via MAPK activation.31 We found, we caused ADNIV. Very few studies have been associated with the believe for the ﬁrst time, that CAPN5 interacts with and functional consequences of release of the SLIT2 C-terminal promotes proteolysis of SLIT2. This provides a novel molecular fragment. In the development of mouse retina, the functional mechanism and therapeutic target for ADNIV pathologies consequences of SLIT2 expression principally occur via its N- caused by CAPN5 mutations. terminal fragment through SLIT2-N/ROBO pathways24 because The SLIT2/ROBO pathway has been associated with retinal SLIT2-C is not associated with ROBO signals.23,26 The secreted neovascularization, proliferative diabetic retinopathy, and RPE full-length SLIT2 and N-terminal SLIT2 enhanced proliferation

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R289W FIGURE 8. CAPN5 mice show an abnormal proliferative RPE layer and increased PKA phosphorylation in RPE cells. Retina sections from 3- month-old WT (n ¼ 8) and CAPN5R289Wmice (n ¼ 9) were H&E stained. (A) Histopathology of CAPN5R289W mouse retinas. Magniﬁcation is 203. Arrows denote the RPE layers. (B) Increased immunoﬂuorescence of p-PKA in CAPN5R289W mice. The black arrows represent the RPE layers and white arrows p-PKA positive retinal epithelial cells. Section thickness was 8 lm. WT (n ¼ 7) mice were the control group. Scale bar: 100 lmfor upper panel, 25 lmforlower enlarged panel. (C) The relative intensity of p-PKA signal was measured and calculated; the mean 6 SEM values were from CAPN5R289W(n ¼ 8) mice and are compared with WT control groups (n ¼ 7) in RPE layers. ***P < 0.001, Student’s t-test was used.

of RPE cells through activation of MAPK.31 Based on these the abnormal proliferation of RPE cells in the CAPN5-induced results, CAPN5 promoted photoreceptor-derived SLIT2 expres- ADNIV model. sion that increased activation of PKA in RPE cells, but the CAPN5 R289W mutant decreased phosphorylation of PKA in photoreceptor cells in vitro and in vivo. This could be because CONCLUSIONS PKA is a specific downstream pathway of activated CAPN5 in In summary, we identified a novel CAPN5 mutation in the photoreceptors but not in RPE cells. Some studies proposed ADNIV family and generated the corresponding knock-in mice. that inhibitors of cAMP upstream of PKA promoted prolifer- We found that CAPN5 regulates cleavage and secretion of ation of RPE cells in vitro.32 However, it is an oversimplification SLIT2 in retinal photoreceptors, which contributes the to say that proliferation of RPE cells is induced by activation or abnormal proliferation of RPE cells. Our study provides a inactivation of PKA. It has also been reported that the novel molecular mechanism and therapeutic target for ADNIV photoreceptor cells release factors that affect neighboring pathologies caused by CAPN5 mutations. cells33; native secreted factors from other cell types could affect abnormal proliferation of the RPE layer.34 Here our Acknowledgments results suggested that the secreted SLIT2-N and SLIT2-C Supported by grants from the Natural Science Foundation of China fragments from photoreceptor cells may increase the activation (81201181 [FG]; 81473295, 81670882 [ZS]), the Natural Science of PKA in RPE cells. Therefore, we propose that photoreceptor Foundation of Zhejiang Province, China (LQ17H120004 [YW]), cells released SLIT2-N and SLIT2-C and that this contributes to Science Technology Project of Zhejiang Province (2017C37176

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