Retina Degeneration of Photoreceptor Cells in Arylsulfatase G- Deficient Mice

Katharina Kruszewski,1 Renate Lullmann-Rauch,¨ 2 Thomas Dierks,3 Udo Bartsch,1 and Markus Damme4

1Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2Anatomical Institute, University of Kiel, Kiel, Germany 3Biochemistry I, Department of Chemistry, Bielefeld University, Bielefeld, Germany 4Department of Biochemistry, University of Kiel, Kiel, Germany

Correspondence: Udo Bartsch, De- PURPOSE. Retinal degeneration is a common feature of several lysosomal storage disorders, partment of Ophthalmology, Univer- including the mucopolysaccharidoses, a group of metabolic disorders that is characterized by sity Medical Center Hamburg- widespread accumulation of due to lysosomal dysfunction. We Eppendorf, Martinistr. 52, 20246 used a new mouse model of IIIE to study the effect of Arylsulfatase G Hamburg, Germany; (ARSG) deficiency on retina integrity. [email protected]. Markus Damme, Department of Bio- METHODS. The retina of Arsg knockout mice aged 1 to 24 months was studied by chemistry, University of Kiel, Ol- immunohistochemistry and Western blot analysis. Electron microscopic analyses were shausenstrasse 40, 24118 Kiel, performed on retinas from 15- and 22-month-old animals. Photoreceptor and microglia cell Germany; numbers and retina thickness were determined to quantitatively characterize retinal [email protected]. degeneration in ARSG-deficient mice. UB and MD are joint senior authors. RESULTS. Arsg knockout mice showed a progressive degeneration of photoreceptor cells Submitted: July 9, 2015 starting between 1 and 6 months of age, resulting in the loss of more than 50% of Accepted: February 8, 2016 photoreceptor cells in 24-month-old mice. Photoreceptor loss was accompanied by reactive Citation: Kruszewski K, Lullmann-¨ astrogliosis, reactive microgliosis that was evident in the outer but not inner retina, and Rauch R, Dierks T, Bartsch U, Damme elevated expression levels of some lysosomal proteins. Electron microscopic analyses of M. Degeneration of photoreceptor retinas revealed no evidence for the presence of storage vacuoles. Of note, expression of cells in Arylsulfatase G-deficient mice. ARSG protein in wild-type mice was detectable only in the RPE which, however, appeared Invest Ophthalmol Vis Sci. morphologically unaffected in knockout mice at the electron microscopic level. 2016;57:1120–1131. DOI:10.1167/ iovs.15-17645 CONCLUSIONS. To our knowledge, this is the first study demonstrating that ARSG deficiency results in progressive photoreceptor degeneration and dysregulation of various lysosomal proteins. Keywords: Arylsulfatase G, retina, neurodegeneration, mucopolysaccharidosis, photoreceptor cells, Sanfilippo syndrome, retinal pigment epithelium

he lysosomal degradative pathway of sulfated glycosamino- acetylglucosaminidase (encoded by NAGLU; MPS IIIB), hepar- T (GAG) comprises a sophisticated hydrolytic net- an-a-glucosaminide N- (encoded by HGSNAT; work of highly specific glycosidases and sulfatases for complete MPS IIIC), and N-acetylglucosamine-6-sulfatase (encoded by degradation of these complex polysaccharides to sulfate and GNS; MPS IIID).3,7 Taking all pathogenic in these four monosaccharides.1–5 For each sulfate residue in different together, MPS III is the most frequently occurring type of positions of the sugar moiety, distinct sulfatases are indispens- MPS with a reported prevalence in different populations of 0.28 able for their desulfation, which in turn is a prerequisite for to 4.1 per 100,000 births.7 We have shown recently that a fifth glycosidic hydrolysis. Pathogenic mutations in genes coding for enzyme is critical for complete degradation of HS glucosamine these hydrolytic lead to impaired degradation of GAGs residues when sulfated in the C3 position of glucosamine: and as a consequence to an accumulation of the corresponding Arylsulfatase G (ARSG), also termed N-sulfoglucosamine-3-O- substrates in lysosomes, a clinical situation described as sulfatase.6,8 We have generated Arsg knockout (KO) mice, and lysosomal storage disorder (LSD). Disorders resulting from have demonstrated accumulation of HS in different organ impaired lysosomal degradation of sulfated GAGs (heparan systems including liver, kidney, and brain.6 Due to its assigned sulfate [HS], dermatan sulfate, chondroitin sulfate, and keratan role in the degradation of HS and the resulting Sanfilippo sulfate) are summarized as mucopolysaccharidoses (MPSs).1–6 syndrome-like pathological alterations, we tentatively assigned One subgroup of the MPSs is Sanfilippo syndrome (MPS type this MPS type as MPS IIIE. Compared to mouse models of the III), which exclusively affects the degradation of HS. Mutations other MPS III subtypes,9–11 Arsg KO mice presented with a in genes coding for four different enzymes, needed for the milder phenotype and a later onset of the disease, with Purkinje removal of sulfated glucosamine residues of HS, are known to cell degeneration in the cerebellum as the major neurological cause MPS III subtypes in humans, including N-sulfoglucos- phenotype.12 Severe ataxia and Purkinje cell degeneration also amine sulfohydrolase (encoded by SGSH; MPS IIIA), N-a- was observed in an American Staffordshire Terrier dog pedigree

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that lacks functional ARSG due to a point in the Arsg Immunohistochemistry of Retina Sections .13 This canine model was assigned as a model of neuronal ceroid lipofuscinosis (NCL) because of the large amounts of Immunohistochemical analyses were performed on retinas from accumulated lipofuscin in neurons. Human patients carrying 1-, 6-, 12-, 19-, and 24-month-old Arsg KO and age-matched wild- pathogenic mutations in ARSG have not been identified until type mice. Animals were killed and eyes were quickly removed now. and fixed overnight in PBS (pH 7.4) containing 4% paraformal- Mucopolysaccharidoses are multisystemic disorders affect- dehyde (PA). After dehydration in an ascending series of sucrose, ing most cell types of the body. However, the impaired cellular eyes were frozen in Tissue-Tek (Sakura Finetek, Zouterwoude, clearance of GAGs and the resulting lysosomal dysfunction is of The Netherlands) and serially sectioned with a cryostat at a particular detrimental significance for postmitotic cells, such as thickness of 25 lm. Central (i.e., in the plane of the optic disc) neurons, as reflected by the profound neurological symptoms retina sections were first blocked in PBS containing 0.1% bovine of most MPS patients. In MPS III patients, neurodegeneration is serum albumin (BSA) and 0.3% Triton X-100 (both from Sigma- the key clinical feature, ultimately leading to premature Aldrich Corp., St. Louis, MO, USA) for 1 hour and then incubated death.3,4,7 The four human MPS III subtypes are similar with primary antibodies (see Table) overnight at room clinically, with typical symptoms, including developmental temperature. After washing with PBS, sections were incubated delay, mild coarse facial features, progressive loss of mental and with Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 4 hours, stained motor functions, and epileptic seizures. Cortical atrophy and with 40,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Corp.) ventricular enlargement are common findings. Patients usually and mounted onto slides. For detection of mannose 6-phosphate die at the end of the second or beginning of the third decade of (M6P)–containing proteins, retina sections were incubated with life, often due to respiratory insufficiencies.2–4,7,14,15 the myc-tagged single-chain antibody fragment scFv M6P-134 In addition to the brain, the retina is affected to a significant followed by polyclonal rabbit anti-myc antibodies (Sigma-Aldrich extent in two prominent groups of LSDs: retinal degeneration Corp.) and Cy3-conjugated donkey anti-rabbit antibodies. To is a characteristic feature of several NCLs16–22 and also is seen visualize cones, sections were stained with biotinylated peanut frequently in MPSs. Ocular involvement was reported in agglutinin (BPA; Vector Laboratories, Burlingame, CA, USA) patients and animal models of the majority of MPS subtypes, overnight at room temperature, followed by Cy3-conjugated including MPS III.9,23–29 Patients with MPS III typically present Streptavidin (Jackson ImmunoResearch) and DAPI. In all with progressive photoreceptor loss closely resembling that experiments sections from Arsg KO and age-matched wild-type occurring in retinitis pigmentosa, whereas the ganglion cells mice were processed in parallel and under identical conditions. and optic nerve usually are unaffected.23–25,28–32 At least 6 animals were analyzed for each antigen, developmen- In this study, we describe degenerative changes in the retina of tal age, and genotype. ARSG-deficient mice, a new mouse model of MPS type III. For immunohistochemical analyses of ARSG expression in Progressive photoreceptor loss in the mouse starts between 1 and RPE cells, the melanin pigment was bleached.35 Sections were 6 months of age and is accompanied by reactive astrogliosis and incubated in 0.05% potassium permanganate (Merck) for 25 microgliosis, and a dysregulation of several lysosomal proteins. minutes, washed with PBS, incubated for 5 minutes in 0.5% We define retinal degeneration as an important and early onset oxalic acid (Carl Roth GmbH, Karlsruhe, Germany) and washed pathological feature of ARSG deficiency. again with PBS before they were incubated with anti-ARSG antibodies. To analyze flat-mounted RPE, eyes were fixed in 4% PA. The retina was removed, and the RPE with attached MATERIALS AND METHODS choroidea blocked in PBS containing 0.1% BSA and 0.3% Triton Animals X-100 and incubated with antibodies to RPE65, OTX2, or ZO-1. Before incubation with anti-ARSG or anti-RPE65 antibodies, Arylsulfatase G knockout (Arsg KO) mice were generated as melanin pigment was bleached as described above. Primary described previously.6 Mice were maintained on a mixed C57BL/ antibodies were detected with Cy2- or Cy3-conjugated second- 6J 129/Ola genetic background and housed according to the ary antibodies, stained with DAPI and mounted onto slides. institutional guidelines of the University Bielefeld, with ad libitum For each antigen, sections from Arsg KO and age-matched access to food and water. Genotyping of mice was performed as wild-type mice were analyzed in parallel and with the same described.6 Retina tissue from CLN7 mutant mice33 was used to microscope settings using an Olympus FV 1000 confocal control the specificity of p62 immunostainings. In all experi- microscope (Olympus, Hamburg, Germany). ments, age-matched C57BL/6J 129/Ola wild-type mice served as a control. All animal work was approved by the local Animal Care Photoreceptor Counts and Retina and Outer Committee and was in accordance with the ARVO Statement for Nuclear Layer Thickness the Use of Animals in Ophthalmic and Vision Research. To quantify the loss of photoreceptor cells in Arsg KO mice, Light and Electron Microscopy central retina sections from mutant and age-matched wild-type mice were stained with anti-recoverin antibodies and DAPI. A For light and electron microscopic analyses, 15- and 22-month- merged confocal image of the entire retina section was old Arsg KO and age-matched wild-type mice were deeply prepared using Photoshop CS6 software (Adobe Systems, anesthetized and transcardially perfused with PBS followed by Inc., San Jose, CA, USA), and photoreceptor nuclei were perfusion with 6% glutaraldehyde (Merck, Darmstadt, Ger- counted at three defined positions corresponding to 25%, 50%, many) in phosphate buffer. Eyes were enucleated, the lenses and 75% of the distance between the optic disc and the were removed, and the bulbs were post-fixed with 2% osmium peripheral margin of the nasal and temporal retina, respec- tetroxide, dehydrated, and embedded in Araldite. Semithin tively. Each area defined for photoreceptor counts covered the sections were stained with toluidine blue. Ultrathin sections outer nuclear layer over a length of 220 lm.38 Statistical werestainedwithuranylacetateandleadcitrate,and analyses of data were performed with the Student’s t-test using examined with a Zeiss EM900 electron microscope (Zeiss, GraphPad software (GraphPad Software, La Jolla, CA, USA). Jena, Germany) equipped with a Megaview III digital camera The thickness of the retina and the outer nuclear layer (i.e., (Albert Tr¨ondle, Moorenweis, Germany). photoreceptor cell bodies and inner and outer photoreceptor

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TABLE. Primary Antibodies Used for Immunohistochemistry

Antigen Dilution Company/Reference Catalog Number

Arylsulfatase G (ARSG) 1:100 R&D Systems, Minneapolis, MN, USA AF4600 Brain-specific homeobox/POU domain protein 1:200 Santa Cruz Biotechnology Inc., Santa Cruz, CA, sc-31984 3A (Brn-3a) USA Cathepsin D 1:4000 Claussen et al.36 n.a. Cluster of differentiation 68 (CD68) 1:1000 AbD Serotec, Kidlington, UK Clone FA 11, MCA1957 Glial fibrillary acidic protein (GFAP) 1:500 Dako Cytomation GmbH, Hamburg, Germany Z0334 Ionized calcium-binding adapter molecule 1 1:200 Wako Chemicals GmbH, Neuss, Germany 019-19741 (Iba1) Lysosomal-associated membrane protein 1 1:200 Developmental Studies Hybridoma Bank, Iowa Clone 1D4B (Lamp1) City, IA, USA Lysosomal-associated membrane protein 2 1:200 Developmental Studies Clone Abl93 (Lamp2) Mannose 6-phosphate (M6P) 1:2000 Muller-L¨¨ onnies et al.34 n.a. Orthodenticle homeobox 2 (OTX2) 1:4000 Millipore, Temecula, CA, USA AB9566 Sequestosome 1, SQSTM1/p62 (p62) 1:300 Enzo Lifesciences BML-PW9860 Protein kinase C alpha (PKCa) 1:500 Santa Cruz Biotechnology, Inc. sc-208 Recoverin (Rec) 1:3000 Merck, Darmstadt, Germany AB5585 Retinal pigment epithelium-specific 65 kDa 1:2000 Neuromics Antibodies, Edina, MN clone 401.8B11.3D9, MO25011 protein (RPE65) Rhodopsin (Rho) 1:5000 Merck Clone Rho 1D4MAB5356 Saposin D 1:2000 Klein et al.37 n.a. Zonula Occludens 1 (ZO-1) 1:100 Invitrogen, Camarillo, CA 40-2200

segments) was measured in central retina sections at nine lysates were loaded on SDS-PAGE gels and blotted on equidistant positions between the optic disc and periphery of the nitrocellulose membranes with a semi-dry blotting apparatus. nasal and temporal retinal halves, respectively. Numbers of Iba1- For Saposin D blotting, membranes were heated up to 1008C and CD68-positive cells with a clearly visible DAPI-positive for 5 minutes immediately after blotting. The same antibodies nucleus were determined in the inner retina (i.e., nerve fiber as indicated for immunohistochemistry were used at the layer, ganglion cell layer, inner plexiform layer, and inner nuclear following dilutions: Saposin D 1:500, Lamp2 1:250, and layer) and outer retina (i.e., outer plexiform layer, photoreceptor Cathepsin D 1:1000. Gapdh (1:1000; Santa Cruz Biotechnology, cell bodies, and inner and outer photoreceptor segments) of Arsg Inc., Santa Cruz, CA, USA) was used as a loading control. After KO and wild-type mice aged 1 to 24 months. The area of the inner incubation with the primary antibodies overnight, blots were and outer retina was measured using Photoshop CS6 software, probed with horseradish peroxidase coupled secondary and the density of positive cells was calculated. Statistical analyses antibodies and subsequently detected by enhanced chemilu- of data were performed with the 2-way ANOVA test followed by a minescence reagent. For quantification, densitometry was Bonferroni post hoc test using GraphPad software. performed using ImageJ software (National Institutes of Health To determine the density of retinal ganglion cells (RGCs), [NIH], Bethesda, MD, USA). Wild-type and age-matched Arsg eyes of 19-month-old Arsg KO and age-matched wild-type mice KO mice at 1 and 19 months of age were used (n ¼ 6 for each (n ¼ 5 for each genotype) were fixed in 4% PA and retinas were genotype). Statistical analysis of data was performed using flat-mounted on nitrocellulose membranes (Sartorius AG, Student’s t-test. G¨ottingen, Germany) as described.39 After blocking in PBS containing 0.1% BSA and 1% Triton X-100, retinas were Determination of b-Hexosaminidase Activity incubated with polyclonal goat anti-Brn-3a antibodies over- night at room temperature. Primary antibodies were detected The specific activity of the lysosomal marker enzyme b- with Cy3-conjugated secondary antibodies, and flat-mounted hexosaminidase was determined as described previously.40 In retinas were stained with DAPI and mounted onto slides. Five brief, approximately 5 lg retina lysates from Arsg KO and age- images were taken from the center to the periphery of the matched wild-type mice (as prepared for Western blots) were superior, inferior, nasal, and temporal retinal quadrants, incubated with the artificial substrate q–nitrophenyl-N-acetyl-b- covering a total area of approximately 1.9 mm2. All Brn-3a– D-glucosaminide in 0.2 M citric acid buffer pH 4.6 for 16 hours positive RGCs visible on these images were counted using at 378C. After addition of 0.4 M glycine/NaOH (pH 10.4) and Adobe Photoshop CS6 software, and the density of RGCs per centrifugation at 13,000g, absorption was measured at 405 nm. mm2 retinal area was calculated. Statistical analysis of data was The activity was normalized to protein concentration of each performed using Student’s t-test. sample to calculate the specific activity.

Western Blotting RESULTS Tissue lysates were prepared by homogenization of whole retinas in 15 volumes (wt/vol) of ice-cold 31 TBS containing Progressive Degeneration of Rod Photoreceptor 1% Triton-X-100 and protease inhibitors. After incubation on Cells in ARSG-Deficient Mice ice for 30 minutes followed by centrifugation at 13,000g, the supernatant was used and protein concentration determined To characterize the retinal phenotype of mice deficient in by Bicinchoninic acid assay (Thermo Fisher Scientific, Inc., ARSG,6,12 we stained central retina sections from Arsg KO and Schwerte, Germany). For Western blots, 20 lg of the retina age-matched wild-type mice with antibodies to glial fibrillary

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FIGURE 1. Expression of GFAP and recoverin and thickness of the retina and the outer nuclear layer of Arsg KO and wild-type mice at different developmental ages. In 1-month-old wild-type (Aa) and Arsg KO mice (Ab), expression of GFAP was restricted to astrocytes located at the vitreal margin of the retina. A similar pattern of GFAP expression was observed in 6- (Ac), 12- (Ae), and 24 (Ag)–month-old wild-type retinas. In 6- (Ad), 12- (Af), and 24 (Ah)–month-old mutants, in comparison, expression of GFAP was strongly elevated in retinal astrocytes and was additionally detectable in Muller¨ cells. Immunostainings with anti-recoverin antibodies revealed a similar thickness of the outer nuclear layer (onl) in 1-month-old wild-type (Ai) and age-matched Arsg KO mice (Aj). In 6- (Al), 12- (An,) and 24 (Ap)–month-old mutants, the thickness of the outer nuclear layer decreased significantly when compared to age-matched wild-type mice ([Ak], [Am], [Ao], respectively). The thickness of the retina and the outer nuclear layer (i.e., photoreceptor cell bodies and inner and outer photoreceptor segments) was measured in central retina sections stained with anti-recoverin antibodies and DAPI (B) at nine equidistant positions (indicated with white lines in [B]) between the optic nerve head (ONH) and the periphery of the nasal and temporal retinal half, respectively. Analyses revealed a similar thickness of the retina and the outer nuclear layer in 1-month-old Arsg KO (gray squares in [Ca] and [Cb], respectively) and age-matched wild-type mice (black circles in [Ca] and [Cb], respectively). In 6-month-old mutants, the thickness of the retina and outer nuclear layer (green triangles in [Ca] and [Cb], respectively) was significantly decreased at all retina positions when compared to 1-month-old mutants. Retina and outer nuclear layer thickness was further decreased in 24-month-old mutants (red triangles in [Ca] and [Cb], respectively) when compared to 6-month-old Arsg KO mice. Each symbol represents the mean value (6SEM) of 6 animals. *P < 0.05; **P < 0.01; ***P < 0.001 according to the 2-way ANOVA followed by a Bonferroni post hoc test. gcl, ganglion cell layer; inl: inner nuclear layer; ipl, inner plexiform layer; M, month; Rec, recoverin. Scale bars: for (Aa–Ah), 50 lm; in (B) 200 lm.

acidic protein (GFAP). In 1-month-old mutants (Fig. 1Ab) and of wild-type mice aged 6 to 24 months (Figs. 1Ac, 1Ae, 1Ag). In wild-type mice (Fig. 1Aa), expression of GFAP was restricted to 6- (Fig. 1Ad), 12- (Fig. 1Af), and 24 (Fig. 1Ah)–month-old Arsg retinal astrocytes located at the vitreal margin of the retinas. A KO mice, in comparison, expression of GFAP was elevated in similar pattern of GFAP expression was observed in the retina retinal astrocytes and became additionally detectable in Muller¨

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Similarly, we observed no significant differences in the thickness of the outer nuclear layer between 1-month-old Arsg KO and wild-type mice, but a progressive thinning of this layer in older Arsg KO mice (Fig. 1Cb). The thickness of the inner retina, in contrast, was similar in mutant and wild-type mice at all ages analyzed (Supplementary Fig. S1), indicating that the retinal dystrophy in Arsg KO mice is mainly or exclusively due to a progressive loss of photoreceptor cells. In line with these results, we found similar densities of PKCa-positive bipolar cells in 24-month-old animals of both genotypes (Supplemen- tary Fig. S1). Furthermore, the density of RGCs in 18-month-old animals did not differ significantly between both genotypes, with 3688 6 165 RGCs/mm2 retina area (mean 6 SEM) in wild-type mice and 3932 6 180 RGCs/mm2 retina area in Arsg KO mutants (n ¼ 5 for each genotype; Supplementary Fig. S1). To further quantify the loss of photoreceptor cells in the mutant, photoreceptor cell nuclei were counted in three areas located at defined positions of the nasal and temporal retina, respectively, each covering the outer nuclear layer over a length of 220 lm. In 1-month-old animals, we found similar numbers of photoreceptor cells in wild-type mice (427.1 6 3.5 photoreceptor cells/area; mean 6 SEM; n ¼ 6) and Arsg KO mutants (434.3 6 9.5; n ¼ 6; Fig. 2A). In older mutants, the number of photoreceptor cells decreased significantly with increasing age of the animals. In 6-, 12-, and 24-month-old mutants, we found 302.1 6 7.6, 258.0 6 8.3, and 179.5 6 6.6 photoreceptor cells/area, respectively (Fig. 2A). Retinas from wild-type mice analyzed for comparison contained 424.8 6 5.3, 404.4 6 4.7, and 404.2 6 5.6 photoreceptor cells/area at the age of 6, 12 and 24 months, respectively (Fig. 2A). To FIGURE 2. Photoreceptor numbers and analyses of rod and cone analyze whether the retinal dystrophy is the result of a photoreceptor cells in Arsg KO and wild-type mice at different developmental ages. Photoreceptor counts in 1-month-old Arsg KO progressive loss of rods or cones or both photoreceptor cell (black bars in [A]) and wild-type mice (gray bars in [A]) revealed types, retina sections were labeled with anti-rhodopsin similar cell numbers in both genotypes. In 6-, 12-, and 24-month-old antibodies or peanut agglutinin. These experiments revealed animals, photoreceptor numbers were significantly lower in Arsg KO the presence of similar numbers of rods and cones with normal mice when compared to age-matched wild-type mice, and decreased inner and outer segments in 1-month-old wild-type (Figs. 2Ba, significantly with increasing age of the mutant. In 1-month-old wild- 2Be) and mutant mice (Figs. 2Bb, 2Bf). In 24-month-old ARSG- type (Ba, Be) and Arsg KO mice (Bb, Bf), the morphology and density deficient mice (Figs. 2Bd, 2Bh), the length of the outer of rod (Ba, Bb) and cone (Be, Bf) photoreceptor cells was similar in segments of rods and cones was shortened when compared to both genotypes. In 24-month-old KO mice, outer segments (os) of Arsg age-matched wild-type mice (Figs. 2Bc, 2Bg). Furthermore, we rod (Bd) and cone (Bh) photoreceptor cells were reduced in length when compared to age-matched wild-type mice ([Bc] for rods, [Bg]for found a similar density of cones in aged Arsg KO and wild-type cones). Note the similar density of cones in mutant (Bh) and wild-type mice (Figs. 2Bh, 2Bg, respectively). (Bg) retinas at this age. All sections were stained with DAPI. Each bar in (A) represents the mean value (6 SEM) from 6 animals. **P < 0.01; Accumulation of Activated Microglial Cells in the ***P < 0.001 according to Student’s t-test. n.s., not significant; mo, month; onl, outer nuclear layer; Rho, rhodopsin. Scale bar: for (Ba– Outer Retina of ARSG-Deficient Mice Bh), 50 lm. Retina sections from mutant and wild-type mice of different developmental ages were stained with antibodies to Iba1 and cells. Reactive astrogliosis in Arsg KO was accompanied by a CD68 to study reactive microgliosis in Arsg KO mice. In 1- progressive thinning of the outer nuclear layer. Analyses of month-old animals, ramified Iba1-positive cells were found in retina sections from 1-month-old Arsg KO and age-matched the ganglion cell layer, inner plexiform layer, inner nuclear wild-type mice stained with anti-recoverin antibodies to label layer, and outer plexiform layer, with no obvious differences in photoreceptor cells revealed a similar thickness of the outer cell density or cell morphology between both genotypes nuclear layer of both genotypes (compare Figs. 1Ai, Aj). In 6- (compare Figs. 3Aa, 3Ab). In older Arsg KO mice, Iba1-positive (Fig. 1Al), 12- (Fig. 1An) and 24- (Fig. 1Ap)–month-old Arsg KO cells with a rounded amoeboid-like morphology became mice, the thickness of the photoreceptor layer decreased with additionally detectable in the subretinal space of Arsg KO retinas (Fig. 3Ad). CD68-positive activated microglia/macro- increasing age of the animals. phages were essentially absent from wild-type retinas (for a 24- Measurements of the retina thickness at 9 equidistant month-old wild-type retina, see Fig. 3Ae) and from 1-month-old positions between the optic disc and periphery of the nasal Arsg KO retinas. In comparison, CD68-positive cells with an and temporal retina, respectively (Fig. 1B), revealed similar amoeboid morphology were found frequently in 6-, 12-, and 24- values for 1-month-old wild-type and age-matched Arsg KO month-old mutant retinas where they were localized mainly in mice at all retinal positions analyzed (Fig. 1Ca). In 6-month-old the subretinal space (Fig. 3Af). Quantitative analyses revealed a mutants, in comparison, retina thickness was significantly similar density of Iba1-positive cells in the inner retina (defined decreased at all retinal positions compared to 1-month-old Arsg as ganglion cell layer, inner plexiform layer, and inner nuclear KO mice (Fig. 1Ca). Retina thickness was further decreased in layer) of Arsg KO mutants and wild-type mice aged 1 to 24 24-month-old mutants compared to 6-month-old Arsg KO mice months, and in the outer retina (defined as outer plexiform (Fig. 1Ca), in line with the immunohistochemical data. layer, photoreceptor cell bodies, and inner and outer photo-

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FIGURE 3. Distribution and density of Iba1-positive and CD68-positive cells in the retina of Arsg KO and wild-type mice. Analyses of 1-month-old animals revealed a similar distribution and density of Iba1-positive cells in wild-type (Aa) and Arsg KO mice (Ab). In 24-month-old Arsg KO retinas (Ad), the number of Iba1-positive cells was significantly increased when compared to age-matched wild-type retinas (Ac), and positive cells were now additionally detectable between the outer nuclear layer (onl) and the RPE. CD68-positive cells were absent from retinas of 24-month-old wild- type mice (Ae) but numerous in retinas of age-matched Arsg KO mice where they were located mainly in the subretinal space (Af). Quantitative analyses revealed similar numbers of Iba1-positive cells in the inner retina (i.e., nerve fiber layer, ganglion cell layer, inner plexiform layer, and inner nuclear layer) of Arsg KO (black bars in [Ba]) and wild-type mice (gray bars in [Ba]) aged 1 to 24 months, and in the outer retina (i.e., outer plexiform layer, photoreceptor cell bodies, and inner and outer photoreceptor segments) of 1-month-old Arsg KO and wild-type mice (Ba). In the outer retina of 6-, 12- and 24-month-old mutants, the number of Iba1-positive cells increased with increasing age of the animals, and was significantly higher than in age-matched wild-type mice (Ba). Cells positive for CD68 were essentially absent from wild-type retinas at all developmental ages analyzed, and were observed only occasionally in the inner retina of Arsg KO mice aged 1 to 24 months and in the outer retina of 1-month-old mutants (Bb). In the outer retina of older Arsg KO mice, the number of CD68-positive cells increased with increasing age of the animals (Bb). Each bar represents the mean value (6SEM) of 6 animals. ***P < 0.001 according to a 2-way ANOVA followed by a Bonferroni post hoc test. Scale bar:for Aa–Af), 50 lm.

receptor segments) of 1-month-old wild-type and Arsg KO mice RPE of aged Arsg KO mice was analyzed by conventional light (Fig. 3Ba). In the outer retina of older Arsg KO mice, however, microscopy (Fig. 5A) and electron microscopy (Fig. 5B) for the number of Iba1-positive cells increased significantly with lysosomal storage, and morphologic or lysosomal abnormali- increasing age of the mutants (Fig. 3Ba). Cells positive for ties. These experiments revealed no obvious morphologic CD68 essentially were absent from wild-type retinas, and only alterations of RPE cells in 15- or 22-month-old ARSG-deficient rarely were observed in the inner retina of the ARSG-deficient mice when compared to age-matched wild-type mice (Fig. 5). mice (Fig. 3Bb). In the outer retina of the mutant, in contrast, Macrophages were observed frequently in the subretinal space CD68-positive cells were found frequently in 6-month-old of mutant retinas (Figs. 5Bb–Bd), but not in the subretinal animals, and their number was significantly increased in 24- space of wild-type retinas (Fig. 5Ba). month-old Arsg KO mice (Fig. 3Bb). Dysregulation of Lysosomal Proteins in Aged Expression of ARSG is Restricted to RPE Cells ARSG-Deficient Retinas Immunohistochemical analyses of the expression pattern of To study the impact of ARSG-deficiency on the expression of ARSG were performed on sections of adult wild-type retinas. lysosomal proteins, we studied the expression pattern of The melanin pigment in RPE cells was bleached before the lysosomal enzymes containing the M6P recognition marker and immunostainings to exclude quenching of the immunofluores- the expression pattern of the lysosomal markers lysosomal- cence. Double immunostainings revealed expression of ARSG associated membrane protein-1 (Lamp1) and lysosomal-associ- in RPE65-positive RPE cells of wild-type retinas (Figs. 4Aa–Af). ated membrane protein-2 (Lamp2), the lysosomal protease In retina sections from Arsg KO mice that were processed in cathepsin D (Ctsd) and Saposin D in retinas of 1- and 24-month- parallel as a negative control, RPE cells were ARSG-negative as old Arsg KO mice and age-matched wild-type mice (Fig. 6). expected (Figs. 4Ag–Al). The weak fluorescence associated No detectable differences in the expression pattern and with photoreceptor outer segments, the outer and inner expression level of M6P and the different lysosomal proteins plexiform layer, and the ganglion cell layer of wild-type retinas were observed between 1-month-old Arsg KO retinas and age- (Fig. 4Ab) also was observed in Arsg KO retinas (Fig. 4Ah), and, matched wild-type retinas (Fig. 6; compare Figs. 6a, 6b for thus, likely represents unspecific background labeling. Immu- M6P; 6e, 6f for Lamp1; 6i, 6j for Lamp2; 6m, 6n for Ctsd; and nostainings of flat-mount preparations of the RPE confirmed 6q, 6r for Saposin D). In 24-month-old animals, in comparison, expression of ARSG in RPE cells (compare Figs. 4Ba, 4Be), and expression levels of M6P were significantly increased in Arsg additionally revealed similar expression levels and expression KO retinas when compared to wild-type retinas, particularly in patterns of RPE65, OTX2, and ZO-1 in wild-type and ARSG- the ganglion cell layer (compare Figs. 6c, 6d). Expression of deficient retinas (compare Figs. 4Bb and 4Bf, 4Bc and 4Bg, and Lamp1 (compare Figs. 6g, 6h) and Lamp2 (compare Figs. 6k, 4Bd and 4Bh, respectively). Given that RPE cells were the only 6l) was only slightly elevated in 24-month-old mutants, retinal cell type with detectable expression levels of ARSG, the whereas immunoreactivity for Ctsd (compare Figs. 6o, 6p)

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FIGURE 4. Expression of ARSG in retinal sections and flat-mounted RPE. In adult wild-type mice, ARSG-immunoreactivity (Ab, Ae) colocalized with RPE65 (Aa, Ad) in RPE cells. In comparison, RPE cells in Arsg KO retinas were ARSG-negative as expected (Ah, Ak). The weak fluorescence of photoreceptor outer segments, outer plexiform layer, inner plexiform layer, and ganglion cell layer in wild-type retinas stained with anti-ARSG antibodies (Ab) also was evident in Arsg KO retinas (Ah), and, thus, likely represents unspecific background labelling. (Ac, Af, Ai, Al) Phase contrast photomicrographs of (Aa, Ab), (Ad, Ae), (Ag, Ah), and (Aj, Ak), respectively, to demonstrate complete bleaching of the melanin pigment in RPE cells. (Ad, Ae, Aj, Ak) Higher magnifications of the RPE shown in (Aa), (Ab), (Ag), and (Ah), respectively. Immunohistochemical analyses of flat-mounted RPE confirmed expression of ARSG in RPE cells (Ba, Be), and revealed similar expression levels and expression patterns of RPE65 (Bb, Bf), OTX2 (Bc, Bg), and ZO-1 (Bd, Bh) in wild-type ([Bb, Bc, Bd], respectively) and ARSG-deficient retinas ([Bf, Bg, Bh], respectively). Sections and flat-mounts were stained with DAPI to label cell nuclei. OTX2, Orthodenticle homeobox 2; RPE65, RPE-specific 65 kDa protein; ZO-1, Zonula Occludens 1. Scale bars: for (Aa–Ac) and (Ag–Ai), 50 lm; for (Ad–Af) and (Aj–Al), 20 lm; for (Ba–Bh), 50 lm.

and Saposin D (compare Figs. 6s, 6t) was strongly increased in (Fig. 7B), it was significantly increased by a factor of Arsg KO retinas when compared to age-matched wild-type approximately 2.5 in 19-month-old Arsg KO animals when retinas. Upregulation of Ctsd was particularly evident in the compared to wild-type controls (Fig. 7B). ganglion cell layer, inner nuclear layer, and outer nuclear layer (Fig. 6p), whereas elevated expression of Saposin D was detected mainly in the ganglion cell layer (Fig. 6t). Further- DISCUSSION more, Lamp1, Lamp2, Ctsd, and Saposin D were accumulated strongly in phagocytotic microglial cells located in the A recent analysis of the Arsg KO mouse provided further subretinal space of 24-month-old Arsg KO retinas (Figs. 6h, insight into the degradative pathway of HS and the endogenous 6l, 6p, 6t). Finally, we also analyzed expression of the substrate of ARSG, 3-O sulfated glucosamine. Moreover, we autophagy adapter protein p62. While p62-immunoreactivity depicted the in vivo relevance of the substrate and its was absent from retina sections of wild-type and ARSG- degradation by ARSG through manifestation of lysosomal deficient mice, it was readily detectable in all layers of CLN7 storage in several tissues, including the liver, kidney, and brain mutant retinas that were processed in parallel as a positive in the absence of ARSG, and identified Purkinje cell degener- control (Supplementary Fig. S2). ation and ataxia as prominent neurologic symptoms in Arsg KO Western blot analyses of retinas from 1-month-old wild-type mice.6,12 In the present study, we extended the phenotypic and Arsg KO mice revealed no significant differences in characterization of the Arsg KO mouse, tentatively assigned as expression levels of Lamp2, Cathepsin D, or Saposin D (Fig. a mouse model of MPS IIIE,6 to an early onset degeneration of 7A), in agreement with the immunhistochemical data. In 19- photoreceptor cells starting between 1 and 6 months of age, month-old mutants, expression levels of Lamp2 were slightly when neuronal loss in the brain is not yet detectable. Retinal but not significantly increased, whereas expression levels of degeneration was accompanied by reactive astrogliosis, the Cathepsin D and Saposin D were strongly elevated in mutant appearance of phagocytic microglia/macrophages in the outer retinas (Fig. 7A). A particular strong increase in expression retina, and elevated expression of several lysosomal proteins. levels was observed for the proteolytically processed forms of Thus, loss of photoreceptor cells is among the earliest Cathepsin D (Fig. 7A). Finally, we have determined the specific phenotypic manifestations of ARSG deficiency in the central activity of the lysosomal marker enzyme b-hexosaminidase. nervous system. While the specific b-hexosaminidase activity was comparable An intriguing question raised by our observations is the between wild-type and Arsg KO mice in 1-month-old animals actual cause of the progressive photoreceptor degeneration in

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revealed essentially normal numbers of cone photoreceptor cells in Arsg KO mice as old as 24 months, demonstrating that rod photoreceptor cells comprise the retinal cell type that is affected primarily in the absence of ARSG. Evidence for a significant loss of other retinal cell types than rod photorecep- tors was not observed in mutant mice at this age, as indicated by the normal numbers of cone photoreceptor cells, bipolar cells, and RGCs, and the normal thickness of the inner retina. Of note, retinal degeneration displays the typical features of a rod-cone dystrophy also in other MPS subtypes, in animal models and in patients.26,28,29,44–46 While immunohistochemical analyses revealed detectable levels of ARSG expression in RPE cells only, elevated levels of M6P, Saposin D, and Cathepsin D were observed mainly in the inner retina, particularly in RGCs. The expression of most lysosomal proteins is coordinated and regulated by the transcription factor EB (TFEB).47 Therefore, RGCs and other retinal cell types might respond to extrinsic pathological stimuli by upregulating TFEB-mediated lysosomal biogenesis or react to intrinsic alterations in the endolysosomal/lysosomal system, such as a subtle accumulation of 3-O sulfated . The latter hypothesis implies weak expression of ARSG below the detection level of our immunohistochemical analyses in retinal cell types other than RPE cells. A characteristic feature of degenerating Purkinje cells in the cerebellum of Arsg KO mice was the presence of large intracellular aggregates that were immunoreactive for ubiquitin FIGURE 5. Light and electron microscopic analysis of ARSG-deficient and p62, an autophagy adapter protein.12 We hypothesized retinas. Analyses of semi-thin sections from 15-month-old wild-type that these aggregates were the result of an impaired clearance (Aa) and Arsg KO (Ab) retinas revealed a dystrophic photoreceptor of damaged lysosomes by autophagy. In the retina, however, layer and a morphologically intact RPE in the mutant. Electron similar p62-positive aggregates were not observed, indicating microscopy confirmed the presence of an intact RPE in 22-month-old Arsg KO mice (Bb). Activated microglia cells with an amoeboid-like that neuronal cell death in the cerebellum and retina follows morphology were observed in the subretinal space of ARSG-deficient different mechanisms. mice (asterisks in [Bb, Bc, Bd]), but not of wild-type mice (Ba). (Bd)is Mucopolysaccharidoses IIIC is caused by mutations in a higher magnification of the microglia cell shown in (Bb). Scale bar: HGSNAT, the gene encoding heparan-a-glucosaminide N- for (Aa, Ab), 20 lm; for (Ba, Bb), 5 lm; for (Bc, Bd), 1 lm. acetyltransferase, and retinal degeneration is among the typical symptoms of MPS IIIC patients.24,31 Interestingly, a recent the Arsg KO mouse. Of interest in this context, we found that study identified novel mutations in HGSNAT in six patients that expression of ARSG in the adult murine retina is confined to presented with retinitis pigmentosa but without any other the RPE. Retinal pigment epithelial cells perform multiple clinical symptoms normally associated with MPS IIIC, such as neurological deterioration or visceral manifestations.29 The functions that are vital for normal photoreceptor cell function mutations led to significantly reduced HGSNAT activities in and photoreceptor cell survival, and loss or dysfunction of RPE these patients, ranging slightly above the level of typical MPS cells results in photoreceptor degeneration.41 However, IIIC patients but considerably below the level of healthy immunohistochemical and electron microscopic analyses of subjects. As none of the patients manifested additional ARSG-deficient retinas did not reveal obvious pathological extraocular symptoms, the authors concluded that tissues alterations of the RPE, such as RPE atrophy or typical storage usually affected by mutations in HGSNAT (especially the brain) vacuoles as they have been observed in the kidney of Arsg KO express sufficient levels of residual enzymatic activity, but that mice and the RPE of animal models or patients of other MPS 6,9,24,26,30,32,42 the retina requires higher levels of HGSNAT activity to maintain variants and MPS III subtypes. Although the normal structure and function.29 Basically similar findings have specific cause of the progressive photoreceptor cell loss in been reported recently for another lysosomal storage disorder, Arsg KO mouse has, thus, to be elucidated, it is tempting to CLN7 disease.29 Similar to MPS IIIC, progressive photoreceptor speculate that heparan sulfate fragments and oligosaccharides loss is a typical feature of CLN7 disease in patients48 and in a released into the extracellular matrix might interfere with the mouse model of this condition.33 Using genome-wide linkage 7 proper function of the RPE or that a subtle lysosomal analysis and exome sequencing, the study identified compound dysfunction causes functional alterations of RPE cells, ulti- heterozygous variants in MFSD8, the gene affected in CLN7 mately resulting in photoreceptor cell death. Of note, the RPE disease, in two families presenting with macular dystrophy is known to significantly contribute to the synthesis and with central cone involvement.49 Characteristic neurologic degradation of all major mucopolysaccharides in the interpho- symptoms normally associated with CLN7 disease, including toreceptor matrix.43 However, we cannot exclude the possi- mental regression, motor impairment or seizures were, bility of a low level expression of ARSG in retinal cell types however, not observed in these patients. Because both families other than RPE cells, and, thus, a direct impact of ARSG carried a severe heterozygous mutation in combination with a deficiency on photoreceptor cells. missense mutation predicted to have a mild effect on the Significant thinning of the outer nuclear layer and protein, it was proposed that there was sufficient residual preferential accumulation of activated microglia cells/macro- activity of MFSD8 in all tissues of the patients, except in the phages in the outer retina suggests that neurodegeneration in retina.49 Together, these studies point to an already high the retina of Arsg KO mice is mainly or exclusively confined to susceptibility of the retina to subtle changes in the lysosomal the photoreceptor cell layer. Analysis of the outer nuclear layer system, and, thus, might provide an explanation for the

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FIGURE 6. Expression of lysosomal proteins in the retina of Arsg KO and age-matched wild-type mice. The distribution and expression levels of M6P, Lamp1, Lamp2, Ctsd, and Saposin D were similar in 1-month-old Arsg KO and wild-type retinas (compare [a, b], [e, f], [i, j], [m, n], and [q, r], respectively). In 24-month-old animals, expression of M6P, Ctsd, and Saposin D was significantly increased in Arsg KO retinas when compared to age-matched wild-type retinas (compare [c, d], [o, p], and [s, t], respectively). Expression levels of Lamp1 and Lamp2, in comparison, were not significantly different between both genotypes at this age (compare [g, h], and [k, l], respectively). Note the accumulation of lysosomal proteins in macrophages (labeled with white arrows in [h, l, p, t]) located between the outer nuclear layer (onl) and the RPE of 24-month-old mutant mice. All sections were stained with DAPI to label cell nuclei. Scale bar: for (a–t), 50 lm.

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FIGURE 7. Immunoblot analysis of lysosomal proteins and b-hexosaminidase activity in retinas of Arsg KO and age-matched wild-type mice. Western blotting of retina lysates from 1-month-old wild-type and Arsg KO animals with antibodies against Lamp2, Cathepsin D, and Saposin D showed no differences in expression levels between genotypes (A). In 19-month-old retina, in comparison, levels of Saposin D and of proteolytically processed forms of Cathepsin D were significantly increased in the mutant when compared to the wild-type, whereas levels of Lamp2 were similar in both genotypes (A). Gapdh was used as a loading control (A). Determination of the specific activity of the lysosomal enzyme b-hexosaminidase revealed a similar specific activity in 1-month-old wild-type and Arsg KO retinas (B). In 19-month-old animals, in comparison, b-hexosaminidase activity was increased approximately 2.5-fold in the mutant when compared to the wild-type retina. *P < 0.05; ***P < 0.001 according to the Student’s t-test. Gapdh, glyceraldehyde-3-phosphate dehydrogenase; dc; double chain; hc: heavy chain; lc: light chain; SapD: Saposin D; sc: single chain.

frequent involvement of the retina in various LSDs and for the helpful in the identification of possible human subjects progressive photoreceptor loss in Arsg KO mice despite the suffering from MPS IIIE caused by mutations in ARSG. absence of detectable lysosomal storage. American Staffordshire Terrier dogs suffering from ataxia have been shown to carry a point mutation in Arsg in a triplet Acknowledgments coding for an in the vicinity of the catalytic domain The authors thank Konrad Sandhoff for the gift of the Saposin D of the protein.13 Evidence was presented that this missense antibody, Thomas Braulke for providing the Cathepsin D and M6P mutation resulted in a significant reduction of ARSG activity.13 antibodies, and Stephan Storch for the CLN7 mutant mice. The In close analogy to Arsg KO mice,6,12 affected dogs showed authors also thank Elke Becker, Sabine Helbing, Stefanie Schlicht- marked Purkinje cell degeneration in the cerebellum.13 ing, and Dagmar Niemeier for the excellent technical support, and However, different from our observations in the mouse model, Marion Knufinke and Christiane Grebe for animal care. retinal degeneration was not observed in this canine model of Supported by the Deutsche Forschungsgemeinschaft Grant DI MPS IIIE.13 Similar to photoreceptor cells, Purkinje cells are 575/6 (TD). affected in various LSDs,40,50–55 indicating that this neuronal cell type also is highly susceptible to lysosomal dysfunction. Disclosure: K. Kruszewski, None; R. L¨ullmann-Rauch, None; T. Thus, it is tempting to speculate that the species-specific Dierks, None; U. Bartsch, None; M. Damme, None differences in the phenotypic expression of ARSG dysfunction are related to the residual enzyme activity in dogs which is References sufficient to maintain photoreceptor cells but not Purkinje cells as opposed to the complete absence of ARSG in the Arsg 1. Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver KO mouse where both nerve cell types are affected. C., ed. The Metabolic and Molecular Bases of Inherited Alternatively, these findings may reflect species-specific Diseases. New York: McGraw-Hill; 2001:3421–3452. differences in the functional relevance of ARSG for photore- 2. Muenzer J. Mucopolysaccharidoses. Adv Pediatr. 1986;33: ceptor cell integrity. 269–302. In conclusion, the present study demonstrates an early onset retinal degeneration in Arsg KO mice that in many 3. Coutinho MF, Lacerda L, Alves S. storage aspects resembles the retinal dystrophy observed in other MPS disorders: a review. Biochem Res Int. 2012;2012:471325. III types and other LSDs. We suggest that the retinal phenotype 4. Muenzer J. Overview of the mucopolysaccharidoses. Rheu- of ARSG-deficient mice described in the present study might be matology (Oxford). 2011;50(suppl 5):v4–v12.

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