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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 glycosaminoglycans due to lysosomal enzyme dysfunction. We Eppendorf, Martinistr. 52, 20246 used a new mouse model of mucopolysaccharidosis 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 glycans (GAG) comprises a sophisticated hydrolytic net- an-a-glucosaminide N-acetyltransferase (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 mutations in these four monosaccharides.1–5 For each sulfate residue in different genes 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 enzymes 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 iovs.arvojournals.org j ISSN: 1552-5783 1120 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/29/2021 Retinal Phenotype of Arsg Knockout Mice IOVS j March 2016 j Vol. 57 j No. 3 j 1121 that lacks functional ARSG due to a point mutation in the Arsg Immunohistochemistry of Retina Sections gene.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