September 2016
Volume 57, Issue 11
Open Access
Retina  |   September 2016
Retinal Degeneration in Mice Deficient in the Lysosomal Membrane Protein CLN7
Author Affiliations & Notes
  • Wanda Jankowiak
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Laura Brandenstein
    Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Simon Dulz
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Christian Hagel
    Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Stephan Storch
    Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Udo Bartsch
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Correspondence: Udo Bartsch, Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; ubartsch@uke.uni-hamburg.de
  • Footnotes
     SS and UB are joint senior authors.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4989-4998. doi:10.1167/iovs.16-20158
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      Wanda Jankowiak, Laura Brandenstein, Simon Dulz, Christian Hagel, Stephan Storch, Udo Bartsch; Retinal Degeneration in Mice Deficient in the Lysosomal Membrane Protein CLN7. Invest. Ophthalmol. Vis. Sci. 2016;57(11):4989-4998. doi: 10.1167/iovs.16-20158.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Neuronal ceroid lipofuscinoses comprise a genetically heterogeneous group of mainly childhood-onset neurodegenerative lysosomal storage disorders. Progressive loss of vision is among the typical clinical symptoms of these fatal disorders. Here, we performed a detailed analysis of retinal degeneration in mice deficient in the lysosomal membrane protein CLN7, a novel animal model of CLN7 disease.

Methods: Immunohistochemical analyses of retinas at different ages were performed to qualitatively and quantitatively characterize retinal degeneration in CLN7-deficient mice. Storage material in mutant retinas was analyzed by electron microscopy, and expression levels of various lysosomal proteins were studied using immunohistochemistry, immunoblot analyses, and quantitative real-time PCR.

Results: We observed an early onset and rapidly progressing degeneration of photoreceptor cells in CLN7-deficient mice, resulting in the loss of more than 70% rod photoreceptors in 4-month-old animals. The number of cone photoreceptors was not detectably altered at this age. Loss of rod photoreceptors was accompanied by reactive astrogliosis and microgliosis. Immunohistochemical and immunoblot analyses revealed accumulation of subunit c of mitochondrial ATP synthase and saposin D in mutant retinas, and electron microscopic analyses demonstrated the presence of curvilinear bodies or fingerprint-like profiles in various cell types of CLN7-deficient retinas. We also found a marked dysregulation of various lysosomal proteins in mutant retinas.

Conclusions: We conclude that the retina of CLN7-deficient mice represents a useful model to elucidate the pathomechanisms ultimately leading to neurodegeneration in CLN7 disease, and to evaluate the efficacy of strategies aimed at developing treatments for this fatal neurodegenerative lysosomal storage disorder.

The neuronal ceroid lipofuscinoses (NCLs) represent a genetically heterogeneous group of neurodegenerative lysosomal storage disorders manifesting mainly in childhood. Neuronal ceroid lipofuscinoses are caused by mutations in more than a dozen different genes (CLN1–CLN8, CLN10–CLN14)13 encoding soluble lysosomal proteins (palmitoyl protein thioesterase 1, tripeptidylpeptidase 1, CLN5, cathepsin D, cathepsin F), lysosomal membrane proteins (CLN3, CLN7, ATPase Type 13A2), membrane proteins of the endoplasmic reticulum (CLN6, CLN8), soluble proteins (progranulin, potassium channel tetramerization domain-containing protein 7), or the cysteine string protein alpha (CSPα) on synaptic vesicles.2 Of note, a recent analysis of patients with NCL lacking mutations in known disease genes has identified three novel candidate NCL genes.4 Neuronal ceroid lipofuscinoses have been categorized based on the affected gene (CLN1–CLN8; CLN10–CLN14 disease) and the clinical presentation into congenital, infantile, late-infantile, juvenile, and adult phenotypes.1,5 These fatal lysosomal storage disorders are characterized by the selective damage and loss of neurons in the brain and the retina, neuroinflammation, and the accumulation of autofluorescent ceroid lipopigments, particularly in neurons containing subunit c of mitochondrial ATP synthase (SCMAS) and/or sphingolipid activator proteins (saposin) A and D.2 Clinically, typical symptoms of patients with NCL include progressive loss of vision, epileptic seizures, psychomotor retardation, and premature death.6 
CLN7 disease, variant late-infantile phenotype (vLINCL; MIM 610951), is caused by mutations in the CLN7/MFSD8 gene, which encodes a polytopic lysosomal membrane protein with putative transporter function.79 Proteomic and immunoblotting analyses using rat, human, or mouse tissues have identified CLN7 as an integral lysosomal membrane protein that is sorted to lysosomes via an N-terminal di-leucine motif.1013 CLN7 is cleaved twice by cysteine proteases in lysosomes.14 Human CLN7/MFSD8 mRNA is ubiquitously expressed and rat Cln7/Mfsd8 mRNA has been shown to be 6-fold and 12-fold more abundant in cultured neurons when compared with cultured astrocytes and microglial cells, respectively.7,15 With only a few exceptions, the more than 35 different mutations in the CLN7/MFSD8 gene identified in patients with CLN7 cause vLINCL and, based on the relatively uniform clinical manifestation, are thought to result in a complete loss of protein function (http://www.ucl.ac.uk/ncl).4,7,8,1622 In addition to the mutations causing vLINCL, a missense mutation in CLN7/MFSD8 affecting a residue located in a cytosolic loop of CLN7 (p.Ala157Pro) was reported to cause a milder disease course with juvenile onset.18 Furthermore, compound heterozygous variants in CLN7/MFSD8 have been identified in patients presenting with autosomal recessive macular dystrophy with central cone involvement but lacking the characteristic hallmarks and severe neurologic symptoms of patients with CLN7 with the vLINCL phenotype (MIM 616170).23 Of interest in the present context, visual impairment has been diagnosed in up to 90% of patients with CLN7 aged between 2 and 7 years,18 and morphologic analyses of retinas from patients with CLN7 who are homozygous for the most common missense mutation p.Thr294Lys have demonstrated the presence of autofluorescent storage material, a marked loss of neurons in the ganglion cell layer and inner nuclear layer, and a complete loss of the photoreceptor cell layer.22 
For most NCL variants, transgenic or naturally occurring mouse models are available that replicate key features of human NCL diseases.24,25 Of note, retinal degeneration is a characteristic phenotypic feature of mouse models of various NCL variants, including CLN1, CLN3, CLN6, CLN8, CLN10, and CLN11 disease.2635 In the present study, we examined the impact of CLN7 deficiency on retinal integrity by using a novel knockout (KO) mouse model of CLN7 disease.36 Using immunohistochemistry, immunoblotting, electron microscopy, and quantitative real-time PCR, we demonstrate that loss of CLN7 leads to an early onset and rapidly progressing degeneration of rod photoreceptor cells that is accompanied by reactive astrogliosis and microgliosis, accumulation of storage material containing SCMAS and saposin D in various retinal cell types, and dysregulation of multiple lysosomal proteins. The combined data indicate that CLN7-deficient mice represent a valuable animal model to study the pathomechanisms ultimately leading to neurodegeneration in CLN7 disease, vLINCL phenotype. 
Material and Methods
Animals and Genotyping
CLN7-deficient mice36 and wild-type mice were kept under standard housing conditions in a 12-hour-light and 12-hour-dark cycle in a specific pathogen-free animal facility at the University Medical Center Hamburg-Eppendorf (Hamburg, Germany) with ad libitum access to food and water. Mice were maintained on a C57BL/6J genetic background and genotyped as described elsewhere.36 All animal experiments were approved by the local authorities (acceptance no. 73/10; ORG532), and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Immunohistochemistry
Cln7 KO and age-matched wild-type mice were killed, and eyes were quickly enucleated. After removal of the cornea, eyes were immersion-fixed in 4% paraformaldehyde (Carl Roth GmbH, Karlsruhe, Germany) in PBS (pH 7.4) overnight at room temperature, dehydrated in an ascending series of sucrose, embedded in TissueTec (Sakura Finetek, Zouterwoude, The Netherlands), frozen, and serially sectioned with a cryostat at a thickness of 25 μm. Immunohistochemical analyses were performed as described.37,38 In brief, central (i.e., in the plane of the optic disc) retina sections were blocked in PBS containing 0.1% bovine serum albumin and 0.3% Triton X-100 (TX-100; both from Sigma-Aldrich, Deisenhofen, Germany), incubated with primary antibodies (Table) overnight at room temperature, washed in PBS, and incubated with Cy3-conjugated secondary antibodies (1:200; all from Jackson Immunoresearch Laboratories, West Grove, PA, USA) overnight. Cone photoreceptors were visualized by incubating sections with biotinylated peanut agglutinin (PNA; 1:5000; Vector Laboratories, Burlingame, CA, USA) followed by Cy3-conjugated streptavidin (1:500; Jackson Immunoresearch Laboratories). All sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 10 minutes to label cell nuclei, washed again, and mounted onto slides. For each antigen, at least three animals of each genotype and developmental age were analyzed. In all experiments, sections from wild-type and age-matched Cln7 KO retinas were processed in parallel and under identical conditions. Sections were analyzed with an Olympus FV 1000 confocal microscope (Olympus, Hamburg, Germany) using the same microscope settings for each antigen to allow for comparison of staining intensities. The number of cluster of differentiation (CD)68-immunoreactive profiles with a diameter of more than 3 μm was determined in central retina sections from 1- (n = 6) and 4-month-old (n = 5) Cln7 KO mice on the nasal and temporal sides of the optic disc. Cluster of differentiation 68–immunoreactive profiles were counted in the outer retina (i.e., outer plexiform layer and photoreceptor layer) and inner retina (i.e., inner nuclear layer, inner plexiform layer and ganglion cell layer), and data were analyzed with the Mann-Whitney rank sum test. 
Table
 
Primary Antibodies
Table
 
Primary Antibodies
Determination of Retina and Outer Nuclear Layer Thickness and of Photoreceptor Numbers
The thickness of the retina and the outer nuclear layer (defined as the layer of photoreceptor cell nuclei without inner and outer photoreceptor segments) was analyzed on merged confocal images of the central retina at nine equidistant positions between the optic disc and the peripheral margin of the temporal and nasal retina, respectively by using Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA, USA).43 Six animals were analyzed for each genotype and developmental age. Statistical analyses of data were performed with the 2-way ANOVA test followed by a Bonferroni post hoc test using Stata Statistical Software: Release 11 (StataCorp LP, College Station, TX, USA). Photoreceptor numbers were determined in central retina sections by counting DAPI-stained photoreceptor cell nuclei encircled by recoverin-immunoreactivity in three equidistant areas between the optic disc and the peripheral margin of the temporal and nasal retina, respectively, each covering the outer nuclear layer over a length of 220 μm.37 The total number of photoreceptor cells in the six retina areas was calculated for six animals of each genotype and developmental age. Numbers of PNA-positive cones were determined in 4-month-old Cln7 KO and age-matched wild-type mice on photomicrographs taken from central retina sections at both sides of the optic disc. Statistical analyses of data were performed using the Student's t-test. 
Electron Microscopy
For electron microscopic analyses, 2-month-old Cln7 KO (n = 3) and age-matched wild-type mice (n = 2) were perfusion-fixed with cacodylate buffer containing 4% paraformaldehyde and 2% glutaraldehyde (Serva, Heidelberg, Germany). Corneas were removed, eyes were postfixed in the same fixative overnight at room temperature, washed in 0.1 M cacodylate buffer (Sigma-Aldrich), incubated for 2 hours in 1% osmium tetroxide (Science Services, Munich, Germany), dehydrated in an ascending series of ethanol, and embedded in Epon 812 (Serva). Ultrathin sections were counterstained with uranyl acetate (Polyscience, Eppelheim, Germany) and lead citrate (Riedel-de Haën, Seelze, Germany), and analyzed with a LEO 912 AB OMEGA electron microscope (Leo Elektronenmikroskopie, Oberkochen, Germany). 
Preparation of Total Protein and Membrane Fractions
For preparation of total protein homogenates, retinas from 2-month-old Cln7 KO and age-matched wild-type mice were homogenized in 10 volumes of lysis buffer containing 0.32 M sucrose, 10 mM HEPES (pH 7.4), 0.02% TX-100, and protease inhibitors, and incubated on ice for 30 minutes. After centrifugation at 14,000g for 10 minutes, protein concentrations of supernatants were determined using the Bradford protein quantification kit (Thermo Fisher Scientific, Schwerte, Germany). For preparation of lysosomal/mitochondrial membrane extracts, 2-month-old Cln7 KO and wild-type retinas were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, and protease inhibitors (Sigma-Aldrich). Homogenization was performed in a Dounce homogenizer using 30 strokes followed by centrifugation at 1500g for 5 minutes at 4°C. The resulting postnuclear supernatants (PNS) were subjected to further centrifugation at 20,000g for 10 minutes. The pellets were homogenized in extraction buffer containing 50 mM Tris-HCl (pH 7.5), 1% TX-100, 1 mM EDTA, and protease inhibitors, and incubated on ice for 30 minutes. After a final centrifugation at 20,000g for 10 minutes, supernatants were used for immunoblot analyses. 
Preparation of detergent-soluble and detergent-insoluble fractions was performed as described.36 Briefly, retinas were homogenized in lysis buffer containing 0.32 M sucrose, 10 mM HEPES (pH 7.4), and protease inhibitors, and centrifuged at 500g for 10 minutes at 4°C to remove cell nuclei. Postnuclear supernatants were adjusted to a final concentration of 1% TX-100, and detergent-soluble and -insoluble fractions were prepared by centrifugation at 13,000g for 15 minutes at 4°C. The supernatants (representing the detergent-soluble fraction) were removed and pellets (representing the detergent-insoluble fraction) were dissolved in 50 mM Tris-HCl (pH 7.5) containing 2% SDS and protease inhibitors. 
Western Blot Analyses
An amount of 20 μg of total protein was solubilized under reducing conditions, and separated by SDS-PAGE (12.5% acrylamide) followed by electrotransfer to nitrocellulose membranes (GE Healthcare Life Sciences, Freiburg, Germany) for 60 minutes at 400 mA. After blocking the membranes with PBS containing 0.05% Tween (PBS-T) and 5% milk powder, membranes were incubated with the following primary antibodies (Table): rabbit anti-mouse cathepsin D, goat anti-mouse cathepsin Z, rabbit anti-human glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), rat anti-mouse lysosome-associated membrane protein 1 (LAMP1), rabbit anti-human SCMAS, goat anti-human saposin D, and mouse anti-chicken α-tubulin for 16 hours at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000; Jackson Immunoresearch Laboratories) in PBS-T containing 5% milk powder. Immunoreactive bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) using a molecular imager (ChemiDoc XRS system; Biorad, Munich, Germany). Determination of molecular masses of immunoreactive proteins was performed by comparison with electrophoretic mobility of prestained protein markers (Peqgold protein marker IV; VWR International, Erlangen, Germany). Each experiment was performed in triplicate on independently prepared retina preparations. 
Quantitative Real-Time PCR
Total RNA from two retinas from 2-month-old Cln7 KO or age-matched wild-type mice was prepared using TRIzol reagent (Invitrogen, Darmstadt, Germany) following the manufacturer's instructions. An amount of 1 μg RNA was transcribed into cDNA using the high-capacity cDNA reverse-transcriptase kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed using TaqMan gene expression assays (Applied Biosystems, Darmstadt, Germany) including predesigned probes and primers sets for mouse cathepsin D (Mm00515587_m1), cathepsin Z (Mm00517687_m1), and β-actin (Mm00607939_s1). Quantitative real-time PCR was performed as described previously44 using the Mx3000P QPCR system (Agilent Technologies, Santa Clara, CA, USA). Expression levels of the analyzed mRNAs were normalized to the amount of β-actin mRNA in the same cDNAs using the comparative CT method (2–ΔΔCT). Experiments were performed in triplicate on independently prepared retina preparations. 
Results
Retinal Degeneration and Reactive Astrogliosis and Microgliosis in Cln7 KO Mice
We have recently reported severe degeneration of the photoreceptor cell layer in 8.5-month-old animals of a hypomorphic mouse model of CLN7 disease.11 In the present study, we performed a detailed analysis of the onset and progression of the retinal pathology in a novel Cln7 KO mouse model that lacks exon 2 of the murine Cln7/Mfsd8 gene and more closely recapitulates the neurologic phenotype observed in human patients with CLN7 with the vLINCL phenotype.36 
To analyze the impact of CLN7 deficiency on retinal morphology, we performed immunohistochemical analyses of glial fibrillary acidic protein (GFAP) expression in central retina sections from Cln7 KO and wild-type mice at different developmental ages. In 0.5-month-old wild-type (Fig. 1a) and age-matched CLN7-deficient animals (Fig. 1b), expression of GFAP was restricted to retinal astrocytes located at the vitreal margin of the retinas (Figs. 1a, 1b). At this age, the outer nuclear layer of both genotypes was similar in thickness (compare Fig. 1a with Fig. 1b). In 1-, 2-, and 4-month-old mutants (Figs. 1d, 1f, 1h, respectively), expression of GFAP was elevated in retinal astrocytes when compared with age-matched wild-type retinas (Figs. 1c, 1e, 1g, respectively), and was additionally detectable in Müller cells. The presence of reactive astrocytes and Müller cells in 1-, 2-, and 4-month-old CLN7-deficient retinas correlated with a progressive thinning of the outer nuclear layer, whereas other retinal layers of the mutant retinas were not detectably affected at these ages (Fig. 1). 
Figure 1
 
Degeneration of photoreceptor cells and reactive astrogliosis in Cln7 KO mice. The outer nuclear layer of 0.5 month old wild-type (a) and Cln7 KO mice (b) was similar in thickness, and expression of GFAP in both genotypes was restricted to retinal astrocytes. In 1-month-old mutant mice (d), the outer nuclear layer was significantly reduced in thickness when compared with age-matched control mice (c), and expression of GFAP was elevated in retinal astrocytes and became additionally detectable in Müller cells (d). With increasing age of the mutant mice, the photoreceptor layer further decreased in thickness, and retinal degeneration was accompanied by reactive astrogliosis (see [f] and [h] for 2- and 4-month-old Cln7 KO mice, respectively). All sections were stained with DAPI to visualize cell nuclei. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; WT, wild-type. Scale bar in (h) (for ah): 100 μm.
Figure 1
 
Degeneration of photoreceptor cells and reactive astrogliosis in Cln7 KO mice. The outer nuclear layer of 0.5 month old wild-type (a) and Cln7 KO mice (b) was similar in thickness, and expression of GFAP in both genotypes was restricted to retinal astrocytes. In 1-month-old mutant mice (d), the outer nuclear layer was significantly reduced in thickness when compared with age-matched control mice (c), and expression of GFAP was elevated in retinal astrocytes and became additionally detectable in Müller cells (d). With increasing age of the mutant mice, the photoreceptor layer further decreased in thickness, and retinal degeneration was accompanied by reactive astrogliosis (see [f] and [h] for 2- and 4-month-old Cln7 KO mice, respectively). All sections were stained with DAPI to visualize cell nuclei. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; WT, wild-type. Scale bar in (h) (for ah): 100 μm.
To further analyze the progression of retinal degeneration in Cln7 KO mice, we determined the thickness of the entire retina and the outer nuclear layer (i.e., layer of photoreceptor cell nuclei) in central retina sections from Cln7 KO and age-matched wild-type mice at different developmental ages (Fig. 2). In 0.5-month-old animals, the thickness of the retina (Fig. 2A) and outer nuclear layer (Fig. 2B) was similar in both genotypes, in agreement with the immunohistochemical results (Fig. 1). In older Cln7 KO mice, the thickness of both the retina and the outer nuclear layer decreased significantly with increasing age of the animals (Fig. 2). Retinal degeneration in CLN7-deficient mice progressed rapidly between 0.5 and 2 months of age, and slowly between the second and fourth month of age (Fig. 2). 
Figure 2
 
Thickness of the retina and outer nuclear layer in Cln7 KO and wild-type mice at different developmental ages. The thickness of the retina (A) and outer nuclear layer (B) was determined in central retina sections at nine equidistant positions of the nasal and temporal retina, respectively. Retina and outer nuclear thickness was similar in 0.5-month-old wild-type and age-matched Cln7 KO mice. In older Cln7 KO mice, the thickness of the retina and outer nuclear layer was significantly reduced when compared with wild-type mice, and progressively decreased with increasing age of the mutant mice. The strongest reduction in the thickness of retina and photoreceptor layer of mutant mice was observed between the first and second postnatal month. Blue symbols represent the mean values (± SEM) of 18 wild-type mice aged 1 (n = 6), 2 (n = 6), and 4 (n = 6) months. All other symbols represent the mean values (± SEM) of six retinas for each genotype and developmental age. *P < 0.05; **P < 0.01; ***P < 0.001 according to the 2-way ANOVA test followed by a Bonferroni post hoc test.
Figure 2
 
Thickness of the retina and outer nuclear layer in Cln7 KO and wild-type mice at different developmental ages. The thickness of the retina (A) and outer nuclear layer (B) was determined in central retina sections at nine equidistant positions of the nasal and temporal retina, respectively. Retina and outer nuclear thickness was similar in 0.5-month-old wild-type and age-matched Cln7 KO mice. In older Cln7 KO mice, the thickness of the retina and outer nuclear layer was significantly reduced when compared with wild-type mice, and progressively decreased with increasing age of the mutant mice. The strongest reduction in the thickness of retina and photoreceptor layer of mutant mice was observed between the first and second postnatal month. Blue symbols represent the mean values (± SEM) of 18 wild-type mice aged 1 (n = 6), 2 (n = 6), and 4 (n = 6) months. All other symbols represent the mean values (± SEM) of six retinas for each genotype and developmental age. *P < 0.05; **P < 0.01; ***P < 0.001 according to the 2-way ANOVA test followed by a Bonferroni post hoc test.
Quantitative analyses of the outer nuclear layer revealed similar numbers of photoreceptor cells in 0.5-month-old CLN7-deficient retinas (2898 ± 39 photoreceptors/area; mean ± SEM) and age-matched wild-type retinas (2859 ± 29 photoreceptors/area; Fig. 3). In older mutants, the number of photoreceptor cells decreased rapidly until the second postnatal month by more than 60% when compared with wild-type mice (Fig. 3). Thereafter, photoreceptor loss in Cln7 KO retinas proceeded at a slower rate. In 4-month-old mutants, we found 789 ± 26 photoreceptors/area as compared with 1045 ± 57 photoreceptors per area in 2-month-old mutants (Fig. 3). To evaluate whether rods or cones or both photoreceptor cell types are affected in Cln7 KO mice, we stained retina sections from 1- and 4-month-old Cln7 KO and age-matched wild-type mice with anti-rhodopsin antibodies or PNA (Fig. 4). Although the number of photoreceptor cells in 4-month-old Cln7 KO mice was reduced by more than 70% when compared with wild-type mice (Fig. 3), the density of cone photoreceptor cells was not detectably reduced at this age when compared with 1-month-old CLN7-deficient retinas or 2-month-old wild-type retinas (Fig. 4), demonstrating preferential degeneration of rod photoreceptor cells in Cln7 KO mice. In fact, determination of cone numbers in temporal and nasal retina regions located close to the optic disc revealed 62.3 ± 1.9 (mean ± SEM) cones per area in 4-month-old Cln7 KO mice and 63.8 ± 2.8 cones per area in age-matched wild-type mice (P = 0.61 according to the Student's t-test). 
Figure 3
 
Photoreceptor numbers in Cln7 KO and wild-type mice at different developmental ages. Retinas from 0.5-month-old wild-type (gray bars) and Cln7 KO mice (black bars) contained similar numbers of photoreceptor cells. In 1-month-old Cln7 KO retinas, in comparison, the number of photoreceptor cells was significantly reduced when compared with wild-type retinas, and further decreased with increasing age of the mutant mice. Each bar represents the mean value (± SEM) of six animals. n.s., not significant. **P < 0.01; ***P < 0.001 according to the Student's t-test.
Figure 3
 
Photoreceptor numbers in Cln7 KO and wild-type mice at different developmental ages. Retinas from 0.5-month-old wild-type (gray bars) and Cln7 KO mice (black bars) contained similar numbers of photoreceptor cells. In 1-month-old Cln7 KO retinas, in comparison, the number of photoreceptor cells was significantly reduced when compared with wild-type retinas, and further decreased with increasing age of the mutant mice. Each bar represents the mean value (± SEM) of six animals. n.s., not significant. **P < 0.01; ***P < 0.001 according to the Student's t-test.
Figure 4
 
Preferential loss of rod photoreceptor cells in Cln7 KO mice. Central sections of CLN7-deficient and wild-type retinas were stained with anti-recoverin antibodies to label all photoreceptor cells (ac), anti-rhodopsin antibodies to label rod photoreceptor cells (df), or PNA to label cone photoreceptor cells (gi). Note the similar number of cone photoreceptor cells in 1- (h) or 4-month-old (i) Cln7 KO retinas and 2-month-old wild-type retinas (g). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (i) (for ai): 100 μm.
Figure 4
 
Preferential loss of rod photoreceptor cells in Cln7 KO mice. Central sections of CLN7-deficient and wild-type retinas were stained with anti-recoverin antibodies to label all photoreceptor cells (ac), anti-rhodopsin antibodies to label rod photoreceptor cells (df), or PNA to label cone photoreceptor cells (gi). Note the similar number of cone photoreceptor cells in 1- (h) or 4-month-old (i) Cln7 KO retinas and 2-month-old wild-type retinas (g). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (i) (for ai): 100 μm.
Reactive microgliosis in Cln7 KO retinas was studied using antibodies to ionized calcium-binding adaptor molecule 1 (IBA1) and CD68 (Fig. 5). Ionized calcium-binding adaptor molecule 1–positive microglial cells in 1- (Fig. 5a) and 4-month-old wild-type retinas (Fig. 5c) displayed a ramified morphology and were mainly located in the inner retina (i.e., ganglion cell layer and inner plexiform layer). In mutant retinas, IBA1-positive cells were increased in number, and IBA1-positive cells with an amoeboid morphology became detectable in the inner nuclear layer, outer plexiform layer, and subretinal space (for 1- and 4-month-old CLN7-deficient retinas, see Figs. 5b, 5d, respectively). Cluster of differentiation 68–positive cells were not detectable in 1- or 4-month-old wild-type retinas (Figs. 5e, 5g, respectively). In 1-month-old mutant retinas, in comparison, CD68-positive cells with an amoeboid morphology were found in the outer plexiform layer and above the outer nuclear layer (Fig. 5f). In 4-month-old Cln7 KO retinas, the number of CD68-positive cells was significantly increased when compared with 1-month-old mutants, and positive cells were particularly numerous in the subretinal space (Fig. 5h). Quantitative analyses revealed a similar number of CD68-immunoreactive profiles in the inner retina of 1- and 4-month-old Cln7 KO mice (3.25 ± 0.43 and 2.50 ± 0.17 (mean ± SEM) immunoreactive profiles per area, respectively; P = 0.13 according to the Mann-Whitney rank sum test). In the outer retina, in comparison, the number of CD68-immunoreactive profiles increased significantly from 8.58 ± 0.72 profiles per area in 1-month-old mutant mice to 12.9 ± 0.96 profiles per area in 4-month-old Cln7 KO mice (P < 0.01). 
Figure 5
 
Reactive microgliosis in the retina of Cln7 KO mice. In 1- (a) and 4-month-old (c) wild-type mice, IBA1-positive cells were present in the ganglion cell layer, inner plexiform layer, and outer plexiform layer. In 1- (b) and 4-month-old Cln7 KO mice (d), the number of IBA1-positive microglia cells was increased when compared to age-matched control animals, and became additionally detectable in the subretinal space (arrowheads in [d]). Cluster of differentiation 68–positive cells were not detectable in retinas of 1- (e) and 4-month-old wild-type mice (g). In 1- (f) and 4-month-old (h) mutant retinas, in comparison, CD68-positive cells were present in the outer retina (i.e., outer plexiform layer, outer nuclear layer, and subretinal space), and their number increased with increasing age of the mutants (compare [f] and [h]). Note the presence of numerous CD68-positive cells in the subretinal space of 4-month-old Cln7 KO retinas (arrowheads in [h]). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (d) (for ad): 100 μm; in (h) (for eh): 50 μm.
Figure 5
 
Reactive microgliosis in the retina of Cln7 KO mice. In 1- (a) and 4-month-old (c) wild-type mice, IBA1-positive cells were present in the ganglion cell layer, inner plexiform layer, and outer plexiform layer. In 1- (b) and 4-month-old Cln7 KO mice (d), the number of IBA1-positive microglia cells was increased when compared to age-matched control animals, and became additionally detectable in the subretinal space (arrowheads in [d]). Cluster of differentiation 68–positive cells were not detectable in retinas of 1- (e) and 4-month-old wild-type mice (g). In 1- (f) and 4-month-old (h) mutant retinas, in comparison, CD68-positive cells were present in the outer retina (i.e., outer plexiform layer, outer nuclear layer, and subretinal space), and their number increased with increasing age of the mutants (compare [f] and [h]). Note the presence of numerous CD68-positive cells in the subretinal space of 4-month-old Cln7 KO retinas (arrowheads in [h]). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (d) (for ad): 100 μm; in (h) (for eh): 50 μm.
Electron Microscopic Analysis of Storage Material in CLN7-Deficient Retinas
Electron microscopic analyses of 2-month-old CLN7-deficient retinas were performed to analyze the distribution and ultrastructure of the storage material (Fig. 6). Large storage bodies were found in photoreceptor cells, interneurons, and retinal ganglion cells of Cln7 KO mice (Figs. 6i–p), but not in retinal cell types of age-matched wild-type mice (Figs. 6a–h). Analysis of mutant retinas at higher magnification identified the storage material as curvilinear bodies in photoreceptor cells (Fig. 6n) and interneurons (Fig. 6o), and as fingerprint-like profiles in retinal ganglion cells (Fig. 6p). 
Figure 6
 
Ultrastructure of storage material in the retina of Cln7 KO mice. Low-power electron micrographs depict the different layers of a 2-month-old wild-type (a, b) and age-matched Cln7 KO retina (i, j). Analyses of the boxed areas in (i) and (j) at higher magnification revealed the presence of storage material in photoreceptor cells (k), interneurons (l), and ganglion cells (m) of mutant mice. Further analyses at higher magnification identified the storage material in Cln7 KO retinas as curvilinear bodies in photoreceptor cells (n) and interneurons (o), and fingerprint-like profiles in ganglion cells (p). Similar pathologic cytoplasmic inclusions were not detectable in retinas of age-matched control mice (ch). (fh) and (np) are higher magnifications of the boxed areas in (ce) and (km), respectively. Scale bar in (j) (for a, b, i, j): 5 μm; in (m) (for ce and km): 1 μm; in (p) (for fh and np): 0.2 μm.
Figure 6
 
Ultrastructure of storage material in the retina of Cln7 KO mice. Low-power electron micrographs depict the different layers of a 2-month-old wild-type (a, b) and age-matched Cln7 KO retina (i, j). Analyses of the boxed areas in (i) and (j) at higher magnification revealed the presence of storage material in photoreceptor cells (k), interneurons (l), and ganglion cells (m) of mutant mice. Further analyses at higher magnification identified the storage material in Cln7 KO retinas as curvilinear bodies in photoreceptor cells (n) and interneurons (o), and fingerprint-like profiles in ganglion cells (p). Similar pathologic cytoplasmic inclusions were not detectable in retinas of age-matched control mice (ch). (fh) and (np) are higher magnifications of the boxed areas in (ce) and (km), respectively. Scale bar in (j) (for a, b, i, j): 5 μm; in (m) (for ce and km): 1 μm; in (p) (for fh and np): 0.2 μm.
Accumulation of SCMAS and Saposin D in CLN7-Deficient Retinas
Lysosomal accumulation of storage material composed of SCMAS and saposins A and D as major protein components is a key feature of NCLs.22 To study the age-dependent presence, localization, and protein composition of the storage material, retinas of Cln7 KO mice were analyzed by immunohistochemistry and immunoblotting. Immunostainings revealed elevated levels of SCMAS in the retina of 1- and 4-month-old Cln7 KO mice (Figs. 7Ab, 7Ad, respectively) when compared with age-matched wild-type mice (Figs. 7Aa, 7Ac, respectively), particularly in the inner nuclear layer and outer plexiform layer. Amounts of saposin D were only slightly elevated in 1-month-old mutant retinas (Fig. 7Af), but markedly increased in 4-month-old CLN7-deficient retinas (Fig. 7Ah) when compared with age-matched wild-type retinas (Figs. 7Ae, 7Ag, respectively). Strongest saposin D-immunoreactivity in 4-month-old mutant retinas was found in the ganglion cell layer and inner and outer plexiform layers (Fig. 7Ah). Analyses of detergent-insoluble fractions by immunoblotting confirmed the immunohistochemical data, and revealed the presence of both SCMAS and saposin D in 2-month-old mutant retinas, but not in age-matched control retinas (Fig. 7B). Furthermore, levels of prosaposin were increased 1.5-fold in Cln7 KO retinas when compared with age-matched wild-type retinas (Fig. 7B). 
Figure 7
 
Elevated amounts of SCMAS and saposin D in the retina of CLN7-deficient retinas. (A) Immunohistochemistry revealed increased amounts of SCMAS in retinas of 1- (b) and 4-month-old (d) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, c, respectively). Elevated SCMAS-immunoreactivity was particularly evident in the inner nuclear layer and outer plexiform layer of mutant mice (b, d). Expression levels of saposin D were slightly elevated in 1-month-old mutant retinas (f), and significantly increased in 4-month-old mutant retinas (h) when compared with age-matched control retinas (e, g, respectively). In 4-month-old Cln7 KO retinas, strongest saposin D-immunoreactivity was found in the ganglion cell layer and inner and outer plexiform layer. All sections were stained with DAPI to visualize cell nuclei. (B) Immunoblot analyses of detergent-insoluble fractions revealed the accumulation of SCMAS and saposin D and increased expression of prosaposin in 2-month-old Cln7 KO retinas when compared with age-matched wild-type retinas. Loading of gels was controlled by Ponceau S staining or α-tubulin immunoblotting. The immunoblots shown are representative examples of three independent experiments. Scale bar in (h) (for ah): 100 μm.
Figure 7
 
Elevated amounts of SCMAS and saposin D in the retina of CLN7-deficient retinas. (A) Immunohistochemistry revealed increased amounts of SCMAS in retinas of 1- (b) and 4-month-old (d) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, c, respectively). Elevated SCMAS-immunoreactivity was particularly evident in the inner nuclear layer and outer plexiform layer of mutant mice (b, d). Expression levels of saposin D were slightly elevated in 1-month-old mutant retinas (f), and significantly increased in 4-month-old mutant retinas (h) when compared with age-matched control retinas (e, g, respectively). In 4-month-old Cln7 KO retinas, strongest saposin D-immunoreactivity was found in the ganglion cell layer and inner and outer plexiform layer. All sections were stained with DAPI to visualize cell nuclei. (B) Immunoblot analyses of detergent-insoluble fractions revealed the accumulation of SCMAS and saposin D and increased expression of prosaposin in 2-month-old Cln7 KO retinas when compared with age-matched wild-type retinas. Loading of gels was controlled by Ponceau S staining or α-tubulin immunoblotting. The immunoblots shown are representative examples of three independent experiments. Scale bar in (h) (for ah): 100 μm.
Dysregulation of Lysosomal Proteins in CLN7-Deficient Retinas
We next studied the impact of CLN7 deficiency on the expression levels of mannose 6-phosphate (M6P) containing lysosomal proteins, cathepsin D (CTSD) and cathepsin Z (CTSZ) and the lysosomal membrane protein LAMP1. Immunohistochemical analyses revealed elevated expression levels of all these lysosomal proteins in retinas of 1- and 4-month-old Cln7 KO mice when compared with retinas of age-matched control mice (Fig. 8). Increased immunoreactivity of the lysosomal proteins was particularly evident in the ganglion cell layer and at the basal and apical margin of the inner nuclear layer of CLN7-deficient retinas (Fig. 8). Quantitative real-time PCR analyses were in line with the immunohistochemical data, and revealed a 1.4-fold increase in Ctsd mRNA levels and a 1.35-fold increase in Ctsz mRNA levels in 2-month-old Cln7 KO retinas when compared with age-matched wild-type retinas (Fig. 9A). To quantify the expression levels of soluble lysosomal enzymes, total protein extracts from 2-month-old Cln7 KO and control retinas were analyzed by CTSD and CTSZ immunoblotting (Fig. 9B). Densitometric evaluation of immunoreactive band intensities revealed a 2-fold and 3-fold increase in CTSD and CTSZ levels, respectively, in CLN7-deficient retinas when compared with wild-type retinas. The amounts of LAMP1 in total extracts of 2-month-old Cln7 KO retinas were increased 2.5-fold when compared with age-matched control retinas (Fig. 9B). 
Figure 8
 
Dysregulation of various lysosomal proteins in the retina of Cln7 KO mice. Immunohistochemical analyses revealed elevated expression levels of M6P-containing soluble lysosomal proteins (ad), LAMP1 (eh), CTSD (il), and CTSZ (mp) in retinas of 1- (b, f, j, n) and 4-month-old (d, h, l, p) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, e, i, m, and c, g, k, o, respectively). At both developmental ages, elevated expression levels of the different lysosomal proteins were particularly evident in the ganglion cell layer and at the basal and apical margin of the inner nuclear layer of CLN7-deficient retinas. All sections were stained with DAPI to visualize cell nuclei. Scale bar in (p) (for ap): 100 μm.
Figure 8
 
Dysregulation of various lysosomal proteins in the retina of Cln7 KO mice. Immunohistochemical analyses revealed elevated expression levels of M6P-containing soluble lysosomal proteins (ad), LAMP1 (eh), CTSD (il), and CTSZ (mp) in retinas of 1- (b, f, j, n) and 4-month-old (d, h, l, p) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, e, i, m, and c, g, k, o, respectively). At both developmental ages, elevated expression levels of the different lysosomal proteins were particularly evident in the ganglion cell layer and at the basal and apical margin of the inner nuclear layer of CLN7-deficient retinas. All sections were stained with DAPI to visualize cell nuclei. Scale bar in (p) (for ap): 100 μm.
Figure 9
 
Lysosomal dysfunction in the retina of Cln7 KO mice. (A) Quantitative real-time PCR revealed increased Ctsd and Ctsz mRNA expression levels in 2-month-old Cln7 KO retinas (black bars) when compared with age-matched wild-type retinas (gray bars). Ctsd and Ctsz mRNA levels were normalized to β-actin mRNA levels, and values of wild-type mice were arbitrarily set to 1. Bars represent mean values (±SD) from three independent experiments. Statistical analysis of data was performed with the Student's t-test (*P < 0.05; **P < 0.01). (B) Immunoblot analyses of lysosomal/mitochondrial membrane extracts from 2-month-old CLN7-deficient and age-matched wild-type retinas revealed elevated amounts of LAMP1 in Cln7 KO mice. Western blotting of total protein homogenates showed increased expression of CTSD and CTSZ in the mutant. α-tubulin or GAPDH Western blotting was performed to control loading.
Figure 9
 
Lysosomal dysfunction in the retina of Cln7 KO mice. (A) Quantitative real-time PCR revealed increased Ctsd and Ctsz mRNA expression levels in 2-month-old Cln7 KO retinas (black bars) when compared with age-matched wild-type retinas (gray bars). Ctsd and Ctsz mRNA levels were normalized to β-actin mRNA levels, and values of wild-type mice were arbitrarily set to 1. Bars represent mean values (±SD) from three independent experiments. Statistical analysis of data was performed with the Student's t-test (*P < 0.05; **P < 0.01). (B) Immunoblot analyses of lysosomal/mitochondrial membrane extracts from 2-month-old CLN7-deficient and age-matched wild-type retinas revealed elevated amounts of LAMP1 in Cln7 KO mice. Western blotting of total protein homogenates showed increased expression of CTSD and CTSZ in the mutant. α-tubulin or GAPDH Western blotting was performed to control loading.
Discussion
Retinal degeneration leading to visual impairment and ultimately blindness during the course of the disease has been reported as a typical neurologic symptom in patients with CLN7 with vLINCL phenotype,7,8,1519,21,22,45 in a naturally occurring CLN7 canine model,46 and in a hypomorphic mouse model of CLN7 disease.11 However, precise information about the onset and progression of the retinal pathology, the affected retinal layers and retinal cell types, and the biochemical alterations associated with CLN7 disease, vLINCL phenotype, are largely missing. In the present study, we therefore performed a detailed analysis of the retinal phenotype of a novel Cln7 KO mouse that was generated by deleting Cln7/Mfsd8 exon 2, resulting in the expression of a truncated, nonfunctional p.Glu23PhefsX16 protein.36 Genetically, Cln7 KO mice closely resemble patients with CLN7 carrying the nonsense mutation p.Arg35X.16,18 In patients with CLN7 of this condition, visual impairment is diagnosed between 4.0 and 5.5 years of age.16,18 
Cln7 KO mice displayed an early onset retinal degeneration, as indicated by the presence of reactive astrocytes, Müller cells and microglia cells, and a reduced thickness of the retina and the photoreceptor cell layer as early as 1 month after birth. Thinning of the retina and photoreceptor layer progressed rapidly with increasing age of the mutant mice, and was particularly pronounced between the first and second postnatal month of age. In line with these data, we observed a rapid and massive loss of photoreceptor cells by more than 60% in 2-month-old mutants and approximately 70% in 4-month-old CLN7-deficient mice when compared with adult wild-type mice. Evidence for a significant loss of retinal cell types other than photoreceptor cells was not observed. Of note, analyses of 4-month-old Cln7 KO retinas revealed normal numbers of cone photoreceptor cells, indicating that retinal degeneration in Cln7 KO mice is mainly, and possibly exclusively, characterized by the loss of rod photoreceptor cells, at least during the first 4 postnatal months. Loss of rod photoreceptors in Cln7 KO mice was evident long before damage and loss of neurons in the brain became detectable,36 and thus represents the earliest phenotypic manifestation of CLN7 deficiency in the central nervous system. Progressive degeneration of photoreceptor cells has also been observed in mouse models of various other lysosomal storage diseases, such as mucolipidosis (ML) II,47 ML IV,48 arylsulfatase G-deficiency,43 CLC7-deficiency,49 and several NCL variants, including CLN6,29 CLN8,28 CLN10,30 and CLN11 disease,31 indicating that photoreceptor cells are particularly vulnerable to lysosomal dysfunction. 
Electron microscopic analyses of Cln7 KO retinas revealed the presence of storage material in essentially all retinal cell types. The ultrastructural appearance of storage material is believed to be characteristic for genetically distinct NCL variants, and typically resembles granular osmiophilic deposits in CLN1 and CLN10 disease, curvilinear bodies in CLN2 disease, fingerprint-like profiles in CLN3 disease, and combinations thereof in other NCL variants.50 In the retina of CLN7-deficient mice, the storage material appeared as curvilinear bodies in photoreceptor cells and retinal interneurons and as fingerprint-like profiles in ganglion cells and thus resembled the appearance of nondegraded material observed in cerebellar neurons of Cln7 KO mice, and in endothelial cells, Schwann cells, and cerebral neurons of patients with CLN7.22,36,51 Curvilinear bodies and fingerprint-like profiles were also observed in skin fibroblasts of patients with CLN7 carrying the nonsense mutation p.Arg35X.16 Immunohistochemical and immunoblot analyses of mutant retinas revealed accumulation of SCMAS and saposin D, in close analogy to the results obtained in the brain of Cln7 KO mice.36 The presence of storage material in essentially all retinal cell types, but degeneration of only rod photoreceptor cells, suggests that the consequences of lysosomal dysfunction and subsequent lysosomal storage on neuronal homeostasis in the retina may be cell-type specific. 
To examine the impact of CLN7 deficiency on lysosomal function, we analyzed the distribution and expression levels of various lysosomal proteins in Cln7 KO retinas. Biochemical and immunohistochemical analyses revealed increased levels of M6P-containing soluble lysosomal enzymes, CTSZ, CTSD, and the lysosomal membrane protein LAMP1 in the retina of 1- and 4-month-old mutant mice, indicating a global upregulation of lysosomal protein biosynthesis. Upregulation of multiple lysosomal proteins has also been observed in the retina of another mouse model of vLINCL: the Cln6/nclf mouse.29 The elevated amounts of Ctsd and Ctsz mRNA in Cln7 KO retinas suggest that transcriptional upregulation rather than impaired turnover contributes to elevated amounts of these lysosomal enzymes. It is possible that lysosomal stress and storage leads to a cell-type–specific dephosphorylation and subsequent nuclear translocation of the transcription factor EB (TFEB). Transcription factor EB has been shown to activate the transcription of lysosomal genes during starvation or lysosomal storage.52 Of note, CTSZ and CTSD were processed to the mature forms, suggesting that loss of CLN7 did not interfere with their proper targeting and proteolytic activation in lysosomes. 
Taken together, increased levels of multiple lysosomal enzymes, accumulation of SCMAS and saposin D, and the presence of storage material suggest that loss of CLN7 leads to lysosomal dysfunction and a reduced degradative capacity in the retina early in the course of the disease. Although the pathologic alterations in the brain of Cln7 KO mice were similar to those observed in the retina, they became detectable much later during development. For instance, storage of SCMAS in the brain became detectable in 3-month-old mutants, elevated levels of lysosomal enzymes and reactive astrogliosis in 5-month-old mutants, and microgliosis in 7-month-old mutants.36 In line with these data, retinal degeneration in Cln7 KO mice started already during the first postnatal month, whereas magnetic resonance imaging revealed atrophy of the olfactory bulb, cerebral cortex, and cerebellum not before 9 months of age.36 Together, these results demonstrate that the retina is more vulnerable to the loss of CLN7 than the brain. Of interest in this context, in a single Dutch patient with CLN7 homozygous for the mutation p.Ala157Pro and presenting with a juvenile onset and protracted course of the disease, visual deterioration was diagnosed as the first clinical symptom of the disease at the age of 11 years.18 A high susceptibility of the retina to CLN7 dysfunction is also suggested by the recent finding that a hypomorphic CLN7 mouse line showed a marked loss of photoreceptor cells, but no neurodegeneration in the brain even in animals as old as 10 months.11 Furthermore, a recent study has identified compound heterozygous variants in CLN7/MFSD8 in two families presenting with autosomal recessive macular dystrophy with central cone involvement, but without the other neurologic symptoms characteristic for vLINCL, such as seizures, mental regression, or motor impairment.23 Affected individuals were compound heterozygous for the missense mutation p.Glu336Gln predicted to have a mild effect on the protein and the severe nonsense mutation p.Glu381X or the severe frame-shift mutation p.Lys333LysfsX3.23 The combined observations suggest a residual activity of CLN7 in the hypomorphic CLN7 mouse line and in the patients with CLN7 with nonsyndromic macular dystrophy with central cone involvement that is sufficient to maintain the integrity of all tissues with the exception of the retina. Whereas retinal degeneration in patients carrying compound heterozygous variants in CLN7/MFS8D and presenting without the typical extraocular symptoms of vLINCL is characterized by an initial loss of cone photoreceptor cells,23 retinal degeneration in Cln7 KO mice is characterized by a preferential degeneration of rod photoreceptor cells. More detailed information about the progression of retinal degeneration in patients with CLN7 with vLINCL phenotype are required to judge whether or not these apparently discrepant findings indicate species-specific differences in the impact of CLN7 dysfunction on retinal morphology. 
Although visual impairment is not the initial neurologic symptom in most patients with CLN7 with the vLINCL phenotype, and patients with CLN7 carrying the missense mutation p.Tyr121Cys have been reported to display no signs of vision loss,20 visual deterioration is among the cardinal symptoms of CLN7 disease. In fact, approximately 90% of patients with CLN7 develop visual failure during the course of the disease.7,16,18,19,21,45,51 The morphologic and biochemical alterations in the retina of patients with CLN7 are, however, largely unknown and limited to morphologic data obtained from postmortem tissues of patients with vLINCL phenotype at the end stage of the disease.15,22 Morphologic analyses of retinas from two patients with CLN7 aged 7 and 9 years and carrying the missense mutation p.Thr294Lys revealed a progressive retinal degeneration, with a complete loss of photoreceptor cells and a marked reduction of neuron numbers in the inner nuclear layer and the ganglion cell layer.22 Similarly, another morphologic analysis of the retina from a patient with CLN7 carrying the intronic c.754+2T>A mutation also showed a marked neuronal degeneration in all retinal layers with maximal neuronal loss in the ganglion cell layer.15 Different from the observations in these patients with CLN722 and a CLN7 canine model,46 we found no evidence for a neuronal depletion of the inner nuclear layer and the ganglion cell layer of Cln7 KO mice up to the age of 4 months. However, accumulation of storage material and elevated expression levels of various lysosomal proteins in CLN7-deficient retinas were particularly evident in the ganglion cell layer, indicative of a major lysosomal dysfunction in retinal ganglion cells. It thus remains to be seen whether degeneration of the ganglion cell layer occurs in Cln7 KO mice older than 4 months. In fact, neuronal loss in the ganglion cell layer was detected at end stages of the disease both in patients with CLN722 and the CLN7 canine model.46 
In summary, this study provides the first comprehensive characterization of the retinal phenotype of a mouse model of CLN7 disease, vLINCL phenotype. Our results identify an early onset and rapidly progressing degeneration of rod photoreceptor cells as a major neurologic phenotype in CLN7-deficient mice. Retinal degeneration is accompanied by reactive astrogliosis and microgliosis, a marked dysregulation of various lysosomal proteins, an accumulation of SCMAS and saposin D, and the presence of cytoplasmic inclusions resembling curvilinear bodies and fingerprint-like profiles in various retinal cell types. We suggest that Cln7 KO mice provide a useful animal model to study the pathomechanisms of CLN7 disease, ultimately leading to neurodegeneration, and to evaluate the efficacy of therapeutic strategies aimed at developing treatments for this rare and fatal lysosomal storage disorder. 
Acknowledgments
The authors thank Irm Hermans-Borgmeyer (University Medical Center Hamburg-Eppendorf, Germany) for providing the C57BL/6J Flp- and Cre-deleter mouse strains, Elizabeth Neufeld (University of California, Los Angeles, CA, USA) for the SCMAS antibody, Konrad Sandhoff (University of Bonn, Bonn, Germany) for the saposin D antibody, Thomas Braulke (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for the M6P and CTSD antibodies, and Vasyl Druchkiv for help with statistical analyses. The authors are also grateful to Elke Becker, Sabine Helbing, and Stefanie Schlichting for excellent technical assistance, and Ali Derin, Susanne Conrad, and Sonja Kaphingst for animal care. 
Supported by grants of the Deutsche Forschungsgemeinschaft (DFG Grant 761/STO3–1, GRK 1459). 
Disclosure: W. Jankowiak, None; L. Brandenstein, None; S. Dulz, None; C. Hagel, None; S. Storch, None; U. Bartsch, None 
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Figure 1
 
Degeneration of photoreceptor cells and reactive astrogliosis in Cln7 KO mice. The outer nuclear layer of 0.5 month old wild-type (a) and Cln7 KO mice (b) was similar in thickness, and expression of GFAP in both genotypes was restricted to retinal astrocytes. In 1-month-old mutant mice (d), the outer nuclear layer was significantly reduced in thickness when compared with age-matched control mice (c), and expression of GFAP was elevated in retinal astrocytes and became additionally detectable in Müller cells (d). With increasing age of the mutant mice, the photoreceptor layer further decreased in thickness, and retinal degeneration was accompanied by reactive astrogliosis (see [f] and [h] for 2- and 4-month-old Cln7 KO mice, respectively). All sections were stained with DAPI to visualize cell nuclei. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; WT, wild-type. Scale bar in (h) (for ah): 100 μm.
Figure 1
 
Degeneration of photoreceptor cells and reactive astrogliosis in Cln7 KO mice. The outer nuclear layer of 0.5 month old wild-type (a) and Cln7 KO mice (b) was similar in thickness, and expression of GFAP in both genotypes was restricted to retinal astrocytes. In 1-month-old mutant mice (d), the outer nuclear layer was significantly reduced in thickness when compared with age-matched control mice (c), and expression of GFAP was elevated in retinal astrocytes and became additionally detectable in Müller cells (d). With increasing age of the mutant mice, the photoreceptor layer further decreased in thickness, and retinal degeneration was accompanied by reactive astrogliosis (see [f] and [h] for 2- and 4-month-old Cln7 KO mice, respectively). All sections were stained with DAPI to visualize cell nuclei. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; WT, wild-type. Scale bar in (h) (for ah): 100 μm.
Figure 2
 
Thickness of the retina and outer nuclear layer in Cln7 KO and wild-type mice at different developmental ages. The thickness of the retina (A) and outer nuclear layer (B) was determined in central retina sections at nine equidistant positions of the nasal and temporal retina, respectively. Retina and outer nuclear thickness was similar in 0.5-month-old wild-type and age-matched Cln7 KO mice. In older Cln7 KO mice, the thickness of the retina and outer nuclear layer was significantly reduced when compared with wild-type mice, and progressively decreased with increasing age of the mutant mice. The strongest reduction in the thickness of retina and photoreceptor layer of mutant mice was observed between the first and second postnatal month. Blue symbols represent the mean values (± SEM) of 18 wild-type mice aged 1 (n = 6), 2 (n = 6), and 4 (n = 6) months. All other symbols represent the mean values (± SEM) of six retinas for each genotype and developmental age. *P < 0.05; **P < 0.01; ***P < 0.001 according to the 2-way ANOVA test followed by a Bonferroni post hoc test.
Figure 2
 
Thickness of the retina and outer nuclear layer in Cln7 KO and wild-type mice at different developmental ages. The thickness of the retina (A) and outer nuclear layer (B) was determined in central retina sections at nine equidistant positions of the nasal and temporal retina, respectively. Retina and outer nuclear thickness was similar in 0.5-month-old wild-type and age-matched Cln7 KO mice. In older Cln7 KO mice, the thickness of the retina and outer nuclear layer was significantly reduced when compared with wild-type mice, and progressively decreased with increasing age of the mutant mice. The strongest reduction in the thickness of retina and photoreceptor layer of mutant mice was observed between the first and second postnatal month. Blue symbols represent the mean values (± SEM) of 18 wild-type mice aged 1 (n = 6), 2 (n = 6), and 4 (n = 6) months. All other symbols represent the mean values (± SEM) of six retinas for each genotype and developmental age. *P < 0.05; **P < 0.01; ***P < 0.001 according to the 2-way ANOVA test followed by a Bonferroni post hoc test.
Figure 3
 
Photoreceptor numbers in Cln7 KO and wild-type mice at different developmental ages. Retinas from 0.5-month-old wild-type (gray bars) and Cln7 KO mice (black bars) contained similar numbers of photoreceptor cells. In 1-month-old Cln7 KO retinas, in comparison, the number of photoreceptor cells was significantly reduced when compared with wild-type retinas, and further decreased with increasing age of the mutant mice. Each bar represents the mean value (± SEM) of six animals. n.s., not significant. **P < 0.01; ***P < 0.001 according to the Student's t-test.
Figure 3
 
Photoreceptor numbers in Cln7 KO and wild-type mice at different developmental ages. Retinas from 0.5-month-old wild-type (gray bars) and Cln7 KO mice (black bars) contained similar numbers of photoreceptor cells. In 1-month-old Cln7 KO retinas, in comparison, the number of photoreceptor cells was significantly reduced when compared with wild-type retinas, and further decreased with increasing age of the mutant mice. Each bar represents the mean value (± SEM) of six animals. n.s., not significant. **P < 0.01; ***P < 0.001 according to the Student's t-test.
Figure 4
 
Preferential loss of rod photoreceptor cells in Cln7 KO mice. Central sections of CLN7-deficient and wild-type retinas were stained with anti-recoverin antibodies to label all photoreceptor cells (ac), anti-rhodopsin antibodies to label rod photoreceptor cells (df), or PNA to label cone photoreceptor cells (gi). Note the similar number of cone photoreceptor cells in 1- (h) or 4-month-old (i) Cln7 KO retinas and 2-month-old wild-type retinas (g). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (i) (for ai): 100 μm.
Figure 4
 
Preferential loss of rod photoreceptor cells in Cln7 KO mice. Central sections of CLN7-deficient and wild-type retinas were stained with anti-recoverin antibodies to label all photoreceptor cells (ac), anti-rhodopsin antibodies to label rod photoreceptor cells (df), or PNA to label cone photoreceptor cells (gi). Note the similar number of cone photoreceptor cells in 1- (h) or 4-month-old (i) Cln7 KO retinas and 2-month-old wild-type retinas (g). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (i) (for ai): 100 μm.
Figure 5
 
Reactive microgliosis in the retina of Cln7 KO mice. In 1- (a) and 4-month-old (c) wild-type mice, IBA1-positive cells were present in the ganglion cell layer, inner plexiform layer, and outer plexiform layer. In 1- (b) and 4-month-old Cln7 KO mice (d), the number of IBA1-positive microglia cells was increased when compared to age-matched control animals, and became additionally detectable in the subretinal space (arrowheads in [d]). Cluster of differentiation 68–positive cells were not detectable in retinas of 1- (e) and 4-month-old wild-type mice (g). In 1- (f) and 4-month-old (h) mutant retinas, in comparison, CD68-positive cells were present in the outer retina (i.e., outer plexiform layer, outer nuclear layer, and subretinal space), and their number increased with increasing age of the mutants (compare [f] and [h]). Note the presence of numerous CD68-positive cells in the subretinal space of 4-month-old Cln7 KO retinas (arrowheads in [h]). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (d) (for ad): 100 μm; in (h) (for eh): 50 μm.
Figure 5
 
Reactive microgliosis in the retina of Cln7 KO mice. In 1- (a) and 4-month-old (c) wild-type mice, IBA1-positive cells were present in the ganglion cell layer, inner plexiform layer, and outer plexiform layer. In 1- (b) and 4-month-old Cln7 KO mice (d), the number of IBA1-positive microglia cells was increased when compared to age-matched control animals, and became additionally detectable in the subretinal space (arrowheads in [d]). Cluster of differentiation 68–positive cells were not detectable in retinas of 1- (e) and 4-month-old wild-type mice (g). In 1- (f) and 4-month-old (h) mutant retinas, in comparison, CD68-positive cells were present in the outer retina (i.e., outer plexiform layer, outer nuclear layer, and subretinal space), and their number increased with increasing age of the mutants (compare [f] and [h]). Note the presence of numerous CD68-positive cells in the subretinal space of 4-month-old Cln7 KO retinas (arrowheads in [h]). All sections were stained with DAPI to visualize cell nuclei. Scale bar in (d) (for ad): 100 μm; in (h) (for eh): 50 μm.
Figure 6
 
Ultrastructure of storage material in the retina of Cln7 KO mice. Low-power electron micrographs depict the different layers of a 2-month-old wild-type (a, b) and age-matched Cln7 KO retina (i, j). Analyses of the boxed areas in (i) and (j) at higher magnification revealed the presence of storage material in photoreceptor cells (k), interneurons (l), and ganglion cells (m) of mutant mice. Further analyses at higher magnification identified the storage material in Cln7 KO retinas as curvilinear bodies in photoreceptor cells (n) and interneurons (o), and fingerprint-like profiles in ganglion cells (p). Similar pathologic cytoplasmic inclusions were not detectable in retinas of age-matched control mice (ch). (fh) and (np) are higher magnifications of the boxed areas in (ce) and (km), respectively. Scale bar in (j) (for a, b, i, j): 5 μm; in (m) (for ce and km): 1 μm; in (p) (for fh and np): 0.2 μm.
Figure 6
 
Ultrastructure of storage material in the retina of Cln7 KO mice. Low-power electron micrographs depict the different layers of a 2-month-old wild-type (a, b) and age-matched Cln7 KO retina (i, j). Analyses of the boxed areas in (i) and (j) at higher magnification revealed the presence of storage material in photoreceptor cells (k), interneurons (l), and ganglion cells (m) of mutant mice. Further analyses at higher magnification identified the storage material in Cln7 KO retinas as curvilinear bodies in photoreceptor cells (n) and interneurons (o), and fingerprint-like profiles in ganglion cells (p). Similar pathologic cytoplasmic inclusions were not detectable in retinas of age-matched control mice (ch). (fh) and (np) are higher magnifications of the boxed areas in (ce) and (km), respectively. Scale bar in (j) (for a, b, i, j): 5 μm; in (m) (for ce and km): 1 μm; in (p) (for fh and np): 0.2 μm.
Figure 7
 
Elevated amounts of SCMAS and saposin D in the retina of CLN7-deficient retinas. (A) Immunohistochemistry revealed increased amounts of SCMAS in retinas of 1- (b) and 4-month-old (d) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, c, respectively). Elevated SCMAS-immunoreactivity was particularly evident in the inner nuclear layer and outer plexiform layer of mutant mice (b, d). Expression levels of saposin D were slightly elevated in 1-month-old mutant retinas (f), and significantly increased in 4-month-old mutant retinas (h) when compared with age-matched control retinas (e, g, respectively). In 4-month-old Cln7 KO retinas, strongest saposin D-immunoreactivity was found in the ganglion cell layer and inner and outer plexiform layer. All sections were stained with DAPI to visualize cell nuclei. (B) Immunoblot analyses of detergent-insoluble fractions revealed the accumulation of SCMAS and saposin D and increased expression of prosaposin in 2-month-old Cln7 KO retinas when compared with age-matched wild-type retinas. Loading of gels was controlled by Ponceau S staining or α-tubulin immunoblotting. The immunoblots shown are representative examples of three independent experiments. Scale bar in (h) (for ah): 100 μm.
Figure 7
 
Elevated amounts of SCMAS and saposin D in the retina of CLN7-deficient retinas. (A) Immunohistochemistry revealed increased amounts of SCMAS in retinas of 1- (b) and 4-month-old (d) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, c, respectively). Elevated SCMAS-immunoreactivity was particularly evident in the inner nuclear layer and outer plexiform layer of mutant mice (b, d). Expression levels of saposin D were slightly elevated in 1-month-old mutant retinas (f), and significantly increased in 4-month-old mutant retinas (h) when compared with age-matched control retinas (e, g, respectively). In 4-month-old Cln7 KO retinas, strongest saposin D-immunoreactivity was found in the ganglion cell layer and inner and outer plexiform layer. All sections were stained with DAPI to visualize cell nuclei. (B) Immunoblot analyses of detergent-insoluble fractions revealed the accumulation of SCMAS and saposin D and increased expression of prosaposin in 2-month-old Cln7 KO retinas when compared with age-matched wild-type retinas. Loading of gels was controlled by Ponceau S staining or α-tubulin immunoblotting. The immunoblots shown are representative examples of three independent experiments. Scale bar in (h) (for ah): 100 μm.
Figure 8
 
Dysregulation of various lysosomal proteins in the retina of Cln7 KO mice. Immunohistochemical analyses revealed elevated expression levels of M6P-containing soluble lysosomal proteins (ad), LAMP1 (eh), CTSD (il), and CTSZ (mp) in retinas of 1- (b, f, j, n) and 4-month-old (d, h, l, p) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, e, i, m, and c, g, k, o, respectively). At both developmental ages, elevated expression levels of the different lysosomal proteins were particularly evident in the ganglion cell layer and at the basal and apical margin of the inner nuclear layer of CLN7-deficient retinas. All sections were stained with DAPI to visualize cell nuclei. Scale bar in (p) (for ap): 100 μm.
Figure 8
 
Dysregulation of various lysosomal proteins in the retina of Cln7 KO mice. Immunohistochemical analyses revealed elevated expression levels of M6P-containing soluble lysosomal proteins (ad), LAMP1 (eh), CTSD (il), and CTSZ (mp) in retinas of 1- (b, f, j, n) and 4-month-old (d, h, l, p) Cln7 KO mice when compared with retinas of age-matched wild-type mice (a, e, i, m, and c, g, k, o, respectively). At both developmental ages, elevated expression levels of the different lysosomal proteins were particularly evident in the ganglion cell layer and at the basal and apical margin of the inner nuclear layer of CLN7-deficient retinas. All sections were stained with DAPI to visualize cell nuclei. Scale bar in (p) (for ap): 100 μm.
Figure 9
 
Lysosomal dysfunction in the retina of Cln7 KO mice. (A) Quantitative real-time PCR revealed increased Ctsd and Ctsz mRNA expression levels in 2-month-old Cln7 KO retinas (black bars) when compared with age-matched wild-type retinas (gray bars). Ctsd and Ctsz mRNA levels were normalized to β-actin mRNA levels, and values of wild-type mice were arbitrarily set to 1. Bars represent mean values (±SD) from three independent experiments. Statistical analysis of data was performed with the Student's t-test (*P < 0.05; **P < 0.01). (B) Immunoblot analyses of lysosomal/mitochondrial membrane extracts from 2-month-old CLN7-deficient and age-matched wild-type retinas revealed elevated amounts of LAMP1 in Cln7 KO mice. Western blotting of total protein homogenates showed increased expression of CTSD and CTSZ in the mutant. α-tubulin or GAPDH Western blotting was performed to control loading.
Figure 9
 
Lysosomal dysfunction in the retina of Cln7 KO mice. (A) Quantitative real-time PCR revealed increased Ctsd and Ctsz mRNA expression levels in 2-month-old Cln7 KO retinas (black bars) when compared with age-matched wild-type retinas (gray bars). Ctsd and Ctsz mRNA levels were normalized to β-actin mRNA levels, and values of wild-type mice were arbitrarily set to 1. Bars represent mean values (±SD) from three independent experiments. Statistical analysis of data was performed with the Student's t-test (*P < 0.05; **P < 0.01). (B) Immunoblot analyses of lysosomal/mitochondrial membrane extracts from 2-month-old CLN7-deficient and age-matched wild-type retinas revealed elevated amounts of LAMP1 in Cln7 KO mice. Western blotting of total protein homogenates showed increased expression of CTSD and CTSZ in the mutant. α-tubulin or GAPDH Western blotting was performed to control loading.
Table
 
Primary Antibodies
Table
 
Primary Antibodies
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