October 2013
Volume 54, Issue 10
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Retinal Cell Biology  |   October 2013
Apoptotic Photoreceptor Loss and Altered Expression of Lysosomal Proteins in the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis
Author Affiliations & Notes
  • Udo Bartsch
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Giovanna Galliciotti
    Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Guillermo F. Jofre
    Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Wanda Jankowiak
    Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Christian Hagel
    Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Thomas Braulke
    Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  • Correspondence: Thomas Braulke, Children's Hospital, Department of Biochemistry, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; braulke@uke.uni-hamburg.de
  • Udo Bartsch, Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; ubartsch@uke.uni-hamburg.de
Investigative Ophthalmology & Visual Science October 2013, Vol.54, 6952-6959. doi:10.1167/iovs.13-12945
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      Udo Bartsch, Giovanna Galliciotti, Guillermo F. Jofre, Wanda Jankowiak, Christian Hagel, Thomas Braulke; Apoptotic Photoreceptor Loss and Altered Expression of Lysosomal Proteins in the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis. Invest. Ophthalmol. Vis. Sci. 2013;54(10):6952-6959. doi: 10.1167/iovs.13-12945.

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

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Abstract

Purpose.: Mutations in the CLN6 gene cause variant late-infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder clinically characterized by progressive loss of vision, dementia, seizures, and early death. Here, we analyzed the time course of photoreceptor loss and the role of lysosomes in nclf mice, an animal model of the human CLN6 disease.

Methods.: Labeling of apoptotic cells, activated astrocytes, and Müller cells, and expression analyses of glial fibrillary acidic protein, rhodopsin, and lysosomal proteins were performed on nclf mice during the course of retinal degeneration. In addition, the distribution and variability of storage material was examined at the ultrastructural level.

Results.: Progressive apoptotic loss of photoreceptor cells was observed in nclf mice, resulting in reduction of the outer nuclear layer to approximately 3 rows of photoreceptor cells at 9 months of age. Onset of reactive gliosis was observed in 1-month-old nclf mice. Ultrastructural analysis revealed lysosomal storage material containing curvilinear and fingerprint-like inclusions in various retinal cell types. Expression levels of soluble mannose 6-phosphate–containing lysosomal enzymes, such as cathepsin D and the lysosomal membrane protein Lamp1, were increased in retinal cells of nclf mice.

Conclusions.: Accumulation of heterogeneous nondegraded macromolecules in dysfunctional lysosomes and autolysosomes impairs photoreceptor cells, ultimately leading to early-onset apoptotic death with subsequent activation of astrocytes and Müller cells in the retina of nclf mice. The defined steps of photoreceptor degeneration suggest that nclf mice might serve as an ideal animal model for experimental therapeutic approaches aimed at attenuating vision loss in neuronal ceroid lipofuscinosis.

Introduction
Neuronal ceroid lipofuscinoses (NCLs) comprise a group of inherited progressive neurodegenerative lysosomal storage disorders of childhood with an incidence of 1:12,500 in the United States. Although the onset of clinical symptoms ranges from prenatal/perinatal, infantile and juvenile to adult, the clinical features of dementia, neuronal and motor deterioration, seizures, and visual failure that lead to blindness and eventually to early death, are common to all NCLs. 1 Thirteen genetically distinct NCL variants have been identified affecting soluble lysosomal proteins (palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, CLN5, cathepsin D and F) modified with mannose 6-phosphate (M6P) residues, lysosomal membrane proteins (CLN3, CLN7, ATP13A2, ClC-7), membrane proteins of the endoplasmic reticulum (ER; CLN6 and CLN8), a secreted protein (progranulin), and the cysteine string protein alpha on synaptic vesicles. 2 The precise function of all these NCL proteins is currently unknown. Ultrastructurally, NCL disorders are characterized by lysosomal accumulation of heterogeneous storage material appearing in curvilinear, fingerprint, or rectilinear profiles or granular osmiophilic deposits. Predominant components of the storage material comprise autofluorescent lipopigments, aggregated proteins, such as subunit c of mitochondrial ATP synthase or sphingolipid activator proteins, cholesterol, sphingolipids, bis(monoacylglycero)phosphate, and dolichol. 35  
The variant late infantile NCL and a few adult NCL forms are caused by mutations in the CLN6 gene encoding a 305–amino acid polytopic ER membrane protein of unknown function. 69 The phenotype of naturally occurring animal models of the CLN6 disease, such as the South Hampshire or Merino sheep and the nclf mouse, closely resemble the human disease, and thus facilitate a more detailed investigation of this disorder. 1013 The nclf mutant mouse displays progressive brain atrophy, neuronal and glial accumulation of lipopigments, increased amounts of electron-dense bodies with rectilinear and fingerprint profiles, and impaired constitutive autophagy culminating in death between 10 and 12 months of age. 14 In addition, photoreceptor cell loss leading to blindness has been reported in nclf mice. 12 At present, it is unclear how mutant NCL proteins affect degradative functions in the lumen of lysosomes catalyzed by the concerted action of more than 60 lysosomal enzymes and accessory proteins. 
In this study, we have examined the effect of mutant CLN6 on photoreceptor and glial cells and the expression levels of lysosomal proteins during the course of retinal degeneration using the nclf mouse as an animal model of variant late infantile NCL. 
Methods
Animals
Mutant mice harboring the c.307insC frameshift mutation in the CLN6 gene (B6.Cg-Cln6 nclf /J; in the following termed nclf mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained on a C57BL/6J genetic background, and housed according to the institutional guidelines of the University Medical Center Hamburg-Eppendorf, with ad libitum access to food and water. The genotype of animals was determined by PCR analysis of DNA from tail biopsies and subsequent sequencing of the PCR product as described elsewhere. 6 In all experiments, age-matched C57BL/6J wild-type mice served as a control. 
Antibodies
M6P-containing proteins were detected by the myc-tagged single-chain antibody fragment scFv M6P-1. 15 Polyclonal antibodies against purified mouse cathepsin D (Ctsd) were raised in rabbits. 16 Polyclonal rabbit anti-glial fibrillary acidic protein (GFAP) antibodies were purchased from DAKO (Glostrup, Denmark), monoclonal mouse anti-rhodopsin antibodies and polyclonal rabbit anti–manganese-dependent superoxide dismutase (MnSOD) from Millipore (Billerica, MA), monoclonal anti-c-myc antibodies (9E10) from Sigma-Aldrich (St. Louis, MO) and polyclonal rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (Gapdh) from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal rat antibody 1D4B against murine lysosomal-associated membrane protein 1 (Lamp1) was obtained from the National Institute of Child Health and Human Development Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). Horseradish peroxidase (HRP)- and Cy3-conjugated goat anti-rabbit, goat anti-mouse, or goat anti-rat IgG were used as secondary antibodies (Jackson ImmunoResearch, West Grove, PA). 
Immunohistochemistry, Photoreceptor Counts, and TUNEL Staining
For immunohistochemical analysis, nclf and age-matched wild-type mice were killed by cervical dislocation; eyes were quickly removed and immersion-fixed in 4% paraformaldehyde (PA) in PBS (pH 7.4) overnight at room temperature. Eyes were cryoprotected in an ascending series of sucrose (7.5%, 15%, and 30%), embedded in Tissue-Tek (Sakura Finetek, Zouterwoude, The Netherlands), frozen, and serially sectioned with a cryostat at a thickness of 25 μm. Cryostat sections of the central retina (i.e., in the plane of the optic disc) were blocked in PBS containing 0.1% bovine serum albumin (Sigma) and 0.3% Triton X-100 (Sigma), and incubated with primary antibodies overnight at room temperature. The following primary and secondary antibodies were used: anti-GFAP (1:500), anti-Ctsd (1:4000), and myc-tagged scFv M6P-1 antibody fragments (1:4000), anti-myc (1:200), and Cy3-conjugated IgG (1:200). All sections were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma) to visualize cell nuclei. After washing with PBS, sections were mounted onto slides and analyzed with an Olympus FV 1000 confocal microscope (Olympus, Hamburg, Germany). For comparison of expression levels, retina sections from both genotypes and all different developmental ages were processed in parallel, and central retina regions (i.e., close to the optic disc) were documented using identical microscope settings. Depending on the antigen, between 4 and 8 animals were analyzed for each genotype and age. 
The number of rows of photoreceptor nuclei was determined in 0.5-, 1-, 4-, and 9-month-old nclf and age-matched wild-type mice (n = 6 for each genotype and age). Photoreceptor rows were counted in confocal images taken from DAPI-stained retinal sections 300 μm apart from the optic disc at a final magnification of ×1200. Statistical analysis of data was performed using the Student's t-test. 
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed to visualize apoptotic cells in retinas from 1- and 4-month-old nclf and wild-type mice (n = 4 for each genotype and age) using the In Situ Cell Death detection Kit, Fluorescein (Roche, Indianapolis, IN). Unfixed eyes were embedded in Tissue-Tek, and cryostat sections from the central retina of 14 μm thickness were mounted onto SuperFrost/Plus slides (Menzel-Glaeser, Braunschweig, Germany). Sections were dried overnight, fixed in 4% PA in PBS for 20 minutes at room temperature, and incubated in PBS containing 0.1% Triton X-100 and 0.1% sodium citrate at 4°C for 2 minutes. Labeling of apoptotic cells and negative control experiments were performed according to the manufacturer's instructions. Sections were mounted onto slides, and retina regions located close to the optic disc were analyzed with an Olympus FV 1000 confocal microscope. 
Electron Microscopy
Retinas from nclf and age-matched wild-type mice (n = 4 for each genotype) were fixed by immersion in 0.36% glutaraldehyde (Serva, Heidelberg, Germany) for 2 hours at room temperature. After washing in 0.1-M cacodylate buffer (Sigma-Aldrich, Seelze, Germany), retinas were incubated in 1% osmium tetroxide (Science Services, Munich, Germany) for 2 hours at room temperature, dehydrated in an ascending series of ethanol, and embedded in Epon 812 (Serva). Semithin sections were stained with toluidine blue, and relevant specimens were further processed for electron microscopic analysis. Ultrathin sections were counterstained with uranyl acetate (Merck, Darmstadt, Germany) and lead citrate (Sigma-Aldrich), and examined with an LEO 912 AB OMEGA electron microscope (Leo Elektronenmikroskopie, Oberkochen, Germany). 
Immunoblot Analysis and Cathepsin D Activity Measurements
For Western blotting, the retinas of one mouse were homogenized in 10 volumes of 0.32-M sucrose in 10 mM HEPES, pH 7.4, containing 0.02% Triton X-100, and protease inhibitors (Sigma), and incubated on ice for 30 minutes. The lysates were centrifuged at 14,000g for 10 minutes. Aliquots of the supernatant (30 μg protein) were solubilized under reducing conditions and subjected to SDS-PAGE (10% acrylamide) followed by electrotransfer to nitrocellulose membranes for 60 minutes at 400 mA. After blocking, membranes were incubated with primary antibodies to GFAP (1:500), rhodopsin (1:500), cathepsin D (1:2000), Lamp1 (1:250), MnSOD (1:1000), or Gapdh (1:1000) for 16 hours at 4°C followed by HRP-conjugated secondary antibodies (1:5000). The immunoreactive bands were visualized by enhanced chemiluminescence and the ChemiDoc XRS system (Bio-Rad, Munich, Germany). Expression of GFAP and rhodopsin was analyzed in three, expression of cathepsin D in two, and expression of Lamp1 in four animals for each age (i.e., 3, 6, and 11 months) and genotype. Cathepsin D activity was measured in three animals for each age (i.e., 3, 6, and 11 months) and genotype using 6-([7-amino-4methylcoumarin-3-acetyl]amino)hexoid acid coupled to hemoglobin (AMCA-Hb) as a substrate as described elsewhere. 17  
Results
Comparison of DAPI-stained retina sections from 2-week-old nclf and wild-type mice revealed no obvious differences in the thickness of the outer nuclear layer between genotypes. The thickness of the photoreceptor cell layer was slightly decreased in 1-month-old nclf mice, and massively reduced in 4-month-old mutants (Fig. 1A). In 9-month-old nclf mice, the outer nuclear layer was reduced to approximately 3 rows of photoreceptor cell nuclei, as opposed to approximately 11 rows found in age-matched wild-type mice (Figs. 1A, 1B). These data were confirmed by Western blotting showing a progressive decrease in rhodopsin expression to almost undetectable levels in 11-month-old nclf mice (Fig. 1C). 
Figure 1
 
Photoreceptor loss and rhodopsin and GFAP immunoblot analysis of nclf retinas from different developmental ages. (A) The thickness of the outer nuclear layer (onl) of 0.5-month-old nclf mice (b) in DAPI-stained sections was similar to that of 1-month-old wild-type mice (a). In 1-month-old mutants (c), the onl was slightly atrophied when compared with wild-type mice. Significant and massive loss of photoreceptor cells was detectable in 4- (d) and 9- (e) month-old nclf retinas, respectively. (B) Although the number of rows of photoreceptor cells in the central retina was similar in 0.5-month-old nclf (filled bars) and age-matched wild-type (open bars) mice, it significantly decreased with increasing age of the mutant. In 9-month-old nclf mice, the onl was composed of approximately 3 rows of photoreceptor cells as opposed to approximately 11 rows of photoreceptors in age-matched wild-type retinas. (C) Levels of GFAP were significantly increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type retinas, whereas levels of rhodopsin decreased with increasing age of mutant mice to undetectable amounts in 11-month-old mutant mice. MnSOD was used as loading control. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; n.s., not significant; opl, outer plexiform layer; wt, wild-type. **P < 0.01; ***P < 0.001. Scale bar in (e) (for [ae]): 100 μm.
Figure 1
 
Photoreceptor loss and rhodopsin and GFAP immunoblot analysis of nclf retinas from different developmental ages. (A) The thickness of the outer nuclear layer (onl) of 0.5-month-old nclf mice (b) in DAPI-stained sections was similar to that of 1-month-old wild-type mice (a). In 1-month-old mutants (c), the onl was slightly atrophied when compared with wild-type mice. Significant and massive loss of photoreceptor cells was detectable in 4- (d) and 9- (e) month-old nclf retinas, respectively. (B) Although the number of rows of photoreceptor cells in the central retina was similar in 0.5-month-old nclf (filled bars) and age-matched wild-type (open bars) mice, it significantly decreased with increasing age of the mutant. In 9-month-old nclf mice, the onl was composed of approximately 3 rows of photoreceptor cells as opposed to approximately 11 rows of photoreceptors in age-matched wild-type retinas. (C) Levels of GFAP were significantly increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type retinas, whereas levels of rhodopsin decreased with increasing age of mutant mice to undetectable amounts in 11-month-old mutant mice. MnSOD was used as loading control. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; n.s., not significant; opl, outer plexiform layer; wt, wild-type. **P < 0.01; ***P < 0.001. Scale bar in (e) (for [ae]): 100 μm.
To determine the onset of retinal degeneration in nclf mice, we performed TUNEL of nclf and age-matched wild-type retinas at two developmental ages. TUNEL labeling revealed a significantly increased number of apoptotic cells in 1-month-old nclf retinas when compared with wild-type controls (Fig. 2). TUNEL-positive cells were still numerous in 4-month-old nclf mice, but hardly detectable in age-matched wild-type animals. At both developmental ages, labeled cells were exclusively found in the outer nuclear layer, indicative of progressive loss of photoreceptor cells in mutant mice (Fig. 2). 
Figure 2
 
Apoptotic cells in retinas of nclf and wild-type mice as revealed by TUNEL staining. In the onl of 1-month-old animals, the number of TUNEL-positive cells was significantly increased in nclf mice (c) when compared with wild-type mice (a). TUNEL-positive cells were still frequent in the onl of 4-month-old nclf retinas (g), but were essentially absent from age-matched wild-type retinas (e). Apoptotic cells were restricted to the onl and absent from other retinal layers (compare [a], [c], [e], and [g] with the corresponding differential interference contrast photomicrographs [b], [d], [f], and [h], respectively). Scale bar in (h) (for [ah]): 100 μm.
Figure 2
 
Apoptotic cells in retinas of nclf and wild-type mice as revealed by TUNEL staining. In the onl of 1-month-old animals, the number of TUNEL-positive cells was significantly increased in nclf mice (c) when compared with wild-type mice (a). TUNEL-positive cells were still frequent in the onl of 4-month-old nclf retinas (g), but were essentially absent from age-matched wild-type retinas (e). Apoptotic cells were restricted to the onl and absent from other retinal layers (compare [a], [c], [e], and [g] with the corresponding differential interference contrast photomicrographs [b], [d], [f], and [h], respectively). Scale bar in (h) (for [ah]): 100 μm.
GFAP-immunohistochemistry of central retinal sections of 2-week-old nclf and wild-type mice revealed exclusive labeling of retinal astrocytes located at the vitreal margin of the retina, without obvious differences in staining intensity between genotypes (Figs. 3A, 3B). In 1-month-old mutants, GFAP expression was strongly increased in retinal astrocytes and was now also detectable in the radially oriented Müller cell processes (Fig. 3D). Pronounced reactive gliosis in retinal astrocytes and Müller cells was also apparent in 4- and 9-month-old nclf mice (Figs. 3F, 3H). In line with these data, immunoblot analysis revealed elevated levels of GFAP in nclf mice aged between 3 and 11 months when compared with wild-type mice (Fig. 1C). 
Figure 3
 
Expression of GFAP in retinas of nclf mice at different developmental ages. In 0.5-month-old nclf (b) and age-matched wild-type mice (a), GFAP immunoreactivity was restricted to retinal astrocytes with no obvious differences in expression levels between genotypes. In retinas of 1- (d), 4- (f), and 9- (h) month-old nclf mice, expression of GFAP was strongly increased in retinal astrocytes when compared with age-matched wild-type retinas (see [c, e, g] for 1-, 4-, and 9-month-old wild-type mice, respectively) and was additionally detectable in Müller cells of mutant retinas. All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h) (for [ah]): 100 μm.
Figure 3
 
Expression of GFAP in retinas of nclf mice at different developmental ages. In 0.5-month-old nclf (b) and age-matched wild-type mice (a), GFAP immunoreactivity was restricted to retinal astrocytes with no obvious differences in expression levels between genotypes. In retinas of 1- (d), 4- (f), and 9- (h) month-old nclf mice, expression of GFAP was strongly increased in retinal astrocytes when compared with age-matched wild-type retinas (see [c, e, g] for 1-, 4-, and 9-month-old wild-type mice, respectively) and was additionally detectable in Müller cells of mutant retinas. All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h) (for [ah]): 100 μm.
Ultrastructural analysis of retinas from 5-month-old nclf mice revealed that the outer nuclear layer was reduced to approximately 6 rows of photoreceptor nuclei (Fig. 4A), confirming the light microscopic observations. Numerous intracellular cytoplasmic inclusions were observed in various retinal cell types throughout all retinal layers. At higher magnifications, photoreceptors and cells of the inner nuclear layer contained storage material of variable shape and with curvilinear inclusions, whereas storage bodies in retinal ganglion cells were densely packed with membranes reminiscent of fingerprint profiles (Fig. 4A). Similar pathological deposits were not detected in retinal cells of age-matched wild-type mice that were analyzed for comparison (Fig. 4B). 
Figure 4
 
Electron microscopic analysis of retinas from nclf (A) and wild-type (B) mice. Low-power magnification images depict the outer nuclear layer (onl in [a, a']), inner nuclear layer (inl in [a, a']), inner plexiform layer (ipl in [b, b']) and ganglion cell layer (gcl in [b, b']) of a 5-month-old nclf (A) and age-matched wild-type (B) mouse. Higher magnifications of the boxed areas in (a, b) demonstrated the presence of cytoplasmic storage material in photoreceptor cells (c), cells of the inner nuclear layer (d) and ganglion cells (e) of nclf mice (A). Further analysis of the boxed areas in (c, d, e) at higher magnification identified the storage material as curvilinear inclusions in photoreceptors (f) and cells of the inner nuclear layer (g), and as fingerprint profiles in ganglion cells (h). No pathological deposits were detected in retinal cells of age-matched wild-type mice (c'h'). Scale bars in (b') (for [a, a', b, b']): 10 μm; in (e') (for [ce, c'e']): 1 μm; in (h') (for [fh, f'h']): 0.2 μm.
Figure 4
 
Electron microscopic analysis of retinas from nclf (A) and wild-type (B) mice. Low-power magnification images depict the outer nuclear layer (onl in [a, a']), inner nuclear layer (inl in [a, a']), inner plexiform layer (ipl in [b, b']) and ganglion cell layer (gcl in [b, b']) of a 5-month-old nclf (A) and age-matched wild-type (B) mouse. Higher magnifications of the boxed areas in (a, b) demonstrated the presence of cytoplasmic storage material in photoreceptor cells (c), cells of the inner nuclear layer (d) and ganglion cells (e) of nclf mice (A). Further analysis of the boxed areas in (c, d, e) at higher magnification identified the storage material as curvilinear inclusions in photoreceptors (f) and cells of the inner nuclear layer (g), and as fingerprint profiles in ganglion cells (h). No pathological deposits were detected in retinal cells of age-matched wild-type mice (c'h'). Scale bars in (b') (for [a, a', b, b']): 10 μm; in (e') (for [ce, c'e']): 1 μm; in (h') (for [fh, f'h']): 0.2 μm.
To examine the impact of lysosomal dysfunction on the retinal dystrophy, the expression level and distribution of total lysosomal enzymes containing the M6P recognition marker was analyzed using a single-chain antibody against M6P residues. In 1- and 9-month-old wild-type mice and age-matched nclf mutants, M6P-containing proteins were similarly distributed and mainly expressed in the retinal ganglion cell layer, the lower and upper margin of the inner nuclear layer, and at the upper margin of the outer nuclear layer (Fig. 5). However, expression levels of M6P-containing proteins were significantly increased in nclf mice at both developmental stages when compared with wild-type mice. Similar to M6P, immunoreactivity of the lysosomal protease cathepsin D was most intense in the ganglion cell layer, the lower and upper margin of the inner nuclear layer, and the upper margin of the outer nuclear layer of both genotypes at 1 and 9 months of age, and was more intense in nclf retinas than in wild-type mice (Fig. 5). The immunohistochemical data were confirmed by elevated levels of cathepsin D expression as demonstrated by immunoblotting and a significant increase in cathepsin D activity in 3-, 6-, and 11-month-old nclf mice (Fig. 6). Finally, we observed increased levels of the lysosomal membrane protein Lamp1 in 6- and 11-month-old, but not in 3-month-old nclf retinas (Fig. 6). Together, data thus demonstrate a significant dysregulation of lysosomal proteins in the retina of nclf mice. 
Figure 5
 
Expression of M6P and cathepsin D in nclf and wild-type retinas. Expression levels of M6P were significantly increased in the ganglion cell layer (gcl), inner nuclear layer (inl) and outer nuclear layer (onl) of 1- (b, b') and 9- (d, d') month-old nclf mutant mice when compared with age-matched wild-type mice (see [a, a'] for 1-month-old and [c, c'] for 9-month-old wild-type mice). Expression levels of cathepsin D expression were similarly increased in a similar pattern in the ganglion cell layer, inner nuclear layer, and outer nuclear layer of 1- (f, f') and 9- (h, h') month-old nclf mice when compared with age-matched wild-type mice (see [e, e'] for 1-month-old and [g, g'] for 9-month-old wild-type mice). All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h') (for [ah']): 100 μm.
Figure 5
 
Expression of M6P and cathepsin D in nclf and wild-type retinas. Expression levels of M6P were significantly increased in the ganglion cell layer (gcl), inner nuclear layer (inl) and outer nuclear layer (onl) of 1- (b, b') and 9- (d, d') month-old nclf mutant mice when compared with age-matched wild-type mice (see [a, a'] for 1-month-old and [c, c'] for 9-month-old wild-type mice). Expression levels of cathepsin D expression were similarly increased in a similar pattern in the ganglion cell layer, inner nuclear layer, and outer nuclear layer of 1- (f, f') and 9- (h, h') month-old nclf mice when compared with age-matched wild-type mice (see [e, e'] for 1-month-old and [g, g'] for 9-month-old wild-type mice). All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h') (for [ah']): 100 μm.
Figure 6
 
Immunoblot analysis of cathepsin D and Lamp1 expression and cathepsin D activity in retinas from nclf and wild-type mice. (A) Immunoblot analysis demonstrates elevated levels of the lysosomal protease cathepsin D (Ctsd) in retinas of 3-, 6-, and 11-month-old nclf mice when compared with age-matched wild-type mice. Expression levels of the lysosomal membrane protein Lamp1 were also increased in 6- and 11-month-old mutant retinas when compared with control retinas. Equal loading was controlled by reprobing blots with anti-Gapdh antibodies. (B) Cathepsin D activity was increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type control retinas (a), in line with the elevated Ctsd levels seen in immunoblots (A). Control measurements performed in the presence of pepstatin A, an inhibitor of aspartic proteinases, revealed only residual enzyme activity (b).
Figure 6
 
Immunoblot analysis of cathepsin D and Lamp1 expression and cathepsin D activity in retinas from nclf and wild-type mice. (A) Immunoblot analysis demonstrates elevated levels of the lysosomal protease cathepsin D (Ctsd) in retinas of 3-, 6-, and 11-month-old nclf mice when compared with age-matched wild-type mice. Expression levels of the lysosomal membrane protein Lamp1 were also increased in 6- and 11-month-old mutant retinas when compared with control retinas. Equal loading was controlled by reprobing blots with anti-Gapdh antibodies. (B) Cathepsin D activity was increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type control retinas (a), in line with the elevated Ctsd levels seen in immunoblots (A). Control measurements performed in the presence of pepstatin A, an inhibitor of aspartic proteinases, revealed only residual enzyme activity (b).
Discussion
Because neurodegeneration is the leading symptom of variant late infantile NCL, pathological alterations in the brain have received most attention in previously performed studies on animal models of this condition. However, visual failure represents the first clinical symptom in affected children at the age of 3 to 6 years. 18 In analogy to the early visual impairment reported for human patients, we observed the first signs of retinal cell loss in the outer nuclear layer in 1-month-old nclf mice. Early-onset photoreceptor loss was accompanied by reactive gliosis of retinal astrocytes and Müller cells. In comparison, proteolipid storage material with rectilinear/fingerprint profiles in neurons and glia and regionally restricted astrogliosis in various brain regions of nclf mice does not become apparent before 5 to 6 months of age. 12,14 We also demonstrated that accumulation of heterogeneous storage material in lysosomes and autolysosomes of various retinal cell types is associated with a general increase in the expression of M6P-containing lysosomal proteins, particularly of the lysosomal protease cathepsin D. 
Rapidly progressing apoptotic loss of photoreceptor cells has been observed in a mouse model of congenital NCL (CLN10) caused by the deficiency of cathepsin D within 25 days after birth. 19 Furthermore, in the CLN3-deficient mouse, a model of juvenile NCL, apoptotic loss of photoreceptor cells becomes apparent by 18 months of age. 20 Photoreceptor degeneration has also been observed in various other mouse models of lysosomal storage diseases, such as CLN8, mucolipidosis type II, mucolipidosis type IV, or autosomal recessive osteopetrosis, 2125 whereas photoreceptor cells in mice or dogs resembling animal models of lysosomal disorders, such as CLN1, CLN2, or mucolipidosis type III, appeared unaffected. 2629 The frequent involvement of photoreceptor cells in these conditions suggests that these cells may exhibit a specific vulnerability for alterations in the lysosomal homeostasis and the subsequent metabolic imbalances. 
The elevated levels of M6P-containing proteins in the ganglion cell layer, inner nuclear layer, and outer nuclear layer of nclf mice are of particular interest in the context of the present study. M6P serves a specific recognition signal on newly synthesized lysosomal enzymes for binding to M6P receptors mediating the efficient segregation of these proteins from the secretory pathway and their targeting to lysosomes. 30 These data are confirmed by the increased expression, in particular of the mature enzymatically active form, and the activity of the lysosomal protease cathepsin D in the same cell layers of nclf mice in comparison with wild-type mice. Whether the increase in M6P-containing enzymes, including cathepsin D, represents an adaptive process regulated by transcriptional activation, or is the result of lysosomal dysfunction affecting the half-lives of lysosomal hydrolases, remains to be investigated. Elevated levels of abundant lysosomal membrane proteins, such as Lamp1, are considered as an indication of an increased number and/or size of lysosomes, 31 most likely as a result of the accumulation of nondegraded storage material. 
The molecular mechanisms leading to photoreceptor degeneration in the different NCL forms are unknown and only limited insights into the molecular mechanisms leading to neurodegeneration in the brain have been provided by studies on the nclf mouse and the OCLN6 South Hampshire sheep, two animal models of CLN6. Neither the expression of a truncated CLN6 protein, nor the extent and distribution of storage material, ER stress, or the activation of unfolded protein response correlate with the regional and temporal pattern of the neurodegenerative processes in these animal models of variant late infantile NCL. 14,32,33 Instead, the localized activation of astrocytes and the disruption of the constitutive autophagy-lysosome degradation pathway of long-lived or aggregate-prone proteins and organelles, such as mitochondria, appears to be more closely related to progressive neurodegeneration in CLN6. 14,34,35 However, neither the LC3-I/LC3-II ratio nor the expression of p62, marker proteins of the autophagic pathway and maturation, were significantly altered in the retina of nclf mice when compared with wild-type controls (G. Galliciotti, unpublished results, 2012). These data suggest that photoreceptor degeneration in these mice is unlikely related to an impaired autophagic pathway. Instead, the progressive loss of photoreceptor cells in nclf mice might be related to an impaired lysosomal degradation of rhodopsin. After light-dependent activation of the G-protein–coupled rhodopsin receptor, rhodopsin is endocytosed and undergoes regular lysosomal turnover. 36 In Drosophila, it has been shown that the perturbation of endocytosis of rhodopsin or its lysosomal delivery results in an accumulation of rhodopsin-arrestin complexes in endosomes, causing photoreceptor cell death by unknown mechanisms. 37 It is therefore tempting to speculate that dysfunctional lysosomes also lead to transient aggregation of similar rhodopsin-arrestin complexes in endosomes of nclf mice, ultimately triggering photoreceptor cell death in this mutant. 
Taken together, nclf mice are characterized by a progressive loss of photoreceptor cells, and thus display phenotypic similarities to human patients affected by variant late infantile NCL. Analysis of retinal degeneration in the nclf mouse allows a better understanding of the pathomechanism of the disease and provides objective biochemical and histologic parameters that can be used to assess the efficacy of novel therapeutic approaches aimed at attenuating visual impairment associated with variant late infantile NCL. 
Acknowledgments
Supported by Nächstenliebe e.V., DFG (FOR885), and the European Union Seventh Framework Programme (FP7/2007-2013) under Grant 281234. 
Disclosure: U. Bartsch, None; G. Galliciotti, None; G.F. Jofre, None; W. Jankowiak, None; C. Hagel, None; T. Braulke, None 
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Figure 1
 
Photoreceptor loss and rhodopsin and GFAP immunoblot analysis of nclf retinas from different developmental ages. (A) The thickness of the outer nuclear layer (onl) of 0.5-month-old nclf mice (b) in DAPI-stained sections was similar to that of 1-month-old wild-type mice (a). In 1-month-old mutants (c), the onl was slightly atrophied when compared with wild-type mice. Significant and massive loss of photoreceptor cells was detectable in 4- (d) and 9- (e) month-old nclf retinas, respectively. (B) Although the number of rows of photoreceptor cells in the central retina was similar in 0.5-month-old nclf (filled bars) and age-matched wild-type (open bars) mice, it significantly decreased with increasing age of the mutant. In 9-month-old nclf mice, the onl was composed of approximately 3 rows of photoreceptor cells as opposed to approximately 11 rows of photoreceptors in age-matched wild-type retinas. (C) Levels of GFAP were significantly increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type retinas, whereas levels of rhodopsin decreased with increasing age of mutant mice to undetectable amounts in 11-month-old mutant mice. MnSOD was used as loading control. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; n.s., not significant; opl, outer plexiform layer; wt, wild-type. **P < 0.01; ***P < 0.001. Scale bar in (e) (for [ae]): 100 μm.
Figure 1
 
Photoreceptor loss and rhodopsin and GFAP immunoblot analysis of nclf retinas from different developmental ages. (A) The thickness of the outer nuclear layer (onl) of 0.5-month-old nclf mice (b) in DAPI-stained sections was similar to that of 1-month-old wild-type mice (a). In 1-month-old mutants (c), the onl was slightly atrophied when compared with wild-type mice. Significant and massive loss of photoreceptor cells was detectable in 4- (d) and 9- (e) month-old nclf retinas, respectively. (B) Although the number of rows of photoreceptor cells in the central retina was similar in 0.5-month-old nclf (filled bars) and age-matched wild-type (open bars) mice, it significantly decreased with increasing age of the mutant. In 9-month-old nclf mice, the onl was composed of approximately 3 rows of photoreceptor cells as opposed to approximately 11 rows of photoreceptors in age-matched wild-type retinas. (C) Levels of GFAP were significantly increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type retinas, whereas levels of rhodopsin decreased with increasing age of mutant mice to undetectable amounts in 11-month-old mutant mice. MnSOD was used as loading control. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; n.s., not significant; opl, outer plexiform layer; wt, wild-type. **P < 0.01; ***P < 0.001. Scale bar in (e) (for [ae]): 100 μm.
Figure 2
 
Apoptotic cells in retinas of nclf and wild-type mice as revealed by TUNEL staining. In the onl of 1-month-old animals, the number of TUNEL-positive cells was significantly increased in nclf mice (c) when compared with wild-type mice (a). TUNEL-positive cells were still frequent in the onl of 4-month-old nclf retinas (g), but were essentially absent from age-matched wild-type retinas (e). Apoptotic cells were restricted to the onl and absent from other retinal layers (compare [a], [c], [e], and [g] with the corresponding differential interference contrast photomicrographs [b], [d], [f], and [h], respectively). Scale bar in (h) (for [ah]): 100 μm.
Figure 2
 
Apoptotic cells in retinas of nclf and wild-type mice as revealed by TUNEL staining. In the onl of 1-month-old animals, the number of TUNEL-positive cells was significantly increased in nclf mice (c) when compared with wild-type mice (a). TUNEL-positive cells were still frequent in the onl of 4-month-old nclf retinas (g), but were essentially absent from age-matched wild-type retinas (e). Apoptotic cells were restricted to the onl and absent from other retinal layers (compare [a], [c], [e], and [g] with the corresponding differential interference contrast photomicrographs [b], [d], [f], and [h], respectively). Scale bar in (h) (for [ah]): 100 μm.
Figure 3
 
Expression of GFAP in retinas of nclf mice at different developmental ages. In 0.5-month-old nclf (b) and age-matched wild-type mice (a), GFAP immunoreactivity was restricted to retinal astrocytes with no obvious differences in expression levels between genotypes. In retinas of 1- (d), 4- (f), and 9- (h) month-old nclf mice, expression of GFAP was strongly increased in retinal astrocytes when compared with age-matched wild-type retinas (see [c, e, g] for 1-, 4-, and 9-month-old wild-type mice, respectively) and was additionally detectable in Müller cells of mutant retinas. All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h) (for [ah]): 100 μm.
Figure 3
 
Expression of GFAP in retinas of nclf mice at different developmental ages. In 0.5-month-old nclf (b) and age-matched wild-type mice (a), GFAP immunoreactivity was restricted to retinal astrocytes with no obvious differences in expression levels between genotypes. In retinas of 1- (d), 4- (f), and 9- (h) month-old nclf mice, expression of GFAP was strongly increased in retinal astrocytes when compared with age-matched wild-type retinas (see [c, e, g] for 1-, 4-, and 9-month-old wild-type mice, respectively) and was additionally detectable in Müller cells of mutant retinas. All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h) (for [ah]): 100 μm.
Figure 4
 
Electron microscopic analysis of retinas from nclf (A) and wild-type (B) mice. Low-power magnification images depict the outer nuclear layer (onl in [a, a']), inner nuclear layer (inl in [a, a']), inner plexiform layer (ipl in [b, b']) and ganglion cell layer (gcl in [b, b']) of a 5-month-old nclf (A) and age-matched wild-type (B) mouse. Higher magnifications of the boxed areas in (a, b) demonstrated the presence of cytoplasmic storage material in photoreceptor cells (c), cells of the inner nuclear layer (d) and ganglion cells (e) of nclf mice (A). Further analysis of the boxed areas in (c, d, e) at higher magnification identified the storage material as curvilinear inclusions in photoreceptors (f) and cells of the inner nuclear layer (g), and as fingerprint profiles in ganglion cells (h). No pathological deposits were detected in retinal cells of age-matched wild-type mice (c'h'). Scale bars in (b') (for [a, a', b, b']): 10 μm; in (e') (for [ce, c'e']): 1 μm; in (h') (for [fh, f'h']): 0.2 μm.
Figure 4
 
Electron microscopic analysis of retinas from nclf (A) and wild-type (B) mice. Low-power magnification images depict the outer nuclear layer (onl in [a, a']), inner nuclear layer (inl in [a, a']), inner plexiform layer (ipl in [b, b']) and ganglion cell layer (gcl in [b, b']) of a 5-month-old nclf (A) and age-matched wild-type (B) mouse. Higher magnifications of the boxed areas in (a, b) demonstrated the presence of cytoplasmic storage material in photoreceptor cells (c), cells of the inner nuclear layer (d) and ganglion cells (e) of nclf mice (A). Further analysis of the boxed areas in (c, d, e) at higher magnification identified the storage material as curvilinear inclusions in photoreceptors (f) and cells of the inner nuclear layer (g), and as fingerprint profiles in ganglion cells (h). No pathological deposits were detected in retinal cells of age-matched wild-type mice (c'h'). Scale bars in (b') (for [a, a', b, b']): 10 μm; in (e') (for [ce, c'e']): 1 μm; in (h') (for [fh, f'h']): 0.2 μm.
Figure 5
 
Expression of M6P and cathepsin D in nclf and wild-type retinas. Expression levels of M6P were significantly increased in the ganglion cell layer (gcl), inner nuclear layer (inl) and outer nuclear layer (onl) of 1- (b, b') and 9- (d, d') month-old nclf mutant mice when compared with age-matched wild-type mice (see [a, a'] for 1-month-old and [c, c'] for 9-month-old wild-type mice). Expression levels of cathepsin D expression were similarly increased in a similar pattern in the ganglion cell layer, inner nuclear layer, and outer nuclear layer of 1- (f, f') and 9- (h, h') month-old nclf mice when compared with age-matched wild-type mice (see [e, e'] for 1-month-old and [g, g'] for 9-month-old wild-type mice). All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h') (for [ah']): 100 μm.
Figure 5
 
Expression of M6P and cathepsin D in nclf and wild-type retinas. Expression levels of M6P were significantly increased in the ganglion cell layer (gcl), inner nuclear layer (inl) and outer nuclear layer (onl) of 1- (b, b') and 9- (d, d') month-old nclf mutant mice when compared with age-matched wild-type mice (see [a, a'] for 1-month-old and [c, c'] for 9-month-old wild-type mice). Expression levels of cathepsin D expression were similarly increased in a similar pattern in the ganglion cell layer, inner nuclear layer, and outer nuclear layer of 1- (f, f') and 9- (h, h') month-old nclf mice when compared with age-matched wild-type mice (see [e, e'] for 1-month-old and [g, g'] for 9-month-old wild-type mice). All sections were counterstained with DAPI to visualize cell nuclei. Scale bar in (h') (for [ah']): 100 μm.
Figure 6
 
Immunoblot analysis of cathepsin D and Lamp1 expression and cathepsin D activity in retinas from nclf and wild-type mice. (A) Immunoblot analysis demonstrates elevated levels of the lysosomal protease cathepsin D (Ctsd) in retinas of 3-, 6-, and 11-month-old nclf mice when compared with age-matched wild-type mice. Expression levels of the lysosomal membrane protein Lamp1 were also increased in 6- and 11-month-old mutant retinas when compared with control retinas. Equal loading was controlled by reprobing blots with anti-Gapdh antibodies. (B) Cathepsin D activity was increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type control retinas (a), in line with the elevated Ctsd levels seen in immunoblots (A). Control measurements performed in the presence of pepstatin A, an inhibitor of aspartic proteinases, revealed only residual enzyme activity (b).
Figure 6
 
Immunoblot analysis of cathepsin D and Lamp1 expression and cathepsin D activity in retinas from nclf and wild-type mice. (A) Immunoblot analysis demonstrates elevated levels of the lysosomal protease cathepsin D (Ctsd) in retinas of 3-, 6-, and 11-month-old nclf mice when compared with age-matched wild-type mice. Expression levels of the lysosomal membrane protein Lamp1 were also increased in 6- and 11-month-old mutant retinas when compared with control retinas. Equal loading was controlled by reprobing blots with anti-Gapdh antibodies. (B) Cathepsin D activity was increased in 3-, 6-, and 11-month-old nclf retinas when compared with age-matched wild-type control retinas (a), in line with the elevated Ctsd levels seen in immunoblots (A). Control measurements performed in the presence of pepstatin A, an inhibitor of aspartic proteinases, revealed only residual enzyme activity (b).
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