Degeneration of Photoreceptor Cells in Arylsulfatase G-Deficient Mice

Kruszewski K, Luellmann-Rauch R, Dierks T, Bartsch U, Damme M. Degeneration of Photoreceptor Cells in Arylsulfatase G-Deficient Mice. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE. 2016;57(3):1120-1131.PURPOSE. Retinal degeneration is a common feature of several lysosomal storage disorders, including the mucopolysaccharidoses, a group of metabolic disorders that is characterized by widespread accumulation of glycosaminoglycans due to lysosomal enzyme dysfunction. We used a new mouse model of mucopolysaccharidosis IIIE to study the effect of Arylsulfatase G (ARSG) deficiency on retina integrity. METHODS. The retina of Arsg knockout mice aged 1 to 24 months was studied by immunohistochemistry and Western blot analysis. Electron microscopic analyses were performed on retinas from 15- and 22-month-old animals. Photoreceptor and microglia cell numbers and retina thickness were determined to quantitatively characterize retinal degeneration in ARSG-deficient mice. RESULTS. Arsg knockout mice showed a progressive degeneration of photoreceptor cells starting between 1 and 6 months of age, resulting in the loss of more than 50% of photoreceptor cells in 24-month-old mice. Photoreceptor loss was accompanied by reactive astrogliosis, reactive microgliosis that was evident in the outer but not inner retina, and elevated expression levels of some lysosomal proteins. Electron microscopic analyses of retinas revealed no evidence for the presence of storage vacuoles. Of note, expression of ARSG protein in wild-type mice was detectable only in the RPE which, however, appeared morphologically unaffected in knockout mice at the electron microscopic level. CONCLUSIONS. To our knowledge, this is the first study demonstrating that ARSG deficiency results in progressive photoreceptor degeneration and dysregulation of various lysosomal proteins

Intravitreally grafted CNTF-NS cells attenuate photoreceptor degeneration in <i>nclf</i> mice.

<p>A CNTF-NS cell clone was grafted into one (a, c, e) and a control-NS cell clone into the contralateral eye (b, d, f) of 14 days old <i>nclf</i> mice. Central retinal sections were stained with anti-recoverin antibodies and DAPI two (a, b), four (c, d) and six (e, f) weeks after transplantation. Note the thicker outer nuclear layer (onl) of CNTF-treated retinas when compared to control retinas at all post-transplantation time points. DAPI, 4’,6-diamidino-2-phenylindole; onl, outer nuclear layer. Bar in f (for a-f): 50 μm.</p

Sustained Neural Stem Cell-Based Intraocular Delivery of CNTF Attenuates Photoreceptor Loss in the <i>nclf</i> Mouse Model of Neuronal Ceroid Lipofuscinosis

<div><p>A sustained intraocular administration of neurotrophic factors is among the strategies aimed at establishing treatments for currently untreatable degenerative retinal disorders. In the present study we have analyzed the neuroprotective effects of a continuous neural stem (NS) cell-based intraocular delivery of ciliary neurotrophic factor (CNTF) on photoreceptor cells in the <i>nclf</i> mouse, an animal model of the neurodegenerative lysosomal storage disorder variant late infantile neuronal ceroid lipofuscinosis (vLINCL). To this aim, we genetically modified adherently cultivated NS cells with a polycistronic lentiviral vector encoding a secretable variant of CNTF together with a Venus reporter gene (CNTF-NS cells). NS cells for control experiments (control-NS cells) were modified with a vector encoding the reporter gene tdTomato. Clonal CNTF-NS and control-NS cell lines were established using fluorescent activated cell sorting and intravitreally grafted into 14 days old <i>nclf</i> mice at the onset of retinal degeneration. The grafted cells preferentially differentiated into astrocytes that were attached to the posterior side of the lenses and the vitreal side of the retinas and stably expressed the transgenes for at least six weeks, the latest post-transplantation time point analyzed. Integration of donor cells into host retinas, ongoing proliferation of grafted cells or adverse effects of the donor cells on the morphology of the host eyes were not observed. Quantitative analyses of host retinas two, four and six weeks after cell transplantation revealed the presence of significantly more photoreceptor cells in eyes with grafted CNTF-NS cells than in eyes with grafted control-NS cells. This is the first demonstration that a continuous intraocular administration of a neurotrophic factor attenuates retinal degeneration in an animal model of neuronal ceroid lipofuscinosis.</p></div

CNTF and reporter gene expression in neural cell types derived from CNTF-NS cells and control-NS cells <i>in vitro</i>.

<p>CNTF-NS (a-c, g-i) and control-NS cells (d-f, j-l) were differentiated into neurons (a-f) or astrocytes (g-l). Note that all MAP-2-positive neurons (b) and GFAP-positive astrocytes (h) derived from CNTF-NS cells co-expressed the reporter gene Venus (a, g) and CNTF (c, i). Neurons (e) and astrocytes (k) derived from control-NS cells, in comparison, expressed the reporter gene tdTomato (d, j) but no detectable levels of the cytokine (f, l). CNTF, ciliary neurotrophic factor; DAPI, 4’,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2. Bar in l (for a-l): 50 μm.</p

Photoreceptor numbers in eyes of <i>nclf</i> mice with grafted CNTF-NS or control-NS cells at different post-transplantation time points.

<p>A CNTF-NS and a control-NS cell line were intravitreally grafted into 14 days old <i>nclf</i> mice and photoreceptor numbers were determined in central retinal sections at six defined positions two, four and six weeks after transplantation. Note that CNTF-treated eyes contained significantly more photoreceptors (filled bars) than the contralateral eyes with grafted control-NS cells (open bars) at all post-transplantation time points. Each bar represents the mean value (±SEM) from six retinas. ***, p<0.001 (Newman-Keuls post hoc test after the mixed two-way ANOVA).</p

Lentiviral vectors and immunoblot analyses of culture supernatants from clonal CNTF-NS and control-NS cell lines.

<p>A lentiviral vector encoding a secretable variant of mouse ciliary neurotrophic factor (CNTF), an internal ribosome entry site (IRES) sequence of the encephalomyocarditis virus and a Venus reporter and a zeocin (ZEO) resistance gene separated by a P2A sequence of porcine teschovirus-1 (2A) under regulatory control of the cytomegalovirus enhancer/chicken ß-actin (CAG) promoter (a) was used to generate CNTF-secreting NS cells. NS cells for control experiments were transduced with a vector containing the CAG promoter, an IRES sequence and a tdTomato (tdTom) reporter gene fused to a blasticidin (BSD) resistance gene (b). Immunoblot analysis (c) of culture supernatants from the newly established CNTF-NS cell clone (clone 2) revealed elevated secretion levels of CNTF when compared to the original clonal CNTF-NS cell line (clone 1). Supernatants from control-NS cell clones (control) lacked detectable levels of the cytokine (c). Recombinant mouse CNTF (rmCNTF) was loaded as a reference. Ψ, packaging signal; cPPT, central polypurine tract; LoxP, recognition site of Cre recombinase; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element.</p

Expression of CNTF and the reporter genes in clonal CNTF-NS and control-NS cell lines.

<p>All cells in the clonal CNTF-NS cell line expressed the reporter gene Venus (a) and showed CNTF-immunoreactivity in the perinuclear region (b). Control-NS cells, in comparison, expressed the reporter gene tdTomato (c) but lacked detectable expression of the cytokine (d). CNTF, ciliary neurotrophic factor; DAPI, 4’,6-diamidino-2-phenylindole. Bar in d (for a-d): 20 μm.</p