Therapeutic effect of stem cell-based glial cell derived neurotrophic factor and ciliary neurotrophic factor on retinal degeneration of CLN7 neuronal ceroid-lipofuscinosis mouse model_Chinese Journal of Ocular Fundus Diseases

Authors：

 Wei Yingying ^1,2 , Udo Bartsch ¹

1. Department of Ophthalmology, University Medical Center Hamburg-Eppendorf, Hamburg 100731, Germany;
2. Changsha Xiangjiang Aier Eye Hospital, Changsha 410011, China;

Corresponding author：

Wei Yingying, Email: 1983430500@qq.com

Keywords：

CLN7 neuronal ceroid lipofuscinosis; Ciliary neurotrophic factor; Glial cell line-derived neurotrophic factor; Lentiviral vector; Neural stem cells; Intraocular stem cell transplantation; Neuroprotection

DOI：

10.3760/cma.j.cn511434-20231212-00482

Video：

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Objective To observe the morphological and functional changes of retinal degeneration in mice with CLN7 neuronal ceroid-lipofuscinosis, and the therapeutic effects of glial cell derived neurotrophic factor (GDNF) and/or ciliary neurotrophic factor (CNTF) based on neural stem cells (NSC) on mouse photoreceptor cells. Methods A total of 100 CLN7 mice aged 14 days were randomly divided into the experimental group and the control group, with 80 and 20 mice respectively. Twenty C57BL/6J mice aged 14 days were assigned as wild-type group (WT group). Mice in control group and WT group did not receive any interventions. At 2, 4, and 6 months of age, immunohistochemical staining was conducted to examine alterations in the distribution and quantity of cones, rod-bipolar cells, and cone-bipolar cells within the retinal of mice while electroretinography (ERG) examination was utilized to record scotopic a and b-waves and photopic b-wave amplitudes. At 14 days of age, the mice in the experimental group were intravitreally injected with 2 μl of CNTF-NSC, GDNF-NSC, and a 1:1 cell mixture of CNTF-NSC and GDNF-NSC (GDNF/CNTF-NSC). Those mice were then subdivided into the CNTF-NSC group, the GDNF-NSC group, and the GDNF/CNTF-NSC group accordingly. The contralateral eyes of the mice were injected with 2 μl of control NSC without neurotrophic factor (NTF) as their own control group. At 2 and 4 months of age, the rows of photoreceptor cells in mice was observed by immunohistochemical staining while ERG was performed to record amplitudes. At 4 months of age, the differentiation of grafted NSC and the expression of NTF were observed. Statistical comparisons between the groups were performed using a two-way ANOVA. Results Compared with WT group, the density of cones in the peripheral region of the control group at 2, 4 and 6 months of age (F=285.10), rod-bipolar cell density in central and peripheral retina (F=823.20, 346.20), cone-bipolar cell density (F=356.30, 210.60) and the scotopic amplitude of a and b waves (F=1 911.00, 387.10) in central and peripheral retina were significantly decreased, with statistical significance (P＜0.05). At the age of 4 and 6 months, the density of retinal cone cells (F=127.30) and b-wave photopic amplitude (F=51.13) in the control group were significantly decreased, and the difference was statistically significant (P＜0.05). Immunofluorescence microscopy showed that the NSC transplanted in the experimental group preferentially differentiated into astrocytes, and stably expressed CNTF and GDNF at high levels. Comparison of retinal photoreceptor nucleus lines in different treatment subgroups of the experimental group at different ages: CNTF-NSC group, at 2 months of age: the whole, central and peripheral regions were significantly different (F=31.73, 75.06, 75.06; P＜0.05); 4 months of age: The difference between the whole area and the peripheral region was statistically significant (F=12.27, 12.27; P＜0.05). GDNF/CNTF-NSC group, 2 and 4 months of age: the whole (F=27.26, 27.26) and the peripheral area (F=16.01, 13.55) were significantly different (P＜0.05). In GDNF-NSC group, there was no statistical significance at all in the whole, central and peripheral areas at different months of age (F=0.00, 0.01, 0.02; P＞0.05). Conclusions CLN7 neuronal ceroid-lipofuscinosis mice exhibit progressively increasing degenerative alterations in photoreceptor cells and bipolar cells with age growing, aligning with both morphological and functional observations. Intravitreal administration of stem cell-based CNTF as well as GDNF/CNTF show therapeutic potential in rescuing photoreceptor cells. Nevertheless, the combined application of GDNF/CNTF-NSC do not demonstrate the anticipated synergistic protective effect. GDNF has no therapeutic effect on the retinal morphology and function in CLN7 neuronal ceroid-lipofuscinosis mice.

Citation： Wei Yingying, Udo Bartsch. Therapeutic effect of stem cell-based glial cell derived neurotrophic factor and ciliary neurotrophic factor on retinal degeneration of CLN7 neuronal ceroid-lipofuscinosis mouse model. Chinese Journal of Ocular Fundus Diseases, 2024, 40(7): 526-537. doi: 10.3760/cma.j.cn511434-20231212-00482 Copy

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2.	Kohlschütter A, Schulz A, Bartsch U, et al. Current and emerging treatment strategies for neuronal ceroid lipofuscinoses[J]. CNS Drugs, 2019, 33(4): 315-325. DOI: 10.1007/s40263-019-00620-8.
3.	Mole SE, Anderson G, Band HA, et al. Clinical challenges and future therapeutic approaches for neuronal ceroid lipofuscinosis[J]. Lancet Neurol, 2019, 18(1): 107-116. DOI: 10.1016/S1474-4422(18)30368-5.
4.	Kousi M, Siintola E, Dvorakova L, et al. Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis[J]. Brain, 2009, 132(Pt 3): 810-819. DOI: 10.1093/brain/awn366.
5.	Brandenstein L, Schweizer M, Sedlacik J, et al. Lysosomal dysfunction and impaired autophagy in a novel mouse model deficient for the lysosomal membrane protein Cln7[J]. Hum Mol Genet, 2016, 25(4): 777-791. DOI: 10.1093/hmg/ddv615.
6.	Fudalej E, Justyniarska M, Kasarełło K, et al. Neuroprotective factors of the retina and their role in promoting survival of retinal ganglion cells: a review[J]. Ophthalmic Res, 2021, 64(3): 345-355. DOI: 10.1159/000514441.
7.	Ohnaka M, Miki K, Gong YY, et al. Long-term expression of glial cell line-derived neurotrophic factor slows, but does not stop retinal degeneration in a model of retinitis pigmentosa[J]. J Neurochem, 2012, 122(5): 1047-1053. DOI: 10.1111/j.1471-4159.2012.07842.x.
8.	Kolomeyer AM, Zarbin MA. Zarbin, trophic factors in the pathogenesis and therapy for retinal degenerative diseases[J]. Surv Ophthalmol, 2014, 59(2): 134-165. DOI: 10.1016/j.survophthal.2013.09.004.
9.	Jung G, Sun J, Petrowitz B, et al. Genetically modified neural stem cells for a local and sustained delivery of neuroprotective factors to the dystrophic mouse retina[J]. Stem Cells Transl Med, 2013, 2(12): 1001-1010. DOI: 10.5966/sctm.2013-0013.
10.	Jankowiak W, Kruszewski K, Flachsbarth K, et al. Sustained neural stem cell-based intraocular delivery of CNTF attenuates photoreceptor loss in the nclf mouse model of neuronal ceroid lipofuscinosis[J/OL]. PLoS One, 2015, 10(5): e0127204[2015-05-20]. https://pubmed.ncbi.nlm.nih.gov/25992714/. DOI: 10.1371/journal.pone.0127204.
11.	Flachsbarth K, Kruszewski K, Jung G, et al. Neural stem cell-based intraocular administration of ciliary neurotrophic factor attenuates the loss of axotomized ganglion cells in adult mice[J]. Invest Ophthalmol Vis Sci, 2014, 55(11): 7029-7039. DOI: 10.1167/iovs.14-15266.
12.	Cuenca N, Fernández-Sánchez L, Campello L, et al. Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases[J]. Prog Retin Eye Res, 2014, 43: 17-75. DOI: 10.1016/j.preteyeres.2014.07.001.
13.	Dulz S, Bassal M, Flachsbarth K, et al. Intravitreal co-administration of GDNF and CNTF confers synergistic and long-lasting protection against injury-induced cell death of retinal ganglion cells in mice[J]. Cells, 2020, 9(9): 2082. DOI: 10.3390/cells9092082.
14.	Flachsbarth K, Jankowiak W, Kruszewski K, et al. Pronounced synergistic neuroprotective effect of GDNF and CNTF on axotomized retinal ganglion cells in the adult mouse[J]. Exp Eye Res, 2018, 176: 258-265. DOI: 10.1016/j.exer.2018.09.006.
15.	Zein WM, Jeffrey BG, Wiley HE, et al. CNGB3-achromatopsia clinical trial with CNTF: diminished rod pathway responses with no evidence of improvement in cone function[J]. Invest Ophthalmol Vis Sci, 2014, 55(10): 6301-6308. DOI: 10.1167/iovs.14-14860.
16.	Wang Y, Zeng W, Lin B, et al. CLN7 is an organellar chloride channel regulating lysosomal function[J/OL]. Sci Adv, 2021, 7(51): eabj9608[2021-12-15]. https://pubmed.ncbi.nlm.nih.gov/34910516/. DOI: 10.1126/sciadv.abj9608.
17.	Jankowiak W, Brandenstein L, Dulz S, et al. Retinal degeneration in mice deficient in the lysosomal membrane protein CLN7[J]. Invest Ophthalmol Vis Sci, 2016, 57(11): 4989-4998. DOI: 10.1167/iovs.16-20158.
18.	Rowe AA, Chen X, Nettesheim ER, et al. Long-term progression of retinal degeneration in a preclinical model of CLN7 Batten disease as a baseline for testing clinical therapeutics[J/OL]. EBioMedicine, 2022, 85: 104314[2022-10-29]. https://pubmed.ncbi.nlm.nih.gov/36374771/. DOI: 10.1016/j.ebiom.2022.104314.
19.	Bartsch U, Galliciotti G, Jofre GF, et al. Apoptotic photoreceptor loss and altered expression of lysosomal proteins in the nclf mouse model of neuronal ceroid lipofuscinosis[J]. Invest Ophthalmol Vis Sci, 2013, 54(10): 6952-6959. DOI: 10.1167/iovs.13-12945.
20.	Mirza M, Volz C, Karlstetter M, et al. Progressive retinal degeneration and glial activation in the CLN6 (nclf) mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation[J/OL]. PLoS One, 2013, 8(10): e75963[2013-10-04]. https://pubmed.ncbi.nlm.nih.gov/24124525/. DOI: 10.1371/annotation/ae907882-62e0-4803-8c00-35b30a649fe9.
21.	Kleine Holthaus SM, Ribeiro J, Abelleira-Hervas L, et al. Prevention of photoreceptor cell loss in a Cln6(nclf) mouse model of Batten disease requires CLN6 gene transfer to bipolar cells[J]. Mol Ther, 2018, 26(5): 1343-1353. DOI: 10.1016/j.ymthe.2018.02.027.
22.	Soldati C, Lopez-Fabuel I, Wanderlingh LG, et al. Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype[J/OL]. EMBO Mol Med, 2021, 13(10): e13742[2021-10-07]. https://pubmed.ncbi.nlm.nih.gov/34411438/. DOI: 10.15252/emmm.202013742.
23.	Chen X, Dong T, Hu Y, et al. AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease[J/OL]. J Clin Invest, 2022, 132(5): e146286[2022-03-01]. https://pubmed.ncbi.nlm.nih.gov/35025759/. DOI: 10.1172/JCI146286.
24.	Lipinski DM, Barnard AR, Singh MS, et al. CNTF gene therapy confers lifelong neuroprotection in a mouse model of human retinitis pigmentosa[J]. Mol Ther, 2015, 23(8): 1308-1319. DOI: 10.1038/mt.2015.68.
25.	Rhee KD, Nusinowitz S, Chao K, et al. CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in Müller glial cells[J/OL]. Proc Natl Acad Sci USA, 2013, 110(47): E4520-4529[2013-11-19]. https://pubmed.ncbi.nlm.nih.gov/24191003/. DOI: 10.1073/pnas.1303604110.
26.	Baranov P, Lin H, McCabe K, et al. A novel neuroprotective small molecule for glial cell derived neurotrophic factor induction and photoreceptor rescue[J]. J Ocul Pharmacol Ther, 2017, 33(5): 412-422. DOI: 10.1089/jop.2016.0121.
27.	García-Caballero C, Lieppman B, Arranz-Romera A, et al. Photoreceptor preservation induced by intravitreal controlled delivery of GDNF and GDNF/melatonin in rhodopsin knockout mice[J]. Mol Vis, 2018, 24: 733-745.
28.	Kimura A, Namekata K, Guo X, et al. Neuroprotection, growth factors and BDNF-TrkB signalling in retinal degeneration[J]. Int J Mol Sci, 2016, 17(9): 1584. DOI: 10.3390/ijms17091584.
29.	Hackett SF, Schoenfeld CL, Freund J, et al. Neurotrophic factors, cytokines and stress increase expression of basic fibroblast growth factor in retinal pigmented epithelial cells[J]. Exp Eye Res, 1997, 64(6): 865-873. DOI: 10.1006/exer.1996.0256.
30.	Bush RA, Lei B, Tao W, et al. Encapsulated cell-based intraocular delivery of ciliary neurotrophic factor in normal rabbit: dose-dependent effects on ERG and retinal histology[J]. Invest Ophthalmol Vis Sci, 2004, 45(7): 2420-2430. DOI: 10.1167/iovs.03-1342.

1. Gardner E, Mole SE. The genetic basis of phenotypic heterogeneity in the neuronal ceroid lipofuscinoses[J/OL]. Front Neurol, 2021, 12: 754045[2021-10-18]. https://pubmed.ncbi.nlm.nih.gov/34733232/. DOI: 10.3389/fneur.2021.754045.
2. Kohlschütter A, Schulz A, Bartsch U, et al. Current and emerging treatment strategies for neuronal ceroid lipofuscinoses[J]. CNS Drugs, 2019, 33(4): 315-325. DOI: 10.1007/s40263-019-00620-8.
3. Mole SE, Anderson G, Band HA, et al. Clinical challenges and future therapeutic approaches for neuronal ceroid lipofuscinosis[J]. Lancet Neurol, 2019, 18(1): 107-116. DOI: 10.1016/S1474-4422(18)30368-5.
4. Kousi M, Siintola E, Dvorakova L, et al. Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis[J]. Brain, 2009, 132(Pt 3): 810-819. DOI: 10.1093/brain/awn366.
5. Brandenstein L, Schweizer M, Sedlacik J, et al. Lysosomal dysfunction and impaired autophagy in a novel mouse model deficient for the lysosomal membrane protein Cln7[J]. Hum Mol Genet, 2016, 25(4): 777-791. DOI: 10.1093/hmg/ddv615.
6. Fudalej E, Justyniarska M, Kasarełło K, et al. Neuroprotective factors of the retina and their role in promoting survival of retinal ganglion cells: a review[J]. Ophthalmic Res, 2021, 64(3): 345-355. DOI: 10.1159/000514441.
7. Ohnaka M, Miki K, Gong YY, et al. Long-term expression of glial cell line-derived neurotrophic factor slows, but does not stop retinal degeneration in a model of retinitis pigmentosa[J]. J Neurochem, 2012, 122(5): 1047-1053. DOI: 10.1111/j.1471-4159.2012.07842.x.
8. Kolomeyer AM, Zarbin MA. Zarbin, trophic factors in the pathogenesis and therapy for retinal degenerative diseases[J]. Surv Ophthalmol, 2014, 59(2): 134-165. DOI: 10.1016/j.survophthal.2013.09.004.
9. Jung G, Sun J, Petrowitz B, et al. Genetically modified neural stem cells for a local and sustained delivery of neuroprotective factors to the dystrophic mouse retina[J]. Stem Cells Transl Med, 2013, 2(12): 1001-1010. DOI: 10.5966/sctm.2013-0013.
10. Jankowiak W, Kruszewski K, Flachsbarth K, et al. Sustained neural stem cell-based intraocular delivery of CNTF attenuates photoreceptor loss in the nclf mouse model of neuronal ceroid lipofuscinosis[J/OL]. PLoS One, 2015, 10(5): e0127204[2015-05-20]. https://pubmed.ncbi.nlm.nih.gov/25992714/. DOI: 10.1371/journal.pone.0127204.
11. Flachsbarth K, Kruszewski K, Jung G, et al. Neural stem cell-based intraocular administration of ciliary neurotrophic factor attenuates the loss of axotomized ganglion cells in adult mice[J]. Invest Ophthalmol Vis Sci, 2014, 55(11): 7029-7039. DOI: 10.1167/iovs.14-15266.
12. Cuenca N, Fernández-Sánchez L, Campello L, et al. Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases[J]. Prog Retin Eye Res, 2014, 43: 17-75. DOI: 10.1016/j.preteyeres.2014.07.001.
13. Dulz S, Bassal M, Flachsbarth K, et al. Intravitreal co-administration of GDNF and CNTF confers synergistic and long-lasting protection against injury-induced cell death of retinal ganglion cells in mice[J]. Cells, 2020, 9(9): 2082. DOI: 10.3390/cells9092082.
14. Flachsbarth K, Jankowiak W, Kruszewski K, et al. Pronounced synergistic neuroprotective effect of GDNF and CNTF on axotomized retinal ganglion cells in the adult mouse[J]. Exp Eye Res, 2018, 176: 258-265. DOI: 10.1016/j.exer.2018.09.006.
15. Zein WM, Jeffrey BG, Wiley HE, et al. CNGB3-achromatopsia clinical trial with CNTF: diminished rod pathway responses with no evidence of improvement in cone function[J]. Invest Ophthalmol Vis Sci, 2014, 55(10): 6301-6308. DOI: 10.1167/iovs.14-14860.
16. Wang Y, Zeng W, Lin B, et al. CLN7 is an organellar chloride channel regulating lysosomal function[J/OL]. Sci Adv, 2021, 7(51): eabj9608[2021-12-15]. https://pubmed.ncbi.nlm.nih.gov/34910516/. DOI: 10.1126/sciadv.abj9608.
17. Jankowiak W, Brandenstein L, Dulz S, et al. Retinal degeneration in mice deficient in the lysosomal membrane protein CLN7[J]. Invest Ophthalmol Vis Sci, 2016, 57(11): 4989-4998. DOI: 10.1167/iovs.16-20158.
18. Rowe AA, Chen X, Nettesheim ER, et al. Long-term progression of retinal degeneration in a preclinical model of CLN7 Batten disease as a baseline for testing clinical therapeutics[J/OL]. EBioMedicine, 2022, 85: 104314[2022-10-29]. https://pubmed.ncbi.nlm.nih.gov/36374771/. DOI: 10.1016/j.ebiom.2022.104314.
19. Bartsch U, Galliciotti G, Jofre GF, et al. Apoptotic photoreceptor loss and altered expression of lysosomal proteins in the nclf mouse model of neuronal ceroid lipofuscinosis[J]. Invest Ophthalmol Vis Sci, 2013, 54(10): 6952-6959. DOI: 10.1167/iovs.13-12945.
20. Mirza M, Volz C, Karlstetter M, et al. Progressive retinal degeneration and glial activation in the CLN6 (nclf) mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation[J/OL]. PLoS One, 2013, 8(10): e75963[2013-10-04]. https://pubmed.ncbi.nlm.nih.gov/24124525/. DOI: 10.1371/annotation/ae907882-62e0-4803-8c00-35b30a649fe9.
21. Kleine Holthaus SM, Ribeiro J, Abelleira-Hervas L, et al. Prevention of photoreceptor cell loss in a Cln6(nclf) mouse model of Batten disease requires CLN6 gene transfer to bipolar cells[J]. Mol Ther, 2018, 26(5): 1343-1353. DOI: 10.1016/j.ymthe.2018.02.027.
22. Soldati C, Lopez-Fabuel I, Wanderlingh LG, et al. Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype[J/OL]. EMBO Mol Med, 2021, 13(10): e13742[2021-10-07]. https://pubmed.ncbi.nlm.nih.gov/34411438/. DOI: 10.15252/emmm.202013742.
23. Chen X, Dong T, Hu Y, et al. AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease[J/OL]. J Clin Invest, 2022, 132(5): e146286[2022-03-01]. https://pubmed.ncbi.nlm.nih.gov/35025759/. DOI: 10.1172/JCI146286.
24. Lipinski DM, Barnard AR, Singh MS, et al. CNTF gene therapy confers lifelong neuroprotection in a mouse model of human retinitis pigmentosa[J]. Mol Ther, 2015, 23(8): 1308-1319. DOI: 10.1038/mt.2015.68.
25. Rhee KD, Nusinowitz S, Chao K, et al. CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in Müller glial cells[J/OL]. Proc Natl Acad Sci USA, 2013, 110(47): E4520-4529[2013-11-19]. https://pubmed.ncbi.nlm.nih.gov/24191003/. DOI: 10.1073/pnas.1303604110.
26. Baranov P, Lin H, McCabe K, et al. A novel neuroprotective small molecule for glial cell derived neurotrophic factor induction and photoreceptor rescue[J]. J Ocul Pharmacol Ther, 2017, 33(5): 412-422. DOI: 10.1089/jop.2016.0121.
27. García-Caballero C, Lieppman B, Arranz-Romera A, et al. Photoreceptor preservation induced by intravitreal controlled delivery of GDNF and GDNF/melatonin in rhodopsin knockout mice[J]. Mol Vis, 2018, 24: 733-745.
28. Kimura A, Namekata K, Guo X, et al. Neuroprotection, growth factors and BDNF-TrkB signalling in retinal degeneration[J]. Int J Mol Sci, 2016, 17(9): 1584. DOI: 10.3390/ijms17091584.
29. Hackett SF, Schoenfeld CL, Freund J, et al. Neurotrophic factors, cytokines and stress increase expression of basic fibroblast growth factor in retinal pigmented epithelial cells[J]. Exp Eye Res, 1997, 64(6): 865-873. DOI: 10.1006/exer.1996.0256.
30. Bush RA, Lei B, Tao W, et al. Encapsulated cell-based intraocular delivery of ciliary neurotrophic factor in normal rabbit: dose-dependent effects on ERG and retinal histology[J]. Invest Ophthalmol Vis Sci, 2004, 45(7): 2420-2430. DOI: 10.1167/iovs.03-1342.

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Therapeutic effect of stem cell-based glial cell derived neurotrophic factor and ciliary neurotrophic factor on retinal degeneration of CLN7 neuronal ceroid-lipofuscinosis mouse model

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