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Kabhilan Mohan, Yu Zhong, Jinpeng Liu, Ahmad Al-Attar, Huijuan Liu, Kyung Jung, Morgan Dow, Jinze Liu, Teresa Fan, Qing Jun Wang, Mark Ellsworth Kleinman; Retinal pigment epithelium atrophy in the Cln3Δex7/8 knock-in mouse model of juvenile neuronal ceroid lipofuscinosis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6030. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten’s disease) is a blinding pigmented retinal dystrophy with homozygous deletion of the exons 7 and 8 of the CLN3 gene in a large population of JNCL patients. Although JNCL is primarily a lysosomal storage disease, the retinal pigment epithelium (RPE) atrophy and the role of autophagy in mediating such atrophy remains unclear. In this study, we aim to characterize RPE pathology and the status of various autophagic markers in the Cln3Δex7/8 knock-in mouse model of JNCL.
Cln3Δex7/8 mutant mice (Jackson Labs) were analyzed for spontaneous rd1/rd8 mutation; 8-month (n=5) and 18-month-old (n=5) mutant mice were compared to age-matched Wild type C57BL/6J mice (8 month, n=4; 18 month; n=3). Fundus photography and autofluorescence were obtained using Topcon TRC-50IX; electroretinograms (ERG; Espion) were obtained under scotopic conditions. RPE/choroid flatmounts were prepared and immuno-stained for the tight junction protein, zona occludens-1 (ZO-1). RNA interference of the CLN3 gene was performed in the RPE-1 cell line (ATCC). Autophagic flux was studied with Western blot for the known markers p62 and LC3II. Autophagy and lysosome gene expression profiles were captured by RNA-Seq and qPCR.
No rd1/rd8 mutations were found in the Cln3Δex7/8 strain. Funduscopic analyses revealed a mottled appearance with extensive hypopigmentation in aged Cln3Δex7/8 mutant mice. Hyper-autofluorescent aggregates were observed in Cln3Δex7/8 mice at both ages. Significant losses in both a- and b-waves of aged mutant mice were observed compared to age-matched controls. ZO-1 immunofluorescence revealed widespread disruption of RPE tight junctions exclusively in the aged mutant mice. CLN3 siRNA treatment of RPE-1 cells led to greatly enhanced autophagic flux as well as autophagy and lysosome gene expression alterations that are consistent with increased autophagic and lysosomal degradation.
We have observed extensive RPE atrophy and gradual loss of retinal function loss in the Cln3Δex7/8 mouse model; interestingly, autofluorescent aggregates occur even in young mutant mice. Cell culture studies show enhanced autophagic activity resulting from CLN3 deficiency. Our data suggest a role for Cln3 function in autophagy which may contribute to RPE degeneration in JNCL and serve as a novel therapeutic in this blinding disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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