Photoreceptor phagosome processing defects and disturbed autophagy in retinal pigment epithelium of Cln3Δex1-6 mice modelling juvenile neuronal ceroid lipofuscinosis (Batten disease)

Human Molecular Genetics, 2015, Vol. 24, No. 24 7060–7074 doi: 10.1093/hmg/ddv406 Advance Access Publication Date: 8 October 2015 Original Article ORIGINAL ARTICLE Photoreceptor phagosome processing defects and Silène T. Wavre-Shapton1,2,†, Alessandra A. Calvi4,†, Mark Turmaine5, Miguel C. Seabra2, Daniel F. Cutler6,7, Clare E. Futter1,* and Hannah M. Mitchison3,* 1 UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK, 2Molecular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK, 3Genetics and Genomic Medicine Programme and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK, 4Nuclear Dynamics and Architecture, Institute of Medical Biology, Singapore 138648, Singapore, 5Faculty of Life Sciences, Division of Biosciences and 6Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK and 7MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, London, UK *To whom correspondence should be addressed at: Genetics and Genomic Medicine Programme and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK. Tel: +44 (0) 20 7905 2866; Fax: +44 (0) 20 7404 6191; Email: (H.M.M.); UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK. Tel: +44 (0) 20 7608 4051; Fax: +44 (0) 20 7608 6810, Email: (C.E.F.) Abstract Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3Δex1-6 null mice, revealing classic ‘fingerprint’ lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3Δex1-6 RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3Δex1-6-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3Δex1-6 RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and † These authors contributed equally to this work. Received: June 24, 2015. Revised and Accepted: September 22, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 7060 disturbed autophagy in retinal pigment epithelium of Cln3Δex1-6 mice modelling juvenile neuronal ceroid lipofuscinosis (Batten disease) Human Molecular Genetics, 2015, Vol. 24, No. 24 | 7061 phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE. Introduction storage material in the photoreceptor and ganglion cell layers (16,19,20). This sequestering of an inner mitochondrial membrane protein component occurs for unknown reasons, but has long been debated to arise from deficient autophagy (21). CLN3 is a ubiquitously expressed, highly conserved glycosylated membrane protein with cytoplasmic C- and N-terminal domains and two cytosolic lysosomal targeting motifs (22–24). It mainly localizes to late endosome and lysosome membranes, with evidence also for localization in the trans-Golgi network (TGN)/post-TGN compartments and for roles in post-Golgi trafficking (25–28). In CLN3-deficient cells there is a markedly reduced exit from the TGN of the cation-independent mannose 6-phosphate receptor that is responsible for delivery of lysosome hydrolases from the TGN to the endocytic pathway (28). CLN3deficient cells have also been reported to display altered trafficking or activity of lysosomal enzymes (25,28–31), altered intralysosomal amino acid transport (32) and a loss of lysosome acidity coinciding with reportedly altered activity of the pH regulator vacuolar H (+)-ATPase (33–35). Correspondingly, CLN3-deficient cells display several faulty lysosome-associated processes, even prior to significant lysosome storage accumulation, notably deficient vesicle fusion and maturation events critical for autophagy (34,36), and deficient endocytosis (27,31,37,38). These suggested protein trafficking and lysosomal maintenance roles indicate involvement of CLN3 in cytoskeleton (cell morphology, migration) (27,38,39), synapse (37,40) and neurotransmission (41,42) functions. Lysosome dysfunction in some other NCL subtypes is already clearly linked to neuronal disease pathology, for example synaptic degeneration, and reduced neuronal plasticity arising from presynaptic abnormalities affecting the synaptic vesicle docking, recycling and neurotransmitter release cycle (43–45). In the eye, the distal tips (10%) of all photoreceptor outer segments (POS) are shed on a daily basis. The phagocytosis and degradation of shed POS by the adjacent RPE cells (46,47) is essential to maintain photoreceptor excitability. Phagosomes must move into the cell body in order to undergo a ‘maturation’ process involving sequential fusions with the endocytic pathway (48,49). Fusion with lysosomes, to form phagolysosomes, delivers membrane and degradative lumenal hydrolase enzymes to the phagosomes for the breakdown and recycling of the internalized POS (49). Failure to phagocytose shed outer segments, such as in the Royal College of Surgeons rat (50,51), leads to rapid photoreceptor cell death, demonstrating the importance of clearance of spent POS by the RPE, and the essential nature of lifelong maintenance of photoreceptors for maintenance of vision. Given the huge phagocytic load of RPE cells they are likely to be particularly sensitive to defects in lysosomal delivery or lysosomal activity. However, no RPE-specific phenotype has ever been described for the NCL diseases. An emerging field of study suggests that autophagy dysfunction can al (...truncated)