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Alterations in EGF-Endocytosis, Lysosomal Enzyme Transport and Maturation of Cathepsins in Juvenile Neuronal Ceroid Lipofuscinosis Fibroblasts

Jaime Carcel Trullols




Background: Juvenile neuronal ceroid lipofuscinosis (JNCL), one of the most frequent forms of the NCL storage diseases, is known to be caused by loss-of-function mutations in ceroid-lipofuscinosis neuronal protein 3 (CLN3), but its cell function has not been fully elucidated. We previously reported increased lysosomal pH in CLN3 deficient cells. In the present study, we analysed the consequences of this effect in the endo-lysosomal pathways in CLN3 cells.
Methods: The present study investigated different endo-lysosomal pathways in control, CLN2, CLN3 human skin fibroblasts under high and low proteolysis conditions. Cell surface biotinylation after EGF (2 ng/mL) stimulation, EGF phosphorylation (Tyr-845), retromer and cation-independent mannose-6-phosphate receptor (CI-MPR) levels and stability, EGF degradation pathways and cathepsin L and D levels were analysed by western blots. Caveolae mediated endocytosis was analysed by flow cytometry. CIMPR subcellular localization was ascertained by immunocytochemistry, confocal microscopy and further image analysis.
Results: Whereas caveolae-mediated endocytosis was not affected in CLN3 cells, clathrin-mediated epidermal growth factor (EGF) internalization was reduced, along with EGF receptor (EGFR) phosphorylation. In addition, cell surface EGFR levels and recycling to the cell membrane were increased. EGFR lysosomal degradation was impaired and our results suggest that the receptor was diverted to proteasomal degradation. We also analysed the machinery responsible for lysosomal hydrolase transport to the lysosome and found increased stability of CIMPR, a major receptor implicated in the transport of hydrolases. The subcellular distribution of the CI-MPR was also altered in CLN3 cells, since it accumulated within the Trans-Golgi network (TGN) and did not progress into the lysosomes. In addition, we found a reduced turnover of retromer subunits, a complex that retrieves the CI-MPR from endosomes to the TGN. Finally and as a possible consequence of these alterations in lysosomal enzyme transport, cathepsin L and D maturation were found suppressed in CLN3 cells.
Conclusion: Altogether, these results point to increased lisosomal pH as a pivotal event causing various alterations in intracellular traffic associated to the development of JNCL disease.


Juvenile neuronal ceroid lipofuscinosis; Lysosomal pH; Clathrin mediated endocytosis; Caveolae-mediated endocytosis; Cathepsin L; Cathepsin D; CI-MPR (CI-MPR); Retromer; Epidermal Growth Factor Receptor.


Williams RE, Mole SE. New nomenclature and classification scheme for the neuronal ceroid lipofuscinoses. Neurology. 2012;79(2):183-91. doi: 10.1212/WNL.0b013e31825f0547.

Isolation of a novel gene underlying Batten disease, CLN3. The International Batten Disease Consortium. Cell. 1995;82(6):949-57. doi: 10.1016/0092-8674(95)90274-0.

Kwon JM, Adams H, Rothberg PG, Augustine EF, Marshall FJ, Deblieck EA, et al. Quantifying physical decline in juvenile neuronal ceroid lipofuscinosis (Batten disease). Neurology. 2011;77(20):1801-7. doi: 10.1212/WNL.0b013e318237f649.

Cooper JD. Moving towards therapies for juvenile Batten disease? Exp Neurol. 2008;211(2):329-31. doi: 10.1016/j. expneurol.2008.02.016.

Järvelä I, Sainio M, Rantamäki T, Olkkonen VM, Carpén O, Peltonen L, et al. Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease. Hum Mol Genet. 1998;7(1):85-90. doi: 10.1093/hmg/7.1.85.

Storch S, Pohl S, Quitsch A, Falley K, Braulke T. C-terminal prenylation of the CLN3 membrane glycoprotein is required for efficient endosomal sorting to lysosomes. Traffic. 2007;8(4):431-44. doi: 10.1111/j.1600-0854.2007.00537.x.

Pearce DA, Ferea T, Nosel SA, Das B, Sherman F. Action of BTN1, the yeast orthologue of the gene mutated in Batten disease. Nat Genet. 1999;22(1):55-8. doi: 10.1038/8861.

Chattopadhyay S, Muzaffar NE, Sherman F, Pearce DA. The yeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alter pH homeostasis. J Bacteriol. 2000;182(22):6418- 23. doi: 10.1128/jb.182.22.6418-6423.2000.

Gachet Y, Codlin S, Hyams JS, Mole SE. btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis. J Cell Sci. 2005;118(Pt 23):5525-36. doi: 10.1242/jcs.02656.

Padilla-López S, Langager D, Chan CH, Pearce DA. BTN1, the Saccharomyces cerevisiae homolog to the human Batten disease gene, is involved in phospholipid distribution. Dis Model Mech. 2012;5(2):191-9. doi: 10.1242/dmm.008490.

Kama R, Robinson M, Gerst JE. Btn2, a Hook1 ortholog and potential Batten disease-related protein, mediates late endosome-Golgi protein sorting in yeast. Mol Cell Biol. 2007;27(2):605-21. doi: 10.1128/mcb.00699-06.

Richardson SC, Winistorfer SC, Poupon V, Luzio JP, Piper RC. Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton. Mol Biol Cell. 2004;15(3):1197-210. doi: 10.1091/mbc.e03-06-0358.

Kama R, Kanneganti V, Ungermann C, Gerst JE. The yeast Batten disease orthologue Btn1 controls endosome-Golgi retrograde transport via SNARE assembly. J Cell Biol. 2011;195(2):203-15. doi: 10.1083/jcb.201102115.

Luiro K, Yliannala K, Ahtiainen L, Maunu H, Järvelä I, Kyttälä A, et al. Interconnections of CLN3, Hook1 and Rab proteins link Batten disease to defects in the endocytic pathway. Hum Mol Genet. 2004;13(23):3017-27. doi: 10.1093/hmg/ddh321.

Uusi-Rauva K, Kyttälä A, van der Kant R, Vesa J, Tanhuanpää K, Neefjes J, et al. Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments. Cell Mol Life Sci. 2012;69(12):2075-89. doi: 10.1007/s00018-011-0913-1.

Persaud-Sawin DA, McNamara JO, 2nd, Rylova S, Vandongen A, Boustany RM. A galactosylceramide binding domain is involved in trafficking of CLN3 from Golgi to rafts via recycling endosomes. Pediatr Res. 2004;56(3):449-63. doi: 10.1203/01.pdr.0000136152.54638.95.

Cao Y, Staropoli JF, Biswas S, Espinola JA, MacDonald ME, Lee JM, et al. Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells. PLoS One. 2011;6(2):e17118. doi: 10.1371/journal.pone.0017118.

Appu AP, Bagh MB, Sadhukhan T, Mondal A, Casey S, Mukherjee AB. Cln3-mutations underlying juvenile neuronal ceroid lipofuscinosis cause significantly reduced levels of Palmitoyl-protein thioesterases-1 (Ppt1)-protein and Ppt1-enzyme activity in the lysosome. J Inherit Metab Dis. 2019;42(5):944-54. doi: 10.1002/jimd.12106.

Fossale E, Wolf P, Espinola JA, Lubicz-Nawrocka T, Teed AM, Gao H, et al. Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis. BMC Neurosci. 2004;5:57. doi: 10.1186/1471-2202-5-57.

Metcalf DJ, Calvi AA, Seaman M, Mitchison HM, Cutler DF. Loss of the Batten disease gene CLN3 prevents exit from the TGN of the mannose 6-phosphate receptor. Traffic. 2008;9(11):1905- 14. doi: 10.1111/j.1600-0854.2008.00807.x.

Braulke T, Bonifacino JS. Sorting of lysosomal proteins. Biochim Biophys Acta. 2009;1793(4):605-14. doi: 10.1016/j. bbamcr.2008.10.016.

Qian M, Sleat DE, Zheng H, Moore D, Lobel P. Proteomics analysis of serum from mutant mice reveals lysosomal proteins selectively transported by each of the two mannose 6-phosphate receptors. Mol Cell Proteomics. 2008;7(1):58- 70. doi: 10.1074/mcp.M700217-MCP200.

Kominami AE. What are the requirements for lysosomal degradation of subunit c of mitochondrial ATPase? IUBMB Life. 2002;54(2):89-90. doi: 10.1080/15216540214307.

Steenhuis P, Froemming J, Reinheckel T, Storch S. Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7. Biochim Biophys Acta. 2012;1822(10):1617-28. doi: 10.1016/j.bbadis.2012.05.015.

Hook V, Funkelstein L, Wegrzyn J, Bark S, Kindy M, Hook G. Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer’s disease. Biochim Biophys Acta. 2012;1824(1):89-104. doi: 10.1016/j. bbapap.2011.08.015.

Vidal-Donet JM, Cárcel-Trullols J, Casanova B, Aguado C, Knecht E. Alterations in ROS activity and lysosomal pH account for distinct patterns of macroautophagy in LINCL and JNCL fibroblasts. PLoS One. 2013;8(2):e55526. doi: 10.1371/ journal.pone.0055526.

Marks DL, Bittman R, Pagano RE. Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells. Histochem Cell Biol. 2008;130(5):819- 32. doi: 10.1007/s00418-008-0509-5.

O’Keefe E, Cuatecasas P. Cholera toxin and membrane gangliosides: binding and adenylate cyclase activation in normal and transformed cells. J Membr Biol. 1978;42(1):61- 79. doi: 10.1007/bf01870394.

Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell. 2008;15(2):209-19. doi: 10.1016/j.devcel.2008.06.012.

Kesarwala AH, Samrakandi MM, Piwnica-Worms D. Proteasome inhibition blocks ligand-induced dynamic processing and internalization of epidermal growth factor receptor via altered receptor ubiquitination and phosphorylation. Cancer Res. 2009;69(3):976-83. doi: 10.1158/0008-5472.can-08-2938.

Arighi CN, Hartnell LM, Aguilar RC, Haft CR, Bonifacino JS. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. J Cell Biol. 2004;165(1):123-33. doi: 10.1083/jcb.200312055.

Seaman MN. Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J Cell Biol. 2004;165(1):111- 22. doi: 10.1083/jcb.200312034.

Vergés M1. Retromer: multipurpose sorting and specialization in polarized transport. Int Rev Cell Mol Biol. 2008;271:153- 98. doi: 10.1016/s1937-6448(08)01204-5.

Banting G, Ponnambalam S. TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphology. Biochim Biophys Acta. 1997;1355(3):209-17. doi: 10.1016/s0167-4889(96)00146-2.

Lieu ZZ, Derby MC, Teasdale RD, Hart C, Gunn P, Gleeson PA. The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network. Mol Biol Cell. 2007;18(12):4979-91. doi: 10.1091/ mbc.e07-06-0622.

Ghosh RN, Mallet WG, Soe TT, McGraw TE, Maxfield FR. An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells. J Cell Biol. 1998;142(4):923-36. doi: 10.1083/ jcb.142.4.923.

Lin SX, Mallet WG, Huang AY, Maxfield FR. Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes. Mol Biol Cell. 2004;15(2):721-33. doi: 10.1091/mbc.e03-07- 0497.

Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol. 2006;7(8):568-79. doi: 10.1038/nrm1985.

Sorkin A, Waters C, Overholser KA, Carpenter G. Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis. J Biol Chem. 1991;266(13):8355-62.

Qureshi YH, Patel VM, Berman DE, Kothiya MJ, Neufeld JL, Vardarajan B, et al. An Alzheimer’s Disease-Linked Loss-of- Function CLN5 Variant Impairs Cathepsin D Maturation, Consistent with a Retromer Trafficking Defect. Mol Cell Biol. 2018;38(20). doi: 10.1128/mcb.00011-18.

Vesa J, Chin MH, Oelgeschlager K, Isosomppi J, DellAngelica EC, Jalanko A, et al. Neuronal ceroid lipofuscinoses are connected at molecular level: interaction of CLN5 protein with CLN2 and CLN3. Mol Biol Cell. 2002;13(7):2410-20. doi: 10.1091/mbc.e02-01-0031.

Huber RJ, Mathavarajah S. Secretion and function of Cln5 during the early stages of Dictyostelium development. Biochim Biophys Acta Mol Cell Res. 2018;1865(10):1437-50. doi: 10.1016/j.bbamcr.2018.07.017.

Maruzs T, Lőrincz P, Szatmári Z, Széplaki S, Sándor Z, Lakatos Z, et al. Retromer Ensures the Degradation of Autophagic Cargo by Maintaining Lysosome Function in Drosophila. Traffic. 2015;16(10):1088-107. doi: 10.1111/tra.12309.

Cárcel-Trullols J, Kovács AD, Pearce DA. Role of the Lysosomal Membrane Protein, CLN3, in the Regulation of Cathepsin D Activity. J Cell Biochem. 2017;118(11):3883-90. doi: 10.1002/jcb.26039.

Awano T, Katz ML, O’Brien DP, Taylor JF, Evans J, Khan S, et al. A mutation in the cathepsin D gene (CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis. Mol Genet Metab. 2006;87(4):341-8. doi: 10.1016/j. ymgme.2005.11.005.

Wavre-Shapton ST, Calvi AA, Turmaine M, Seabra MC, Cutler DF, Futter CE, et al. Photoreceptor phagosome processing defects and disturbed autophagy in retinal pigment epithelium of Cln3Deltaex1-6 mice modelling juvenile neuronal ceroid lipofuscinosis (Batten disease). Hum Mol Genet. 2015;24(24):7060-74. doi: 10.1093/hmg/ddv406.

Koike M, Nakanishi H, Saftig P, Ezaki J, Isahara K, Ohsawa Y, et al. Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons. J Neurosci. 2000;20(18):6898-906.

Canuel M, Libin Y, Morales CR. The interactomics of sortilin: an ancient lysosomal receptor evolving new functions. Histol Histopathol. 2009;24(4):481-92. doi: 10.14670/hh-24.481.

Felbor U, Kessler B, Mothes W, Goebel HH, Ploegh HL, Bronson RT, et al. Neuronal loss and brain atrophy in mice lacking cathepsins B and L. Proc Natl Acad Sci U S A. 2002;99(12):7883-8. doi: 10.1073/pnas.112632299.

Sevenich L, Pennacchio LA, Peters C, Reinheckel T. Human cathepsin L rescues the neurodegeneration and lethality in cathepsin B/L double-deficient mice. Biol Chem. 2006;387(7):885-91. doi: 10.1515/bc.2006.112.

Xiang B, Fei X, Zhuang W, Fang Y, Qin Z, Liang Z. Cathepsin L is involved in 6-hydroxydopamine induced apoptosis of SH-SY5Y neuroblastoma cells. Brain Res. 2011;1387:29-38. doi: 10.1016/j.brainres.2011.02.092.

Haft CR, de la Luz Sierra M, Bafford R, Lesniak MA, Barr VA, Taylor SI. Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes. Mol Biol Cell. 2000;11(12):4105-16. doi: 10.1091/mbc.11.12.4105.

Göstring L, Chew MT, Orlova A, Höidén-Guthenberg I, Wennborg A, Carlsson J, et al. Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects. Int J Oncol. 2010;36(4):757-63. doi: 10.3892/ijo_00000551.

Lauer S, Goldstein B, Nolan RL, Nolan JP. Analysis of cholera toxin-ganglioside interactions by flow cytometry. Biochemistry. 2002;41(6):1742-51. doi: 10.1021/bi0112816.

Martin de Llano JJ, Andreu EJ, Pastor A, de la Guardia M, Knecht E. Electrothermal atomic absorption spectrometric diagnosis of familial hypercholesterolemia. Anal Chem. 2000;72(11):2406-13. doi: 10.1021/ac991287p.

Gao YS, Hubbert CC, Yao TP. The microtubule-associated histone deacetylase 6 (HDAC6) regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation. J Biol Chem. 2010;285(15):11219-26. doi: 10.1074/jbc. M109.042754.


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