Subcellular trafficking of proteolipid protein (PLP/DM20) and novel mechanisms of ER retention in Pelizaeus-Merzbacher disease PhD Thesis in partial fulfilment of the requirements for the degree “Doctor of Philosophy (PhD)/Dr. rer. nat.” in the Neuroscience Program at the Georg August University Göttingen, Faculty of Biology submitted by Ajit Singh Dhaunchak born in Chandigarh, India 29. May 2006 DECLARATION I hereby declare that I prepared the Ph.D. thesis “Subcellular trafficking of proteolipid protein (PLP/DM20) and novel mechanisms of ER retention in Pelizaeus-Merzbacher disease” on my own and with no other sources and aids than quoted. I gratefully acknowledge specific help by Dr. Eva-Maria Krämer (standardization of transfection protocols), Mrs. Annette Fahrenholz (immuno-histochemistry of murine spinal cord), Mrs. Gudrun Fricke-Bode (primary oligodendrocyte cultures), and Mrs. Ulli Bode (selection of ES cells). Dr. Hauke Werner contributed equally to the experimental treatment of rumpshaker mice with Turmeric. Two introductory figures were composed of images kindly provided by Christian Humml, Dr. Petra Hirrlinger, Dr. Gesine Saher, Foteini Orfaniotou, Dr. Wiebke Möbius, and Susanne Quintes. Ajit Singh Dhaunchak Göttingen, 29. May 2006 ACKNOWLEDGEMENT I am sincerely grateful to Prof. Klaus-Armin Nave who not only gave me an opportunity to work on such an amazing project under terrific conditions but also granted a high degree of freedom during the work. During these years I have learned a lot from him and I want to learn a lot more. I am indebted to Prof. Ari Helenius, Prof. Reinhard Jahn and Prof. Harald Neumann for their advice and discussions. I am grateful to entire PLP and friends group, especially Dr. Mikael Simons and Katarina Trajkovic for comments, discussions and support. I owe many thanks to Dr. Markus H Schwab, Dr. Sandra Goebbels and especially to Dr. Kanwar Vikas Singh Rana who kindled an interest of molecular neurobiology in me and helped me during initial phase of my PhD. Many thanks to Mrs. Endo Gabriele for always keeping me away from bureaucratic hurdles and creating a friendly atmosphere in the whole department. I would also like to thank the co-ordination team from Neuroscience Graduate Program Dr. Steffen Burkhardt, Dr. Simone Cardoso de Oliviera, Sandra Drube, Dr. Dorothee Wegener and Prof. Michael Hörner for their support in administrative matters during the last four years. Special thanks to Dr. Johannes Hirrlinger for his expertise in establishing live cell imaging setup in the department. Ulli Bode for her help in a day to day life in the department. I would also like to thank Ingo and an entire CNS myelin group, Hauke, Gesine, Celia, Anke, Susanne, Thorsten and not to mention the warm atmosphere maintained by younger generation PhD students of the north wing including Amit, Schanila, Foteini, Alex and Jan. I am grateful to Patricia and others for taking a close look on my thesis and posters. Many thanks to people who provided with images for composite of two introductory figures, acknowledged in the declaration. I am also grateful to Hajo Horn, Rolf Merker and Markus Born for their help in rescuing me from day to day PC crashes. I owe special thanks to my dearest friends Neelabh Shankar, Dr. Kanwar Vikas Singh Rana and Anshul Awasthi for always being there for me. I would like to thank my dear friend Rajesh for watching my favourite Hindi movie along for at least fifty times. My deepest gratitude goes to my loving Parents and to Katja for always being beside me and for their unconditional love and support. Table of contents TABLE OF CONTENTS LIST OF FIGURES.....................................................................................................................................VII 1 SUMMARY............................................................................................................................................1 2 INTRODUCTION.................................................................................................................................3 2.1 General Introduction.......................................................................................................................4 2.2 Myelin............................................................................................................................................5 2.3 Human PLP 1 and mouse Plp1 gene transcripts............................................................................8 2.4 PLP conservation during evolution and topology in myelin membranes.....................................10 2.5 Mutations associated with Plp gene.............................................................................................12 2.6 Sorting of membrane proteins and mechanisms of ER retention.................................................13 2.7 Oligodendrocytes are polarized cells............................................................................................14 2.8 A therapeutic approach toward a mouse model of PMD..............................................................18 3 MATERIAL AND METHODS...........................................................................................................19 3.1 Material Used...............................................................................................................................20 3.1.1 Kits, chemicals and protocol source........................................................................................20 3.1.2 Solutions and buffers...............................................................................................................20 3.1.2.1 Molecular biology buffers.............................................................................................................20 3.1.2.2 Protein biochemistry buffers.........................................................................................................21 3.1.2.3 Immunocytochemistry buffers......................................................................................................24 3.1.3 Bacterial and cell culture media..............................................................................................25 3.1.3.1 LB-Medium...................................................................................................................................25 3.1.3.2 Buffers and media for Cell Culture...............................................................................................26 3.1.4 Bacterial strains and cell lines used.........................................................................................27 3.1.5 Plasmids...................................................................................................................................28 3.1.6 Antibodies and Enzymes.........................................................................................................28 3.1.6.1 Antibodies.....................................................................................................................................28 3.1.6.2 Enzyme.........................................................................................................................................29 3.1.7 DNA and Protein Markers.......................................................................................................29 3.1.8 Oligonucleotides......................................................................................................................29 3.2 Methods........................................................................................................................................30 3.2.1 Molecular biological techniques..............................................................................................30 3.2.1.1 Maintenance of bacterial glycerol stocks......................................................................................30 3.2.1.2 Transformation of bacteria............................................................................................................30 3.2.1.3 Plasmid isolation of E. coli...........................................................................................................30 3.2.1.4 Enzymatic modification and manipulation of DNA......................................................................31 3.2.1.5 Generation of PLP-myc, PLP-EGFP , truncated and myc replacement chimeras.........................33 3.2.1.6 Site-directed mutagenesis of DNA................................................................................................34 3.2.1.7 Generation of the PLP-EGFP transgenic “Knock-in” mice...........................................................37 IV Table of contents 3.2.2 Protein-biochemical methods..................................................................................................39 3.2.2.1 SDS-poly-acrylamide gel electrophoresis.....................................................................................39 3.2.2.2 Western Blot-analysis...................................................................................................................39 3.2.2.3 Lysis of COS-7 and oli-neu cells...................................................................................................40 3.2.2.4 Protein biotinylation......................................................................................................................40 3.2.2.5 Oxidation and reduction assay......................................................................................................40 3.2.2.6 Co-immunoprecipitation...............................................................................................................41 3.2.2.7 S35 labeling of proteins and radioimmunoassay...........................................................................41 3.2.3 Cell culture..............................................................................................................................41 3.2.3.1 COS-7 and OLN93 cell culture.....................................................................................................41 3.2.3.2 Oli-neu cell culture........................................................................................................................42 3.2.3.3 Hybridoma cell culture..................................................................................................................42 3.2.3.4 Transient transfection of COS-7 and oli-neu cells.........................................................................42 3.2.3.5 Stable transfection of oli-neu cells................................................................................................43 3.2.4 Immunocytochemistry.............................................................................................................43 3.2.4.1 Immunocytochemistry of living cells............................................................................................43 3.2.4.2 Immunocytochemistry of fixed cells.............................................................................................43 3.2.5 Confocal analysis.....................................................................................................................44 4 RESULTS.............................................................................................................................................45 4.1 Cysteine mediated cross links cause Pelizaeus-Merzbacher Disease...........................................46 4.1.1 Video microscopy of EGFP-tagged PLP in oligodendrocytes.................................................47 4.1.2 Trafficking differences between mutant PLP and DM20 isoforms.........................................50 4.1.3 The role of disulfide bridges in PLP folding...........................................................................54 4.1.4 Genetic uncoupling of protein misfolding and ER retention...................................................56 4.1.5 ER retention of mutant PLP/DM20 and its rescue by removal of cysteines............................58 4.1.6 Misfolded PLP forms abnormal dimers and unspecific aggregates.........................................63 4.1.7 ER retention of PLP/DM20 chimeras can be rescued by removal of cysteines.......................67 4.2 Quality control of transmembrane domain assembly in PLP.......................................................70 4.2.1 Spastic Paraplegia 2 (SPG2) a mild form of PMD..................................................................71 4.2.2 Truncated PLP transmembranes are retained in the ER..........................................................71 4.2.3 N or C termini deletions did not alter PLP localization...........................................................73 4.2.4 Perturbations proximal to TM3 retain both PLP and DM20 in the ER...................................74 4.2.5 Self assembly of transmembrane domains...............................................................................74 4.2.6 Truncated transmembranes associate with calnexin................................................................75 4.3 Conformation sensitive and compartment specific epitope: evidence that PLP matures within the ER.................................................................................................................................................78 4.3.1 Wildtype PLP masks 3F4 epitope during its exit from ER......................................................79 4.3.2 Adult CNS myelin presents a complete overlapping avidity to 3F4 and A431.......................82 4.3.3 A novel 16kDa PLP proteolytic cleavage product...................................................................84 4.4 From transfected oligodendrocytes to PLP-EGFP expressing transgenic “knock-in” mice.........86 V Table of contents 4.4.1 PLP accumulates in endosomes/lysosomes (E/L)....................................................................87 4.4.2 Association of PLP with cholesterol........................................................................................87 4.4.3 Directed trafficking of rapidly moving PLP-EGFP+ endo/lysosomes in primary oligodendrocytes......................................................................................................................89 4.4.4 Generation of an in vivo tool to study myelination, demyelination and remyelination...........94 4.5 A therapeutic approach towards a mouse model of Pelizaeus-Merzbacher disease; treatment of rumpshaker mice with Turmeric..................................................................................................96 4.5.1 Curcumin is an active constituent of Turmeric........................................................................97 4.5.2 Curcumin treatment of stable cell line expressing PLPmsd-EGFP............................................97 4.5.3 Treatment of rumpshaker mice with Turmeric........................................................................98 5 DISCUSSION.....................................................................................................................................100 5.1 Quality Control of Polytopic Membrane Proteins......................................................................101 5.1.1 Luminal quality control in PLP/DM20 trafficking: an implication to various membrane/secretory protein related diseases.........................................................................101 5.1.2 Self assembly of PLP/DM20 tetraspans................................................................................105 5.2 Conformation sensitive epitope of PLP and polarized oligodendrocytes...................................107 5.2.1 3F4 and 010 label mutually exclusive compartments of premyelinating oligodendrocytes..107 5.2.2 Oligodendrocytes are polarized cells.....................................................................................108 5.3 Treatment of Rumpshaker mice with Turmeric..........................................................................110 6 REFERENCES...................................................................................................................................111 Appendix A: Abbreviations..........................................................................................................................121 Appendix B: Publications.............................................................................................................................123 VI List of figures LIST OF FIGURES Figure 1: Cells of the central nervous system....................................................................................................4 Figure 2: Myelin ultrastructure and major myelin proteins...............................................................................6 Figure 3: Nodal, paranodal, juxtaparanodal and internodal organization in CNS and PNS..............................7 Figure 4: Topology of PLP/DM20 in myelin membranes.................................................................................9 Figure 5: Conservation of PLP among different species.................................................................................11 Figure 6: Protein sorting and domain organization in polarized cells.............................................................15 Figure 7: Structure of PLP/DM20 and mutations associated with Pelizaeus-Merzbacher disease..................48 Figure 8: Subcellular distribution of PLPwt and PLPmsd fused to EGFP, in oli-neu cells.................................49 Figure 9: ER retention in oli-neu cells distinguishes PMD-associated isoforms of PLP, DM20, and chimeras...........................................................................................................................................51 Figure 10: Kyte and Doolitle hydropathy plot of PLP, DM20 and DM20LSAT-HPDK.........................................53 Figure 11: Length and position of TM3 determine ER retention or release of mutant DM20........................54 Figure 12: The function of extracellular disulfide-bridges in PLP folding and cell surface expression..........55 Figure 13: PLP cysteine mutants that reach the cell surface also accumulate in endo/lysosomes...................57 Figure 14: Uncoupling of protein folding, ER exit, and the wild-type conformation of PLP.........................58 Figure 15: Unpaired Cys200 causes ER retention and dimerization of a PMD mutant PLPC219Y......................59 Figure 16: PMD-causing PLP mutations can be rescued by the replacement of cysteines.............................62 Figure 17: PMD-causing PLP mutations rescued by the replacement of cysteines.........................................63 Figure 18: Cysteine-mediated PLP crosslinks.................................................................................................64 Figure 19: Cysteine-mediated PLP crosslinks in COS-7 cells.........................................................................66 Figure 20: ER lectins associate with mutant and wt PLP with a same affinity...............................................67 Figure 21: Outer disulfide bond governs the local and global folding of PLP/DM20 chimera.......................68 Figure 22: All truncated PLPs are retained in the ER, when expressed individually......................................72 Figure 23: Neither N and C termini nor IC2 but TM assembly monitors surface expression of PLP.............73 Figure 24: Prerequisite for an exit from the ER is proper alignment and masking of TMs in the bilayer.......76 Figure 25: Truncated PLPs associate with calnexin with an equal affinity.....................................................77 Figure 26: Oligomeric PLP masks 3F4 epitope at the cell surface..................................................................80 Figure 27: Maturation of PLP completes in pre-myelin E/Ls whereas PLPC00,219S matures at the cell surface81 VII List of figures Figure 28: Identical avidity of 3F4 and A431 antibody towards MDL and IPL embedded PLP epitopes......83 Figure 29: A novel 16kDa myelin PLP proteolytic cleavage product.............................................................84 Figure 30: Association of PLP with cholesterol..............................................................................................88 Figure 31: Co-immunopreipitation from oli-neu stably expressing PLPwt-EGFP and PLPmsd-EGFP.............89 Figure 32: cAMP treatment induces process outgrowth and redistributes PLP to the cell surface.................90 Figure 33: Highly mobile and directed endo/lysosomes in primary oligodendrocytes....................................92 Figure 34: Strategy for targeted homologus recombination of Plp gene in mouse ES cells............................95 Figure 35: Treatment of PLPmsd-EGFP expressing cells with curcumin.........................................................98 Figure 36: Survival of rumpshaker mice treated with turmeric.......................................................................99 Figure 37: Proposed mechanism of ER retention..........................................................................................102 VIII Summary 1 SUMMARY 1 Summary Missense mutations that predict the misfolding of membrane proteins have been associated with a number of neurogenetic diseases. However, it is not known how apparently minor changes in the amino acid sequence of an extracellular loop or a transmembrane domain lead to complete ER retention with complex loss- and gain-of-function effects. I have chosen PLP/DM20, a highly conserved and abundant tetraspan myelin protein, associated with Pelizaeus-Merzbacher disease (PMD), as a model system. By expressing wildtype and mutant PLP isoforms in glial cells, surprising molecular properties became apparent, including the ability to self-assemble from two truncated PLP polypeptides, and to form conformation sensitive epitope that become masked as the protein matures in the ER. With respect to human disease, it was possible to identify a novel molecular mechanism by which missense mutations cause ER retention of misfolded PLP. Unexpectedly, pairs of cysteines within an extracellular loop of PLP/DM20 play a critical role. Multiple disease- causing mutations require the presence of cysteines such that misfolded PLP/DM20 is efficiently retained in the ER. Replacing cysteines by serine completely prevents ER retention and restores normal trafficking of mutant PLP/DM20. This demonstrates a novel pathological mechanism by which missense mutations greatly reduce the efficiency of intramolecular disulfide bridging. When exposed by misfolding to the ER lumen, unpaired cysteines engage in alternative oxidations that lead to abnormal intermolecular crosslinks. Since extracellular cysteines are a feature of many membrane proteins, this novel pathomechanism is likely to contribute to a diverse group of genetic diseases. To monitor the expression and subcellular trafficking of PLP in vivo, a transgenic “knock-in” mouse in being generated that will express a PLP-EGFP fusion protein under control of the endogenous promoter. In an attempt to develop a cure for Pelizaeus-Merzbacher disease (PMD), we treated a genuine animal model (rumpshaker mice) with Turmeric. The active constituent of this herbal drug (Curcumin) is a non-toxic Ca2+–adenosine triphosphatase pump inhibitor, and known to release membrane proteins from ER retention. In a pilot experiment, we extended the lifespan of rumpshaker mice from 20 to 60 days. These promising data suggest that a therapeutic strategy should be developed for PMD, using turmeric and our in vitro and in vivo models. 2