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Hallmark cellular pathology of Alzheimer’s disease induced by mutant human tau expression in cultured Aplysia neurons

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Abstract

The mechanisms underlying neurodegenerative diseases are the outcome of pathological alterations of evolutionary conserved molecular and cellular cascades. For this reason, Drosophila and C. elegans serve as useful model systems to study various aspects of neurodegenerative diseases. Here, we introduce the advantageous use of cultured Aplysia neurons (which express over 100 disease-related gene homologs shared with mammals), as a platform to study cell biological processes underlying the generation of tauopathy. Using live confocal imaging to follow cytoskeletal elements, autophagosomes, lysosomes, anterogradely and retrogradely transported organelles, complemented with electron microscopy, we demonstrate that the expression of mutant human tau in cultured Aplysia neurons leads to the development of hallmark Alzheimer disease (AD) pathologies. These include a reduction in the number of microtubules and their redistribution, impaired organelle transport, a dramatic accumulation of macro-autophagosomes and lysosomes, compromised neurite morphology and degeneration. Our study demonstrates the accessibility of the platform for long-term live imaging and quantification of subcellular pathological cascades leading to tauopathy. Based on the present study, it is conceivable that this system can also be used to screen for reagents that alter the pathological cascades.

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References

  1. Baas PW, Qiang L (2005) Neuronal microtubules: when the MAP is the roadblock. Trends Cell Biol 15(4):183–187

    Article  CAS  PubMed  Google Scholar 

  2. Ballatore C, Lee VM, Trojanowski JQ (2007) Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat Rev Neurosci 8(9):663–672

    Article  CAS  PubMed  Google Scholar 

  3. Boland B, Kumar A, Lee S, Platt FM, Wegiel J, Yu WH, Nixon RA (2008) Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer’s disease. J Neurosci 28(27):6926–6937

    Article  CAS  PubMed  Google Scholar 

  4. Brandt R, Leger J, Lee G (1995) Interaction of tau with the neural plasma membrane mediated by tau’s amino-terminal projection domain. J Cell Biol 131(5):1327–1340

    Article  CAS  PubMed  Google Scholar 

  5. Brunden KR, Trojanowski JQ, Lee VM (2009) Advances in tau-focused drug discovery for Alzheimer’s disease and related tauopathies. Nat Rev Drug Discov 8(10):783–793

    Article  CAS  PubMed  Google Scholar 

  6. Dias-Santagata D, Fulga TA, Duttaroy A, Feany MB (2007) Oxidative stress mediates tau-induced neurodegeneration in Drosophila. J Clin Invest 117(1):236–245

    Article  CAS  PubMed  Google Scholar 

  7. Erez H, Malkinson G, Prager-Khoutorsky M, De Zeeuw CI, Hoogenraad CC, Spira ME (2007) Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J Cell Biol 176(4):497–507

    Article  CAS  PubMed  Google Scholar 

  8. Erez H, Spira ME (2008) Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J Comp Neurol 507(2):1019–1030

    Article  PubMed  Google Scholar 

  9. Fass E, Shvets E, Degani I, Hirschberg K, Elazar Z (2006) Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes. J Biol Chem 281(47):36303–36316

    Article  CAS  PubMed  Google Scholar 

  10. Fulga TA, Elson-Schwab I, Khurana V, Steinhilb ML, Spires TL, Hyman BT, Feany MB (2007) Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo. Nat Cell Biol 9(2):139–148

    Article  CAS  PubMed  Google Scholar 

  11. Gitler D, Spira ME (1998) Real time imaging of calcium-induced localized proteolytic activity after axotomy and its relation to growth cone formation. Neuron 20:1123–1135

    Article  CAS  PubMed  Google Scholar 

  12. Gumy LF, Tan CL, Fawcett JW (2009) The role of local protein synthesis and degradation in axon regeneration. Exp Neurol 223(1):28–37

    Article  PubMed  CAS  Google Scholar 

  13. Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A (2009) A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 11(12):1433–1437

    Article  CAS  PubMed  Google Scholar 

  14. He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93

    Article  CAS  PubMed  Google Scholar 

  15. Jaworski J, Hoogenraad CC, Akhmanova A (2008) Microtubule plus-end tracking proteins in differentiated mammalian cells. Int J Biochem Cell Biol 40(4):619–637

    Article  CAS  PubMed  Google Scholar 

  16. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728

    Article  CAS  PubMed  Google Scholar 

  17. Kamber D, Erez H, Spira ME (2009) Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone. Exp Neurol 219(1):112–125

    Article  CAS  PubMed  Google Scholar 

  18. Kandel ER (2009) The biology of memory: a forty-year perspective. J Neurosci 29(41):12748–12756

    Article  CAS  PubMed  Google Scholar 

  19. Kandel ER (2001) The molecular biology of memory storage: a dialog between genes and synapses. Biosci Rep 21(5):565–611

    Article  CAS  PubMed  Google Scholar 

  20. Knudsen B, Kohn AB, Nahir B, McFadden CS, Moroz LL (2006) Complete DNA sequence of the mitochondrial genome of the sea-slug, Aplysia californica: conservation of the gene order in Euthyneura. Mol Phylogenet Evol 38(2):459–469

    Article  CAS  PubMed  Google Scholar 

  21. Kretzschmar D (2005) Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases? Invert Neurosci 5(3–4):97–109

    Article  CAS  PubMed  Google Scholar 

  22. Lee G, Newman ST, Gard DL, Band H, Panchamoorthy G (1998) Tau interacts with src-family non-receptor tyrosine kinases. J Cell Sci 111(Pt 21):3167–3177

    CAS  PubMed  Google Scholar 

  23. Lee JA, Lim CS, Lee SH, Kim H, Nukina N, Kaang BK (2003) Aggregate formation and the impairment of long-term synaptic facilitation by ectopic expression of mutant huntingtin in Aplysia neurons. J Neurochem 85(1):160–169

    Article  CAS  PubMed  Google Scholar 

  24. Link CD (2005) Invertebrate models of Alzheimer’s disease. Genes Brain Behav 4(3):147–156

    Article  CAS  PubMed  Google Scholar 

  25. Lu B, Vogel H (2009) Drosophila models of neurodegenerative diseases. Annu Rev Pathol 4:315–342

    Article  CAS  PubMed  Google Scholar 

  26. Malkinson G, Fridman ZM, Kamber D, Dormann A, Shapira E, Spira ME (2006) Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles. Brain Cell Biol 35(1):57–73

    Article  CAS  PubMed  Google Scholar 

  27. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15(3):1101–1111

    Article  CAS  PubMed  Google Scholar 

  28. Morfini GA, Burns M, Binder LI, Kanaan NM, LaPointe N, Bosco DA, Brown RH Jr, Brown H, Tiwari A, Hayward L, Edgar J, Nave KA, Garberrn J, Atagi Y, Song Y, Pigino G, Brady ST (2009) Axonal transport defects in neurodegenerative diseases. J Neurosci 29(41):12776–12786

    Article  CAS  PubMed  Google Scholar 

  29. Moroz LL, Edwards JR, Puthanveettil SV, Kohn AB, Ha T, Heyland A, Knudsen B, Sahni A, Yu F, Liu L, Jezzini S, Lovell P, Iannucculli W, Chen M, Nguyen T, Sheng H, Shaw R, Kalachikov S, Panchin YV, Farmerie W, Russo JJ, Ju J, Kandel ER (2006) Neuronal transcriptome of Aplysia: neuronal compartments and circuitry. Cell 127(7):1453–1467

    Article  CAS  PubMed  Google Scholar 

  30. Nakagawa H, Koyama K, Murata Y, Morito M, Akiyama T, Nakamura Y (2000) EB3, a novel member of the EB1 family preferentially expressed in the central nervous system, binds to a CNS-specific APC homologue. Oncogene 19(2):210–216

    Article  CAS  PubMed  Google Scholar 

  31. Nixon RA (2007) Autophagy, amyloidogenesis and Alzheimer disease. J Cell Sci 120(Pt 23):4081–4091

    Article  CAS  PubMed  Google Scholar 

  32. Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM (2005) Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol 64(2):113–122

    PubMed  Google Scholar 

  33. Nixon RA, Yang DS, Lee JH (2008) Neurodegenerative lysosomal disorders: a continuum from development to late age. Autophagy 4(5):590–599

    CAS  PubMed  Google Scholar 

  34. Perez F, Diamantopoulos GS, Stalder R, Kreis TE (1999) CLIP-170 highlights growing microtubule ends in vivo. Cell 96(4):517–527

    Article  CAS  PubMed  Google Scholar 

  35. Rami A (2009) Review: autophagy in neurodegeneration: firefighter and/or incendiarist? Neuropathol Appl Neurobiol 35(5):449–461

    Article  CAS  PubMed  Google Scholar 

  36. Ravikumar B, Futter M, Jahreiss L, Korolchuk VI, Lichtenberg M, Luo S, Massey DC, Menzies FM, Narayanan U, Renna M, Jimenez-Sanchez M, Sarkar S, Underwood B, Winslow A, Rubinsztein DC (2009) Mammalian macroautophagy at a glance. J Cell Sci 122(Pt 11):1707–1711

    Article  CAS  PubMed  Google Scholar 

  37. Rodriguez OC, Schaefer AW, Mandato CA, Forscher P, Bement WM, Waterman-Storer CM (2003) Conserved microtubule-actin interactions in cell movement and morphogenesis. Nat Cell Biol 5(7):599–609

    Article  CAS  PubMed  Google Scholar 

  38. Sahly I, Erez H, Khoutorsky A, Shapira E, Spira ME (2003) Effective expression of the green fluorescent fusion proteins in cultured Aplysia neurons. J Neurosci Methods 126(2):111–117

    Article  CAS  PubMed  Google Scholar 

  39. Schacher S, Proshansky E (1983) Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment. J Neurosci 3(12):2403–2413

    CAS  PubMed  Google Scholar 

  40. Shemesh OA, Erez H, Ginzburg I, Spira ME (2008) Tau-induced traffic jams reflect organelles accumulation at points of microtubule polar mismatching. Traffic 9(4):458–471

    Article  CAS  PubMed  Google Scholar 

  41. Shemesh OA, Spira ME (2010) Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy. Acta Neuropathol 119(2):235–248

    Article  CAS  PubMed  Google Scholar 

  42. Shulman JM, Shulman LM, Weiner WJ, Feany MB (2003) From fruit fly to bedside: translating lessons from Drosophila models of neurodegenerative disease. Curr Opin Neurol 16(4):443–449

    Article  PubMed  Google Scholar 

  43. Spira ME, Dormann A, Ashery U, Gabso M, Gitler D, Benbassat D, Oren R, Ziv NE (1996) Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro. J Neurosci Methods 69(1):91–102

    Article  CAS  PubMed  Google Scholar 

  44. Spira ME, Oren R, Dormann A, Gitler D (2003) Critical calpain-dependent ultrastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons. J Comp Neurol 457(3):293–312

    Article  PubMed  Google Scholar 

  45. Stamer K, Vogel R, Thies E, Mandelkow E, Mandelkow EM (2002) Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J Cell Biol 156(6):1051–1063

    Article  CAS  PubMed  Google Scholar 

  46. Stepanova T, Slemmer J, Hoogenraad CC, Lansbergen G, Dortland B, De Zeeuw CI, Grosveld F, van Cappellen G, Akhmanova A, Galjart N (2003) Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein). J Neurosci 23(7):2655–2664

    CAS  PubMed  Google Scholar 

  47. Yu WH, Cuervo AM, Kumar A, Peterhoff CM, Schmidt SD, Lee JH, Mohan PS, Mercken M, Farmery MR, Tjernberg LO, Jiang Y, Duff K, Uchiyama Y, Naslund J, Mathews PM, Cataldo AM, Nixon RA (2005) Macroautophagy—a novel Beta-amyloid peptide-generating pathway activated in Alzheimer’s disease. J Cell Biol 171(1):87–98

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This study was supported by Grant No. 137/08 from the Israel Science Foundation (ISF), and Grant No. 300000-4955 from the Israel Ministry of Health. Parts of this work were conducted at the Charles E. Smith Family and Prof. Elkes Laboratory for Collaborative Research in Psychobiology. We thank Dr. E. Shapira, A. Dormann and H. Glickstein for technical help in preparing mRNAs, and complementary electron micrographs. The LC3-GFP construct was kindly given to us by Prof. Zvulun Elazar (Weizmann Institute). M.E. Spira is Levi DeViali Professor of Neurobiology.

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Authors and Affiliations

  1. Department of Neurobiology, Institute of Life Science, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel

    Or A. Shemesh & Micha E. Spira

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  1. Or A. Shemesh
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  2. Micha E. Spira
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Correspondence to Micha E. Spira.

Electronic supplementary material

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401_2010_689_MOESM1_ESM.jpg

Supplementary material 1. Formation of MT-swirls underlies axonal swelling and transport defects in tau overexpressing neurons. The figure depicts phenotype B as defined in the text. The figure shows a neuron microinjected with EB3-GFP and mutant cerulean-tau mRNA. 96 hours later swelling of a ~ 50µm long axonal segment was observed (a). Imaging of this compartment (yellow rectangle in a) revealed that the EB3-GFP comet tails form a “MT-swirl” (b, see Supporting Movie 3) and that the EB3-GFP distribution fits the tau distribution (c). Color coding of 10 frames from a time-lapse imaging sequence of SR101 labeled organelles taken at an interval of 6.4 s revealed that a large fraction of SR101 labeled vesicles are stationary (d, coded white, and the limited spectrum coding for motility). Electron micrographs of the swelling (e) reveal that the MTs are densely packed (g), and autophagosomes “entrapped” within the dense MT swirl (f). MT-microtubules, AV –autophagosomes, mit-mitochondria. Scale bars: 100 µm in a, 10 µm in d relating to a-d, 10 µm in e, 1000 nm in f, g. (JPEG 11612 kb)

401_2010_689_MOESM2_ESM.jpg

Supplementary material 2. The density of LC3-GFP- and lysotracker-labeled organelles in control and mutant-human tau expressing neurons. 96h post culturing, 3 control neurons and 3 mt-human tau expressing neurons were injected with LC3-GFP mRNA. 24 h later the neurons were incubated with 50 nM lysotracker and imaged as described in Fig. 6. Thereafter, within a standard axonal AOI of 500µm2 (5µm * 100µm), the number of LC3-GFP labeled organelles (“control”, green), and lysotracker-labeled organelles (“control” red) were counted. Note the increased number of organelles. (JPEG 1512 kb)

401_2010_689_MOESM3_ESM.jpg

Supplementary material 3. Size and transport velocity of LC3-GFP- and lysotracker-labeled organelles in control and mutant-human tau expressing neurons. Data shown here were collected from the experiments described in S1. (a, b,) the size, (c, d), the transport velocity. Control (blue) and mt-human tau expressing neurons (red). (a, c) lysotracker labeled organelles (b, d) LC3-GFP labeled organelles. (JPEG 6955 kb)

401_2010_689_MOESM4_ESM.jpg

Supplementary material 4. Co-localization of lysotracker- and LC3-GFP-labeled organelles in control- and mt-human tau expressing neurons. Control neurons and mt-human tau expressing neurons grown in culture for 96h were labeled with lysotracker (red) and GFP LC3 (green, as described in figure 6). To avoid the problem of switching the emission spectrum of the lysotracker (see text and methods) only the first 1-5 image scans were analyzed. (a, b) The cell body of control and mt-human tau expressing neurons respectively. (c, d) the axons of a control and mt-human-tsau expressing neurons respectively. Note that the number and distribution of organelles that co- express lysotracker and LC3-GFP is larger in the mt-human tau expressing neurons. For quantitative analysis see supplementary material 4. Scale bars: 10 µm in a and b, 10 µm in d relating to c, d. (JPEG 7893 kb)

401_2010_689_MOESM5_ESM.jpg

Supplementary material 5. Co-localization of lysotracker- and LC3-GFP-labelef organelles in the cell body, axon hillock and axon in control and mt-human tau expressing neurons. The figure relates to the data displayed in supplementary material 4. Control and mt-human tau expressing neurons were labeled by LC3-GFP and lysotracker as described in the text. Fluorograms demonstrating co-localizations (see methods) were produced for the soma (a), axon hillock (b) and axon (c). For each fluorogram, a Pearson’s coefficient was calculated giving a numerical estimate of colocalization (Pearson’s coefficients are given on the graphs). The same procedure was repeated for the soma (d), axon hillock (e) and axon (f) of a tau expressing neuron.

Movie S1. Control neuron- uniform MT polar orientation and retrograde transport. The movie relates to Fig. 1. (Upper panel, green) EB3-GFP comet tails in a control axon, 100-200µm away from cell body, 72 h following mRNA microinjection. (Lower panel, red) retrograde transport of SR101 labeled vesicles along the same location of the same axon. The video is composed of 30 images, taken at intervals of 5.7 seconds. The frames are shown at a rate of 7/s. Scale bar: 10 µm. (MPEG 21537 kb)

401_2010_689_MOESM7_ESM.mpeg

Movie S2. MT polar orientation and retrograde transport in phenotype-A; mutant human tau expressing neuron. The movie relates to Fig. 2. (Upper panel, green) EB3-GFP comet tails in a tau expressing axon, 100-200µm away from cell body, 72 h following cerulean-tau and EB3-GFP mRNA microinjection. (Lower panel, red) SR101 labeled vesicles along the same location of the same axon. Note that SR101 transport takes place only along the submembrane domain. The video is composed of 30 images, taken at intervals of 5.7 seconds. The frames are shown at a rate of 7/s. Scale bar: 10 µm. (MPEG 19804 kb)

401_2010_689_MOESM8_ESM.mpeg

Movie S3. MT polarity and retrograde transport in phenotype-B; mt-human tau expressing neuron. The movie relates to supplementary material 3. (Upper panel, green) EB3-GFP comet tails in a tau expressing axon, 50-150µm away from cell body, 96 h following cerulean-tau and EB3-GFP mRNA microinjection. (Lower panel, red) SR101 labeled vesicles within the “MT swirl”. The video is composed of 30 images, taken at intervals of 6.4 seconds. The frames are shown at a rate of 7/s. Scale bar: 10 µm. (MPEG 43216 kb)

Movie S4. Lysotracker and LC3-GFP labeled organelles in a control neuron. The movie relates to Fig. 6. (Upper panel, green) LC3-GFP labeled organelles along a 150µm segment extending from the axon hillock to the axon, 120 h after culturing. (Middle panel, red) lysotracker labeled organelles . (Lower panel) a merged image, created by overlaying the upper and middle panel. The video contains 25 images, taken at intervals of 7.8 seconds. The frames are shown at a rate of 7/s. Scale bar: 10 µm. (MPEG 31143 kb)

401_2010_689_MOESM10_ESM.mpeg

Movie S5. Lysotracker and LC3-GFP labeled organelles in a mt-human tau expressing neuron. The movie relates to Fig. 6. (Upper panel, green) LC3-GFP labeled organelles along a 150µm segment extending distally from the axon hillock, 120 h after culturing and 96h from cerulean tau mRNA microinjection. (Middle panel, red) lysotracker labeled organelles. (Lower panel) a merged image, created by overlaying the upper and middle panel. The video contains 20 images, taken at intervals of 7.8 seconds. The frames are shown at a rate of 7/s. Scale bar: 10 µm. (MPEG 27297 kb)

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Shemesh, O.A., Spira, M.E. Hallmark cellular pathology of Alzheimer’s disease induced by mutant human tau expression in cultured Aplysia neurons. Acta Neuropathol 120, 209–222 (2010). https://doi.org/10.1007/s00401-010-0689-7

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