Acta Neuropathologica

, Volume 120, Issue 2, pp 209–222

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

Original Paper

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.

Keywords

Autophagosomes Lysosomes Tauopathy Alzheimer’s disease Axoplasmic transport Aplysia 

Supplementary material

401_2010_689_MOESM1_ESM.jpg (11.3 mb)
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 (139 kb)
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 (1.5 mb)
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 (6.8 mb)
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 (7.7 mb)
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 (19.3 mb)
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 (42.2 mb)
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 (26.7 mb)
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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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Neurobiology, Institute of Life ScienceThe Hebrew University of JerusalemJerusalemIsrael

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