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Dynein motors transport activated Trks to promote survival of target-dependent neurons

Abstract

Mutations that alter dynein function are associated with neurodegenerative diseases, but it is not known why defects in dynein-dependent transport impair neuronal survival. Here we show that dynein function in axons is selectively required for the survival of neurons that depend on target-derived neurotrophins. Stimulation of axon terminals with neurotrophins causes internalization of neurotrophin receptors (Trks). Using real-time imaging of fluorescently tagged Trks, we show that dynein is required for rapid transport of internalized, activated receptors from axon terminals to remote cell bodies. When dynein-based transport is inhibited, neurotrophin stimulation of axon terminals does not support survival. These studies indicate that defects in dynein-based transport reduce trafficking of activated Trks and thereby obstruct the prosurvival effect of target-derived trophic factors, leading to degeneration of target-dependent neurons.

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Figure 1: Trk activity in the cell body is required for survival responses to target-derived neurotrophins.
Figure 2: BDNF induces internalization and retrograde movement of TrkB.
Figure 3: FRAP assay shows retrograde transport of TrkB-GFP.
Figure 4: Inhibition of clathrin-dependent endocytosis blocks BDNF-induced TrkB internalization and transport, and prevents retrograde survival signals.
Figure 5: Introduction of exogenous dynamitin into axons selectively disrupts retrograde transport.
Figure 6: Retrograde transport of phosphorylated Trk requires receptor internalization and dynein-based transport.
Figure 7: Inhibition of dynein selectively blocks survival responses to target-derived neurotrophins.
Figure 8: Immobilized ligand stimulates internalization and transport of activated Trks, resulting in a dynein-dependent retrograde survival response.

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Acknowledgements

We thank R. Vallee, M. Lin and P. Silver for plasmids and antibodies; and M. Greenberg, A. Hans, C. Stiles, J. Trinidad, L.-H. Tsai, F. Watson and R. Witt for discussions. This work was supported by grants from the NIH (NS35148 and NS49381), a Quan fellowship (to H.M.H.) and the Claudia Adams Barr Investigator Award (to R.A.S.).

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Correspondence to Rosalind A Segal.

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Supplementary information

Supplementary Video 1

BDNF induces movement of TrkB-GFP positive puncta. TrkB-GFP expressing neurons were stimulated with BDNF and visualized every two minutes. Many GFP-positive puncta move rapidly toward the cell body following BDNF stimulation. (MOV 3046 kb)

Supplementary Video 2

Vehicle control stimulation of TrkB-GFP expressing neurons induces little movement of GFP-positive puncta. TrkB-GFP expressing neurons were control stimulated and visualized every two minutes. Little movement of GFP-positive puncta is observed. (MOV 1127 kb)

Supplementary Fig. 1

Kinesin inhibitor does not affect retrograde transport or phosphorylation of Trk. (a) DRG neurons were treated with the kinesin inhibitor monastrol or vehicle control (DMSO). Fluorescent WGA was added to cell bodies or distal axons for 12 hours. Monastrol inhibits anterograde, but not retrograde, transport of WGA. (b) TrkB-GFP expressing DRG neurons were treated with monastrol or vehicle, then used for the FRAP transport assay. Monastrol has no effect on BDNF-induced retrograde transport of TrkB, although the vehicle (DMSO) slightly decreases transport (compare with Fig. 2b). (c) DRG neurons were treated with monastrol or vehicle, stimulated with neurotrophin at distal axons for 20 min, then fixed and immunostained for phospho-Trk. In control-treated cultures, neurotrophin stimulation caused a 1.86 ± 0.10 fold increase in axon fluorescence, and a 1.19 ± 0.03 fold increase in cell body fluorescence. In monastrol-treated cultures, neurotrophin stimulation caused a 1.90 ± 0.06 fold increase in axon fluorescence, and a 1.29 ± 0.06 fold increase in cell body fluorescence. P < 0.005 in all conditions. (PDF 353 kb)

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Heerssen, H., Pazyra, M. & Segal, R. Dynein motors transport activated Trks to promote survival of target-dependent neurons. Nat Neurosci 7, 596–604 (2004). https://doi.org/10.1038/nn1242

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