Tsc2-Rheb signaling regulates EphA-mediated axon guidance

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Abstract

Tuberous sclerosis complex is a disease caused by mutations in the TSC1 or TSC2 genes, which encode a protein complex that inhibits mTOR kinase signaling by inactivating the Rheb GTPase. Activation of mTOR promotes the formation of benign tumors in various organs and the mechanisms underlying the neurological symptoms of the disease remain largely unknown. We found that Tsc2 haploinsufficiency in mice caused aberrant retinogeniculate projections that suggest defects in EphA receptor–dependent axon guidance. We also found that EphA receptor activation by ephrin-A ligands in neurons led to inhibition of extracellular signal–regulated kinase 1/2 (ERK1/2) activity and decreased inhibition of Tsc2 by ERK1/2. Thus, ephrin stimulation inactivates the mTOR pathway by enhancing Tsc2 activity. Furthermore, Tsc2 deficiency and hyperactive Rheb constitutively activated mTOR and inhibited ephrin-induced growth cone collapse. Our results indicate that TSC2-Rheb-mTOR signaling cooperates with the ephrin-Eph receptor system to control axon guidance in the visual system.

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Figure 1: Increased mTOR activity in Tsc2+/− retina in vivo.
Figure 2: Tsc2+/− mice have defects in ipsilateral retinogeniculate projections.
Figure 3: Tsc2+/− mice have defects in topographic mapping of contralateral projections in dLGN.
Figure 4: Tsc2 deficiency and hyperactive Rheb inhibit ephrin-A–induced growth cone collapse.
Figure 5: Inactivation of ERK1/2 and mTOR pathway in ephrin stimulated neurons.
Figure 6: Ephrin inhibits axonal protein synthesis and the local translation of a beta-actin reporter in growth cones.
Figure 7: Tsc2 is required for ephrin-A1–stimulated mTOR inactivation.
Figure 8: Regulation of TSC2 (Ser664) phosphorylation by ephrin-A1 is involved in growth cone dynamics.

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Acknowledgements

We thank B. Stevens and D. Shafer for assistance with multi-threshold LGN analysis, J. Twiss and T. Merianda for assistance with fluorescence recovery after photobleaching and M. Bear, E. Osterweil and D. Krueger for assistance with metabolic labeling experiments. The Tsc2loxP/loxP mice were a gift from M. Gambello, and the Tsc1loxP/loxP mice were gift of D. Kwiatkowski. We are also grateful to B. Stevens, C. Chen, Z. He and members of the Sahin laboratory for critical reading of the manuscript and to L. Mariani, A. Sadowski and S. Goldman for technical assistance. This work was supported in part by grants from the US National Institutes of Health (NS58956 to M.S. and HD025938 to E.B.P.), the John Merck Scholars Fund, Tuberous Sclerosis Alliance, the Manton Foundation and Children's Hospital Boston Translational Research Program to M.S. and the Children's Hospital Boston Mental Retardation and Developmental Disabilities Research Center (P01 HD18655). D.N. is supported by a Mentor Based Postdoctoral Fellowship from Autism Speaks. A.D.N. is supported by a grant from the Hearst Foundation. H.B. is supported by an Howard Hughes Medical Institute Research Training Fellowship for Medical Students.

Author information

D.N. performed most of the experiments. A.D.N. carried out the preliminary biochemistry experiments on the relationship between Eph and Tsc pathways. D.N., I.K., T.H. and M.S. conducted and analyzed the retinal projection experiments. J.M.H. and H.B. performed the quantification on the growth cone collapse assays and immunocytochemical staining. S.D. provided guidance with the initial experiments on the Tsc2+/ mice. P.P.P. provided reagents and troubleshooting for experiments on Tsc2 Ser664 phosphorylation. S.C. and E.B.P. contributed unpublished preliminary data on the Eph-Tsc interaction. M.S. supervised the project. D.N., E.B.P. and M.S. wrote and edited the manuscript.

Correspondence to Mustafa Sahin.

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Nie, D., Di Nardo, A., Han, J. et al. Tsc2-Rheb signaling regulates EphA-mediated axon guidance. Nat Neurosci 13, 163–172 (2010) doi:10.1038/nn.2477

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