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Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy

Abstract

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke1. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia2 and the tolerance conferred by ischemic preconditioning (IPC)3, would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

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Figure 1: Hamartin expression is selectively induced in the CA3 hippocampal area following ischemia.
Figure 2: Hamartin regulates neuronal susceptibility to OGD-induced cell death.
Figure 3: Resistance of CA3 neurons to ischemia is mediated by upregulation of hamartin in vivo.
Figure 4: Hamartin promotes neuronal survival by inhibiting mTORC1 and inducing productive autophagy.

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Acknowledgements

This work was supported by the UK Medical Research Council grant G0500495 and by the Dunhill Medical Trust. A.M.B. is a senior investigator of the UK National Institute for Health Research (NIHR) and received funding from Fondation Leducq for neurovascular coupling. G.H. was funded through the NIHR Integrated Academic Training Programme and Oxford University Clinical Academic Graduate School. B.K. and C.W.D. were supported by the NIHR Biomedical Research Centre. S.N. was supported by the Deutsche Forschungsgemeinschaft. G.T. and S.M.W. received funding from National Health Service Blood and Transplant and the NIHR under its Programme Grants Scheme (NIHR Programmes RP-PG-0310-10001 and -10003). We would also like to thank R. Deacon from the Department of Experimental Psychology, University of Oxford, for providing us with the open-field apparatus and for his guidance with the behavioral experiments. We thank E. Martin Rendon for her input concerning the lentiviral vectors and J. Peeling for his evaluation of the manuscript. We dedicate this paper to the memory of our colleague and mentor, John P. MacManus.

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M.P. initiated and designed the study, carried out the proteomic and biochemical analyses, performed part of the in vitro and in vivo shRNA lentiviral studies and part of the in vitro overexpression studies and their analysis, and wrote the manuscript. G.H. carried out the subcellular fractionation and immunoblotting experiments for Figures 1 and 4. M.X. produced the overexpression lentiviral particles and helped with the analysis of long-lived protein degradation assays. L.C.H. carried out the rat surgeries for the IPC studies and contributed to the development of the subcellular fractionation protocol. S.N. assisted with the rat surgeries and immunofluorescence experiments and contributed in the interpretation of the proteomic data sets. G.T. assisted with the cortical culture experiments. S.M.W. supervised the National Health Service Blood and Transplant collaborative studies and critically reviewed and edited the manuscript. C.W.D. assisted with the IPA. R.C. assisted in the behavioral testing. Z.Z. carried out the rat surgeries to generate the tissue for the proteomic experiments and time course studies. M.M.M. and M.J.A.W. assisted with the design and conduct of the lentiviral overexpression experiments. B.K. supervised and helped with the proteomic analysis. K.V. contributed to the primary culture lentiviral studies, assisted with the in vivo shRNA experiments and designed, carried out and assisted in the analysis of protein degradation, autophagy and necrosis assays and supervised the collaboration. A.M.B. initiated and supervised the whole project. All authors edited the manuscript.

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Correspondence to Alastair M Buchan.

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Supplementary Figures 1–13, Supplementary Tables 1–9 and Supplementary Results and Discussion (PDF 3125 kb)

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Papadakis, M., Hadley, G., Xilouri, M. et al. Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy. Nat Med 19, 351–357 (2013). https://doi.org/10.1038/nm.3097

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