Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia (original) (raw)

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Acknowledgements

We are indebted to the patients and their families for their participation in this project. We thank A. Knehr, N. Todt, E. Jasovskaja, B. Schmoll, A.-K. Rikardsson, A. Birve, A.-C. Nilsson and the Ulm Neurology biobank team for technical assistance. We also thank the many physicians who provided samples for this study, particularly L. Brättström (Karlskrona Hospital, Sweden), S. Saker (Généthon cell and DNA bank), S. Forlani (ICM DNA and cell bank), and E. LeGuern and C. Cazeneuve (APHP, Hôpital Pitié-Salpêtrière). We also thank P. Sarvari and D.Y.R. Stainier for early studies on the role of TBK1 in Zebrafish neurodegeneration. The authors would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about. This work was supported by grants from the German Federal Ministry of Education and Research (STRENGTH consortium, Sophia and BiomarkAPD and BMBF; 01GI0704, German network for ALS research (MND-NET), O1GI1007A Competence net neurodegenerative dementias/Frontotemporal lobar degeneration consortium (FTLDc)), the German Research Foundation (SYNERGY excellence cluster), the Charcot Foundation for ALS Research (A.C.L., J.H.W.), the virtual Helmholtz Institute “RNA-Dysmetabolismus in ALS and FTD” and the DFG-funded Swabian ALS Registry, the LOEWE Centrum for Gene and Cell therapy and LOEWE Ub-Net (I.D. and B.R.), the Swedish Research Council, the Swedish Brain Power Foundation, the Swedish Brain Research Foundation and the Ulla-Carin Lindquist Foundation, the Hållsten Research Foundation, the Swedish Association for the Neurologically Disabled, the Knut and Alice Wallenberg Foundation, by the Association pour la Recherche sur la Sclérose latérale amyotrophique et autres maladies du motoneurone (ARSla, France, contract R13132DD) and the Association française contre les myopathies (AFM, France, contract R11038DD to S.M.).

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Author notes

1. Axel Freischmidt, Thomas Wieland, Benjamin Richter and Wolfgang Ruf: These authors contributed equally to this work.
2. Tim M Strom, Peter M Andersen and Jochen H Weishaupt: These authors jointly directed this work.

Authors and Affiliations

1. Department of Neurology, Ulm University, Ulm, Germany
   Axel Freischmidt, Wolfgang Ruf, Kathrin Müller, Nicolai Marroquin, Annemarie Hübers, Patrick Weydt, Johannes Dorst, Marisa S Feiler, Markus Otto, Karin M Danzer, Albert C Ludolph, Peter M Andersen & Jochen H Weishaupt
2. Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany
   Thomas Wieland, Elisabeth Graf, Peter Lichtner, Thomas Meitinger & Tim M Strom
3. Institute of Biochemistry II, Goethe University Medical School, Frankfurt, Germany
   Benjamin Richter, Veronique Schaeffer & Ivan Dikic
4. Department of Pharmacology and Clinical Neurosience, Umeå University, Umeå, Sweden
   Frida Nordin, Ulrika Nordström & Peter M Andersen
5. Institute of Physiology and Institute of Molecular Medicine, University of Lisbon, Lisbon, Portugal
   Susana Pinto & Mamede de Carvalho
6. Department of Neurology, Karolinska Hospital Huddinge, Stockholm, Sweden
   Rayomond Press
7. Institut du Cerveau et de la Moelle épinière (ICM), CNRS UMR7225, Inserm U1127, Sorbonne Universités, Université Pierre et Marie (UPMC) P6 UMRS1127, Paris, France
   Stéphanie Millecamps
8. Centre Hospitalier Territorial Gaston Bourret, Noumea, New Caledonia
   Nicolas Molko
9. Centre de référence SLA, Hôpital Neurologique Pierre Wertheimer, CHU de Lyon, Bron, France
   Emilien Bernard
10. Centre de Référence Maladies Neuromusculaires et SLA, Hôpital Archet, CHU de Nice, France
    Claude Desnuelle & Marie-Hélène Soriani
11. Institute of Anatomy and Cell Biology, Ulm University, Faculty of Medicine, Ulm, Germany
    Stefan Putz & Tobias M Boeckers
12. Charité University Hospital, Humboldt-University, Berlin, Germany
    Thomas Meyer
13. Department of Neurology, Technical University of Munich, Munich, Germany
    Andrea S Winkler & Juliane Winkelman
14. Department of Neurosciences, Hospital de Santa Maria-CHLN, Lisbon, Portugal
    Mamede de Carvalho
15. Institute of Pathology–Laboratory of Neuropathology, Ulm University, Ulm, Germany
    Dietmar R Thal
16. Department of Medical Biosciences, Umeå University, Umeå, Sweden
    Thomas Brännström
17. Institute of Human Genetics, Ulm University, Ulm, Germany
    Nicolai Marroquin & Alexander E Volk
18. Institute of Human Genetics, University Clinic Hamburg-Eppendorf, Hamburg, Germany
    Alexander E Volk
19. University of Oulu Biocenter, Faculty of Biochemistry and Molecular Medicine, Oulu, Finland
    Petri Kursula
20. Department of Biomedicine, University of Bergen, Bergen, Norway
    Petri Kursula
21. Institute of Human Genetics, Technische Universität München, Munich, Germany
    Thomas Meitinger & Tim M Strom
22. SyNergy, Munich Cluster for Systems Neurology, Ludwig Maximilians Universität München, Munich, Germany
    Thomas Meitinger

Authors

1. Axel Freischmidt
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2. Thomas Wieland
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3. Benjamin Richter
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4. Wolfgang Ruf
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5. Veronique Schaeffer
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6. Kathrin Müller
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7. Nicolai Marroquin
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8. Frida Nordin
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9. Annemarie Hübers
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10. Patrick Weydt
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11. Susana Pinto
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12. Rayomond Press
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13. Stéphanie Millecamps
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14. Nicolas Molko
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15. Emilien Bernard
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16. Claude Desnuelle
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17. Marie-Hélène Soriani
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18. Johannes Dorst
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19. Elisabeth Graf
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20. Ulrika Nordström
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21. Marisa S Feiler
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22. Stefan Putz
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23. Tobias M Boeckers
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24. Thomas Meyer
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25. Andrea S Winkler
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26. Juliane Winkelman
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27. Mamede de Carvalho
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28. Dietmar R Thal
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29. Markus Otto
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30. Thomas Brännström
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31. Alexander E Volk
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32. Petri Kursula
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33. Karin M Danzer
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34. Peter Lichtner
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35. Ivan Dikic
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36. Thomas Meitinger
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37. Albert C Ludolph
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38. Tim M Strom
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39. Peter M Andersen
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40. Jochen H Weishaupt
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Contributions

T.M.S., P.M.A., A.C.L., J.H.W., T. Meitinger, I.D., T.W., A.F., B.R., W.R., V.S., T. Brännström, P.K., P.L., A.E.V. and D.R.T. designed experiments, and analyzed and interpreted data. K.M., N.M., E.G., U.N., M.S.F., K.M.D., D.R.T., T. Brännström, A.E.V. and P.L. performed and analyzed experiments. F.N., A.H., P.W., S. Pinto, R.P., S.M., N.M., E.B., C.D., M.-H.S., J.D., T. Meyer, A.S.W., J.W., M.d.C., M.O., A.C.L., P.M.A. and J.H.W. clinically characterized patients. J.H.W., P.M.A., T.M.S., T. Meitinger, I.D., B.R. and A.C.L. wrote the manuscript. S. Putz and T.M.B. provided patient-derived cell lines.

Corresponding authors

Correspondence toPeter M Andersen or Jochen H Weishaupt.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quantile-quantile plots showing the expected −log10 p-values for different burden tests.

CMC tests were used to compare 251 case and 827 control exomes. The 95% confidence interval is shown in grey. This gene is located in a non-unique region.

Supplementary Figure 3 Neuropathological findings in the ALS/FTD case with TBK1 p.690-713del mutation (patient V-5 of family 11 in Fig. 2).

(a) Micrograph of a section from right temporal lobe showing microvacuolation and gliosis (Hematoxylin/Eosin). (b) Micrograph showing Type B TDP-43+ perinuclear inclusion in neurons in lamina III of the temporal lobe (polyclonal anti-TDP43, Proteintech). (c) Micrograph showing p62+ perinuclear inclusions in lamina III in the right parahippocampal gyrus (monoclonal anti-p62, BD Biosciences).

Supplementary Figure 4 TBK1 expression in patient-derived LCLs, fibroblasts and keratinocytes at mRNA and protein level.

(a) qRT-PCR measurement of TBK1 mRNA abundance in LCLs, fibroblasts and keratinocytes of 3-5 healthy controls and respective TBK1 mutant cell lines normalized to TBP. The bars indicate mean ± s.d. (b) PCR products amplified from LCL-derived cDNA using oligonucleotides spanning Exon 3 and 5 of TBK1 mRNA separated on a 1% agarose gel. The arrowhead marks a band exclusively observed in LCLs carrying the direct TBK1 splice site mutation c.358+2T>C. Sequencing of this band reveals the retention of the first 226 bp of the Intron separating Exon 4 and 5 in the mature mRNA and usage of an alternative splice site. The molecular size marker is indicated in bp. (c) PCR products amplified from fibroblast-derived cDNA using oligonucleotides spanning Exon 17/18 and 21 of TBK1 mRNA separated on a 1% agarose gel. The arrowhead marks a band exclusively observed in fibroblasts carrying the TBK1 c.2138+2T>C mutation. Sequencing of this band reveals the predicted skipping of Exon 20 in the mature mRNA. The molecular size marker is indicated in bp. (b, c) are representative images out of 2 replicates. (d, e) Western blots comparing TBK1 protein expression of LCLs (d) and fibroblasts (e) from healthy controls and respective TBK1 mutant cell lines. Western blots were repeated three times The arrowhead in (e) marks a band corresponding to the expression of a truncated TBK1 protein missing amino acids 690-713. ß-Actin is shown as a loading control. Molecular weights are indicated in kDa (All TBK1- and Actin-bands in (d) result from the same Western blot but were re-arranged for better presentation).

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Supplementary Figure 5 Uncropped IB images corresponding to Supplementary Figure 4 and 5

Supplementary Figure 6 Functional analysis of TBK1 missense variance

(a) Co-immunoprecipitation (Co-IP): Flag-TBK1-WT or the indicated mutants were expressed in HEK293T cells. The fusion proteins were purified and immobilized from HEK293T cell lysates using anti-Flag beads followed by incubation with recombinant GST-OPTN. Co-precipitated GST-OPTN was detected with an antibody against OPTN (top panel). Protein levels of immobilized Flag-TBK1 WT were analyzed by immunoblotting (anti-Flag; lower panel). Flag-beads only (IgG only) were used as a negative control. WT (wild-type); KD (kinase-dead TBK1 mutation; p.Lys38Ala). (b) GST pull-down assay (upper panel): Flag-TBK1-WT or the indicated mutants from HEK293T cell lysates (as in a) were incubated with recombinant, immobilized GST-OPTN. Equal GST-OPTN protein levels were confirmed by Ponceau S staining. Arrowhead points to full-length GST-OPTN. Flag-TBK1 protein expression was analyzed by immunoblotting (lower panel). (c) Co-IP of myc-IRF3 and Flag-TBK1-WT or the indicated mutants from HEK293T cells using anti-Flag agarose. (Co-)Immunoprecipitated proteins were analyzed by IB with the indicated antibodies (upper panel). Asterisk points to heavy IgG bands. Flag-TBK1 and myc-IRF3 protein expression was analyzed by IB with the indicated antibodies (lower panel). IB images are representatives of three repetitions.

Supplementary Figure 7 Rare ALS-associated missense variants identified in our study (MAF < 0.0001) mapped onto the 3D structure* of human TBK1.

Shown is a TBK1 dimer, in which one monomer is white, and the second one is coloured according to domain structure (kinase domain, blue; ubiquitin-like domain, orange; coiled-coil domain 1, green). The N and C termini of both monomers are labelled, the additional ends of the protein chain relate to missing loops in the crystal structure. Displayed variants are p.Arg47His (R47H), p.Tyr105Ser (Y105S), p.Ile305Thr (I305T), p.Arg308Gln (R308Q), p.Arg357Gln (R357Q), p.Met559Arg (M559R), p.Ala571Val (A571V), p.Met598Val (M598V) and p.Glu643del (E643del). The variants selected for functional studies based on 3D structure modeling are highlighted in red. Met559 is buried inside a bundle of 3 helices, and the p.Met559Arg substitution must induce at least a local structural rearrangement, which could lead to effects on overall folding and dimerization. Arg308 is at the interface between the kinase and ubiquitin-like domains, and the p.Arg308Gln mutation will break its salt bridge to Asp304. Arg357 is located within the ubiquitin-like domain, but also at the dimer interface, and it is heavily involved in a salt bridge network. The p.Arg357Gln substitution must affect this network of interactions central to TBK1 dimerization. The locations of the C termini are indicated; note that the very C-terminal domain after residue 657 (containing the p.Glu696Lys mutation) was not included in the crystal structure. The p.Glu696Lys mutation was nevertheless included in the functional analysis due to its position in the functionally important coiled-coil domain, as was also the p.Arg47His variant because of its location in the kinase domain and its high evolutionary conservation. * Larabi, A. et al. Crystal structure and mechanism of activation of TANK-binding kinase 1. Cell Rep 3, 734–46 (2013).

Supplementary Figure 8 Uncropped IB images corresponding to Figure 3

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Freischmidt, A., Wieland, T., Richter, B. et al. Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia.Nat Neurosci 18, 631–636 (2015). https://doi.org/10.1038/nn.4000

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• Received: 11 March 2015
• Accepted: 17 March 2015
• Published: 24 March 2015
• Issue Date: May 2015
• DOI: https://doi.org/10.1038/nn.4000