Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS


Expansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders, including Huntington disease [caused by expanded CAG repeats (CAGr) in the HTT gene], and amyotrophic lateral sclerosis [ALS, possibly caused by expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene], of which the molecular mechanisms remain unclear. Here, we demonstrated that lowering the Drosophila homologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and the shortened life-span in Drosophila expressing expanded CAGr or expanded G4C2r. Expression of human tau (htau4R) restored the disease-related phenotypes that had been mitigated by the loss of dtau, suggesting an evolutionarily-conserved role of tau in neurodegeneration. We further revealed that G4C2r expression increased tau accumulation by inhibiting autophagosome–lysosome fusion, possibly due to lowering the level of BAG3, a regulator of autophagy and tau. Taken together, our results reveal a novel mechanism by which expanded G4C2r causes neurodegeneration via an evolutionarily-conserved mechanism. Our findings provide novel autophagy-related mechanistic insights into C9ORF72-ALS and possible entry points to disease treatment.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 2011, 72: 245–256.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR, et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011, 72: 257–268.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Mizielinska S, Gronke S, Niccoli T, Ridler CE, Clayton EL, Devoy A, et al. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science 2014, 345: 1192–1194.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Mori K, Weng SM, Arzberger T, May S, Rentzsch K, Kremmer E, et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 2013, 339: 1335–1338.

    CAS  PubMed  Google Scholar 

  5. 5.

    Zhang K, Daigle JG, Cunningham KM, Coyne AN, Ruan K, Grima JC, et al. Stress granule assembly disrupts nucleocytoplasmic transport. Cell 2018, 173: 958–971 e917.

    Google Scholar 

  6. 6.

    Fay MM, Anderson PJ, Ivanov P. ALS/FTD-associated C9ORF72 repeat RNA promotes phase transitions in vitro and in cells. Cell Rep 2017, 21: 3573–3584.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lee YB, Chen HJ, Peres JN, Gomez-Deza J, Attig J, Stalekar M, et al. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep 2013, 5: 1178–1186.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Fernandez-Nogales M, Cabrera JR, Santos-Galindo M, Hoozemans JJ, Ferrer I, Rozemuller AJ, et al. Huntington’s disease is a four-repeat tauopathy with tau nuclear rods. Nat Med 2014, 20: 881–885.

    CAS  PubMed  Google Scholar 

  9. 9.

    TheHuntington’sDiseaseCollaborativeResearchGroup. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993, 72: 971–983.

  10. 10.

    King A, Al-Sarraj S, Troakes C, Smith BN, Maekawa S, Iovino M, et al. Mixed tau, TDP-43 and p62 pathology in FTLD associated with a C9ORF72 repeat expansion and p.Ala239Thr MAPT (tau) variant. Acta Neuropathol 2013, 125: 303–310.

  11. 11.

    Ballatore C, Lee VM, Trojanowski JQ. Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat Rev Neurosci 2007, 8: 663–672.

    CAS  PubMed  Google Scholar 

  12. 12.

    Andorfer C, Kress Y, Espinoza M, de Silva R, Tucker KL, Barde YA, et al. Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms. J Neurochem 2003, 86: 582–590.

    CAS  PubMed  Google Scholar 

  13. 13.

    Li C, Gotz J. Tau-based therapies in neurodegeneration: opportunities and challenges. Nat Rev Drug Discov 2017, 16: 863–883.

    CAS  PubMed  Google Scholar 

  14. 14.

    Schoch KM, DeVos SL, Miller RL, Chun SJ, Norrbom M, Wozniak DF, et al. Increased 4R-Tau induces pathological changes in a Human-Tau mouse model. Neuron 2016, 90: 941–947.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Guo JL, Buist A, Soares A, Callaerts K, Calafate S, Stevenaert F, et al. The dynamics and turnover of Tau aggregates in cultured cells: Insights into therapies for tauopathies. J Biol Chem 2016, 291: 13175–13193.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Jo C, Gundemir S, Pritchard S, Jin YN, Rahman I, Johnson GV. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52. Nat Commun 2014, 5: 3496.

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Ittner LM, Fath T, Ke YD, Bi M, van Eersel J, Li KM, et al. Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia. Proc Natl Acad Sci U S A 2008, 105: 15997–16002.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Reddy PH. Abnormal tau, mitochondrial dysfunction, impaired axonal transport of mitochondria, and synaptic deprivation in Alzheimer’s disease. Brain Res 2011, 1415: 136–148.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Lu MH, Zhao XY, Yao PP, Xu DE, Ma QH. The mitochondrion: A potential therapeutic target for Alzheimer’s disease. Neurosci Bull 2018, 34: 1127–1130.

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Ito K, Sass H, Urban J, Hofbauer A, Schneuwly S. GAL4-responsive UAS-tau as a tool for studying the anatomy and development of the Drosophila central nervous system. Cell Tissue Res 1997, 290: 1–10.

    CAS  PubMed  Google Scholar 

  21. 21.

    Guo C, Pan Y, Gong Z. Correction to: Recent advances in the genetic dissection of neural circuits in Drosophila. Neurosci Bull 2019, 35: 1138.

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Lu B, Al-Ramahi I, Valencia A, Wang Q, Berenshteyn F, Yang H, et al. Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance. Nat Neurosci 2013, 16: 562–570.

    CAS  PubMed  Google Scholar 

  23. 23.

    Yao Y, Cui X, Al-Ramahi I, Sun X, Li B, Hou J, et al. A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity. Elife 2015, 4.

  24. 24.

    Yu M, Fu Y, Liang Y, Song H, Yao Y, Wu P, et al. Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington’s disease models. Cell Res 2017, 27: 1441–1465.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Al-Ramahi I, Giridharan SSP, Chen YC, Patnaik S, Safren N, Hasegawa J, et al. Inhibition of PIP4Kgamma ameliorates the pathological effects of mutant huntingtin protein. Elife 2017, 6.

  26. 26.

    Burnouf S, Gronke S, Augustin H, Dols J, Gorsky MK, Werner J, et al. Deletion of endogenous Tau proteins is not detrimental in Drosophila. Sci Rep 2016, 6: 23102.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Xu Z, Poidevin M, Li X, Li Y, Shu L, Nelson DL, et al. Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration. Proc Natl Acad Sci U S A 2013, 110: 7778–7783.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Wittmann CW, Wszolek MF, Shulman JM, Salvaterra PM, Lewis J, Hutton M, et al. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 2001, 293: 711–714.

    CAS  PubMed  Google Scholar 

  29. 29.

    Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, Houlden H, et al. Association of missense and 5’-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 1998, 393: 702–705.

    CAS  PubMed  Google Scholar 

  30. 30.

    Liu F, Gong CX. Tau exon 10 alternative splicing and tauopathies. Mol Neurodegener 2008, 3: 8.

    PubMed  PubMed Central  Google Scholar 

  31. 31.

    Himmler A. Structure of the bovine tau gene: alternatively spliced transcripts generate a protein family. Mol Cell Biol 1989, 9: 1389–1396.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Himmler A, Drechsel D, Kirschner MW, Martin DW, Jr. Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains. Mol Cell Biol 1989, 9: 1381–1388.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Heidary G, Fortini ME. Identification and characterization of the Drosophila tau homolog. Mech Dev 2001, 108: 171–178.

    CAS  PubMed  Google Scholar 

  34. 34.

    Adams SJ, Crook RJ, Deture M, Randle SJ, Innes AE, Yu XZ, et al. Overexpression of wild-type murine tau results in progressive tauopathy and neurodegeneration. Am J Pathol 2009, 175: 1598–1609.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Zhu M, Zhang S, Tian X, Wu C. Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification. Autophagy 2017, 13: 1924–1938.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Roberson ED, Halabisky B, Yoo JW, Yao J, Chin J, Yan F, et al. Amyloid-beta/Fyn-induced synaptic, network, and cognitive impairments depend on tau levels in multiple mouse models of Alzheimer’s disease. J Neurosci 2011, 31: 700–711.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Alonso AC, Grundke-Iqbal I, Iqbal K. Alzheimer’s disease hyperphosphorylated tau sequesters normal tau into tangles of filaments and disassembles microtubules. Nat Med 1996, 2: 783–787.

    CAS  PubMed  Google Scholar 

  38. 38.

    Lasagna-Reeves CA, de Haro M, Hao S, Park J, Rousseaux MW, Al-Ramahi I, et al. Reduction of Nuak1 decreases Tau and reverses phenotypes in a tauopathy mouse model. Neuron 2016, 92: 407–418.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Iijima-Ando K, Sekiya M, Maruko-Otake A, Ohtake Y, Suzuki E, Lu B, et al. Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer’s disease-related tau phosphorylation via PAR-1. PLoS Genet 2012, 8: e1002918.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Rapoport M, Dawson HN, Binder LI, Vitek MP, Ferreira A. Tau is essential to beta -amyloid-induced neurotoxicity. Proc Natl Acad Sci U S A 2002, 99: 6364–6369.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Roberson ED, Scearce-Levie K, Palop JJ, Yan F, Cheng IH, Wu T, et al. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer’s disease mouse model. Science 2007, 316: 750–754.

    CAS  PubMed  Google Scholar 

  42. 42.

    Vossel KA, Zhang K, Brodbeck J, Daub AC, Sharma P, Finkbeiner S, et al. Tau reduction prevents Abeta-induced defects in axonal transport. Science 2010, 330: 198.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Lee S, Wang JW, Yu W, Lu B. Phospho-dependent ubiquitination and degradation of PAR-1 regulates synaptic morphology and tau-mediated Abeta toxicity in Drosophila. Nat Commun 2012, 3: 1312.

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Fu H, Possenti A, Freer R, Nakano Y, Villegas NCH, Tang M, et al. A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology. Nat Neurosci 2019, 22: 47–56.

    CAS  PubMed  Google Scholar 

  45. 45.

    Jackson GR, Wiedau-Pazos M, Sang TK, Wagle N, Brown CA, Massachi S, et al. Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 2002, 34: 509–519.

    CAS  PubMed  Google Scholar 

  46. 46.

    Wang Y, Mandelkow E. Tau in physiology and pathology. Nat Rev Neurosci 2016, 17: 5–21.

    PubMed  Google Scholar 

  47. 47.

    Guthrie CR, Kraemer BC. Proteasome inhibition drives HDAC6-dependent recruitment of tau to aggresomes. J Mol Neurosci 2011, 45: 32–41.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Feuillette S, Miguel L, Frebourg T, Campion D, Lecourtois M. Drosophila models of human tauopathies indicate that Tau protein toxicity in vivo is mediated by soluble cytosolic phosphorylated forms of the protein. J Neurochem 2010, 113: 895–903.

    CAS  PubMed  Google Scholar 

  49. 49.

    Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 2007, 3: 452–460.

    CAS  PubMed  Google Scholar 

  50. 50.

    Li Z, Wang C, Wang Z, Zhu C, Li J, Sha T, et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature 2019, 575: 203–209.

    CAS  PubMed  Google Scholar 

  51. 51.

    Mahr A, Aberle H. The expression pattern of the Drosophila vesicular glutamate transporter: a marker protein for motoneurons and glutamatergic centers in the brain. Gene Expr Patterns 2006, 6: 299–309.

    CAS  PubMed  Google Scholar 

  52. 52.

    DeVorkin L, Gorski SM. Monitoring autophagic flux using Ref(2)P, the Drosophila p62 ortholog. Cold Spring Harb Protoc 2014, 2014: 959–966.

    PubMed  Google Scholar 

  53. 53.

    Chen CS, Chen WN, Zhou M, Arttamangkul S, Haugland RP. Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy. J Biochem Biophys Methods 2000, 42: 137–151.

    CAS  PubMed  Google Scholar 

  54. 54.

    Gamerdinger M, Hajieva P, Kaya AM, Wolfrum U, Hartl FU, Behl C. Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3. EMBO J 2009, 28: 889–901.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Kathage B, Gehlert S, Ulbricht A, Ludecke L, Tapia VE, Orfanos Z, et al. The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1. Biochim Biophys Acta Mol Cell Res 2017, 1864: 62–75.

    CAS  PubMed  Google Scholar 

  56. 56.

    Ji C, Tang M, Zeidler C, Hohfeld J, Johnson GV. BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes. Autophagy 2019, 15: 1199–1213.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Lei Z, Brizzee C, Johnson GV. BAG3 facilitates the clearance of endogenous tau in primary neurons. Neurobiol Aging 2015, 36: 241–248.

    CAS  PubMed  Google Scholar 

  58. 58.

    Arndt V, Dick N, Tawo R, Dreiseidler M, Wenzel D, Hesse M, et al. Chaperone-assisted selective autophagy is essential for muscle maintenance. Curr Biol 2010, 20: 143–148.

    CAS  PubMed  Google Scholar 

  59. 59.

    Bieniek KF, Murray ME, Rutherford NJ, Castanedes-Casey M, DeJesus-Hernandez M, Liesinger AM, et al. Tau pathology in frontotemporal lobar degeneration with C9ORF72 hexanucleotide repeat expansion. Acta Neuropathol 2013, 125: 289–302.

    CAS  PubMed  Google Scholar 

  60. 60.

    He H, Huang W, Wang R, Lin Y, Guo Y, Deng J, et al. Amyotrophic Lateral Sclerosis-associated GGGGCC repeat expansion promotes Tau phosphorylation and toxicity. Neurobiol Dis 2019, 130: 104493.

    CAS  PubMed  Google Scholar 

  61. 61.

    Bolkan BJ, Kretzschmar D. Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila. Dev Neurobiol 2014, 74: 1210–1225.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Berger Z, Roder H, Hanna A, Carlson A, Rangachari V, Yue M, et al. Accumulation of pathological tau species and memory loss in a conditional model of tauopathy. J Neurosci 2007, 27: 3650–3662.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Yamada K, Patel TK, Hochgrafe K, Mahan TE, Jiang H, Stewart FR, et al. Analysis of in vivo turnover of tau in a mouse model of tauopathy. Mol Neurodegener 2015, 10: 55.

    PubMed  PubMed Central  Google Scholar 

  64. 64.

    Khlistunova I, Biernat J, Wang Y, Pickhardt M, von Bergen M, Gazova Z, et al. Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs. J Biol Chem 2006, 281: 1205–1214.

    CAS  PubMed  Google Scholar 

  65. 65.

    Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, et al. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 1994, 78: 761–771.

    CAS  PubMed  Google Scholar 

  66. 66.

    Wong E, Cuervo AM. Integration of clearance mechanisms: the proteasome and autophagy. Cold Spring Harb Perspect Biol 2010, 2: a006734.

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 2008, 456: 264–268.

    CAS  PubMed  Google Scholar 

  68. 68.

    Lee MJ, Lee JH, Rubinsztein DC. Tau degradation: the ubiquitin-proteasome system versus the autophagy-lysosome system. Prog Neurobiol 2013, 105: 49–59.

    CAS  PubMed  Google Scholar 

  69. 69.

    Behl C. Breaking BAG: The co-chaperone BAG3 in health and disease. Trends Pharmacol Sci 2016, 37: 672–688.

    CAS  PubMed  Google Scholar 

Download references


We thank Prof. Peng Jin and Ranhui Duan for providing the G4C2 Drosophila, Prof. L. Partridge, Haihuai He, and Peng Lei for providing the dtau-knockout Drosophila, Prof. Yongqin Zhang for providing related tool Drosophila stocks and UAS-htau4R [28], and Prof. Shouqing Luo for providing the Lamp1-mcherry vector. This work was supported by the National Natural Science Foundation of China (81925012 and 31961130379) and a Newton Advanced Fellowship (NAF_R1_191045).

Author information



Corresponding authors

Correspondence to Jian Wang or Lixiang Ma or Boxun Lu.

Ethics declarations

Conflict of interest

The authors claim no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1956 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wen, X., An, P., Li, H. et al. Tau Accumulation via Reduced Autophagy Mediates GGGGCC Repeat Expansion-Induced Neurodegeneration in Drosophila Model of ALS. Neurosci. Bull. 36, 1414–1428 (2020). https://doi.org/10.1007/s12264-020-00518-2

Download citation


  • ALS
  • C9orf72
  • G4C2
  • Huntington disease