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TBC1D24 regulates axonal outgrowth and membrane trafficking at the growth cone in rodent and human neurons

Abstract

Mutations in TBC1D24 are described in patients with a spectrum of neurological diseases, including mild and severe epilepsies and complex syndromic phenotypes such as Deafness, Onycodystrophy, Osteodystrophy, Mental Retardation and Seizure (DOORS) syndrome. The product of TBC1D24 is a multifunctional protein involved in neuronal development, regulation of synaptic vesicle trafficking, and protection from oxidative stress. Although pathogenic mutations in TBC1D24 span the entire coding sequence, no clear genotype/phenotype correlations have emerged. However most patients bearing predicted loss of function mutations exhibit a severe neurodevelopmental disorder. Aim of the study is to investigate the impact of TBC1D24 knockdown during the first stages of neuronal differentiation when axonal specification and outgrowth take place. In rat cortical primary neurons silenced for TBC1D24, we found defects in axonal specification, the maturation of axonal initial segment and action potential firing. The axonal phenotype was accompanied by an impairment of endocytosis at the growth cone and an altered activation of the TBC1D24 molecular partner ADP ribosylation factor 6. Accordingly, acute knockdown of TBC1D24 in cerebrocortical neurons in vivo analogously impairs callosal projections. The axonal defect was also investigated in human induced pluripotent stem cell-derived neurons from patients carrying TBC1D24 mutations. Reprogrammed neurons from a patient with severe developmental encephalopathy show significant axon formation defect that were absent from reprogrammed neurons of a patient with mild early onset epilepsy. Our data reveal that alterations of membrane trafficking at the growth cone induced by TBC1D24 loss of function cause axonal and excitability defects. The axonal phenotype correlates with the disease severity and highlight an important role for TBC1D24 in connectivity during brain development.

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References

  1. 1.

    Balestrini S, Milh M, Castiglioni C, Lüthy K, Finelli M, Taylor JC. TBC1D24 genotype – phenotype correlation Epilepsies and other neurologic features. Neurology. 2016;87:77–85.

  2. 2.

    Azaiez H, Booth KT, Bu F, Huygen P, Shibata SB, Shearer AE, et al. TBC1D24 mutation causes autosomal-dominant nonsyndromic hearing Loss. Hum Mutat. 2014;35:819–23.

  3. 3.

    Rehman AU, Santos-Cortez RLP, Morell RJ, Drummond MC, Ito T, Lee K, et al. Mutations in TBC1D24, a gene associated with epilepsy, also cause nonsyndromic deafness DFNB86. Am J Human Genet. 2014;94:144–52.

  4. 4.

    Zhang L, Hu L, Chai Y, Pang X, Yang T, Wu H. A Dominant mutation in the stereocilia-expressing gene TBC1D24 is a probable cause for nonsyndromic hearing impairment. Hum Mutat. 2014;35:814–8.

  5. 5.

    Finelli MJ, Oliver PL. TLDc proteins: new players in the oxidative stress response and neurological disease. Mamm Genome. 2017;28:395–406.

  6. 6.

    Fischer B, Lüthy K, Paesmans J, De Koninck C, Maes I, Swerts J, et al. Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function. Nat Struct Mol Biol. 2016;23:965–73.

  7. 7.

    Uytterhoeven V, Kuenen S, Kasprowicz J, Miskiewicz K, Verstreken P. Loss of Skywalker reveals synaptic endosomes as sorting stations for synaptic vesicle proteins. Cell. 2011;145:117–32.

  8. 8.

    Fernandes AC, Uytterhoeven V, Kuenen S, Wang YC, Slabbaert JR, Swerts J, et al. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration. J Cell Biol. 2014;207:453–62.

  9. 9.

    Falace A, Filipello F, La Padula V, Vanni N, Madia F, De Pietri Tonelli D, et al. TBC1D24, an ARF6-interacting protein, is mutated in familial infantile myoclonic epilepsy. Am J Hum Genet. 2010;87:365–70.

  10. 10.

    Falace A, Buhler E, Fadda M, Watrin F, Lippiello P, Pallesi-Pocachard E, et al. TBC1D24 regulates neuronal migration and maturation through modulation of the ARF6-dependent pathway. Proc Natl Acad Sci USA. 2014;111:2337–42.

  11. 11.

    Milh M, Falace A, Villeneuve N, Vanni N, Cacciagli P, Assereto S, et al. Novel compound heterozygous mutations in TBC1D24 cause familial malignant migrating partial seizures of infancy. Hum Mutat. 2013;34:869–72.

  12. 12.

    Finelli MJ, Sanchez-Pulido L, Liu KX, Davies KE, Oliver PL. The evolutionarily conserved Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain is neuroprotective against oxidative stress. J Biol Chem. 2016;291:2751–63.

  13. 13.

    Barry J, Gu Y, Jukkola P, O’Neill B, Gu H, Mohler PJ, et al. Ankyrin-G directly binds to kinesin-1 to transport voltage-gated Na + channels into axons. Dev Cell. 2014;28:117–31.

  14. 14.

    McCormick DA, Shu Y, Yu Y. Neurophysiology: hodgkin and huxley model — still standing? Nature. 2007;445:E1–2.

  15. 15.

    Naundorf B, Wolf F, Volgushev M. Unique features of action potential initiation in cortical neurons. Nature. 2006;440:1060–3.

  16. 16.

    Namba T, Kibe Y, Funahashi Y, Nakamuta S, Takano T, Ueno T, et al. Pioneering axons regulate neuronal polarization in the developing cerebral cortex. Neuron. 2014;81:814–29.

  17. 17.

    Witteveen JS, Willemsen MH, Dombroski TC, van Bakel NH, Nillesen WM, van Hulten JA, et al. Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity. Nat Genet. 2016;48:877–87.

  18. 18.

    Hernández-Deviez DJ, Roth MG, Casanova JE, Wilson JM. ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase. Mol Biol Cell. 2004;15:111–20.

  19. 19.

    Eva R, Crisp S, Marland JRK, Norman JC, Kanamarlapudi V, ffrench-Constant C, et al. ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons. J Neurosci. 2012;32:10352–64.

  20. 20.

    Franssen EHP, Zhao R-R, Koseki H, Kanamarlapudi V, Hoogenraad CC, Eva R, et al. Exclusion of integrins from CNS axons is regulated by Arf6 activation and the AIS. J Neurosci. 2015;35:8359–75.

  21. 21.

    Vitriol EA, Zheng JQ. Growth cone travel in space and time: the cellular ensemble of cytoskeleton, adhesion, and membrane. Neuron. 2012;73:1068–81.

  22. 22.

    Sivadasan R, Hornburg D, Drepper C, Frank N, Jablonka S, Hansel A, et al. C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons. Nat Neurosci. 2016;19:1610–8.

  23. 23.

    Bellani S, Mescola A, Ronzitti G, Tsushima H, Tilve S, Canale C, et al. GRP78 clustering at the cell surface of neurons transduces the action of exogenous alpha-synuclein. Cell Death Differ. 2014;21:1971–83.

  24. 24.

    Ageta-Ishihara N, Miyata T, Ohshima C, Watanabe M, Sato Y, Hamamura Y, et al. Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation. Nat Commun. 2013;4:1–11.

  25. 25.

    Bonanomi D, Fornasiero EF, Valdez G, Halegoua S, Menegon A, Valtorta F. Identification of a developmentally regulated pathway of membrane retrieval in neuronal growth cones. J Cell Sci. 2009;121(Pt 22):3757–69.

  26. 26.

    Fruscione F, Valente P, Sterlini B, Romei A, Baldassari S, Fadda M, et al. PRRT2 controls neuronal excitability by negatively modulating Na + channel 1.2/1.6 activity. Brain. 2018;141:1000–16.

  27. 27.

    Campeau PM, Kasperaviciute D, Lu JT, Burrage LC, Kim C, Hori M, et al. The genetic basis of DOORS syndrome: an exome-sequencing study. Lancet Neurol. 2014;13:44–58.

  28. 28.

    Lozano R, Herman K, Rothfuss M, Rieger H, Bayrak-Toydemir P, Aprile D, et al. Clinical intrafamilial variability in lethal familial neonatal seizure disorder caused by TBC1D24 mutations. Am J Med Genet, Part A. 2016;170:3207–14.

  29. 29.

    Corbett MA, Bahlo M, Jolly L, Afawi Z, Gardner AE, Oliver KL, et al. A focal epilepsy and intellectual disability syndrome is due to a mutation in TBC1D24. Am J Hum Genet. 2010;87:371–5.

  30. 30.

    Yin J, Chen W, Chao ES, Soriano S, Wang L, Wang W, et al. Otud7a knockout mice recapitulate many neurological features of 15q13.3 microdeletion syndrome. Am J Human Genet. 2018;102:296–308.

  31. 31.

    Weckhuysen S, Mandelstam S, Suls A, Audenaert D, Deconinck T, Claes LRF, et al. KCNQ2 encephalopathy: Emerging phenotype of a neonatal epileptic encephalopathy. Ann Neurol. 2012;71:15–25.

  32. 32.

    Miceli F, Soldovieri MV, Ambrosino P, De Maria M, Migliore M, Migliore R, et al. Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits. J Neurosci. 2015;35:3782–93.

  33. 33.

    Devaux J, Abidi A, Roubertie A, Molinari F, Becq H, Lacoste C, et al. A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression-burst enhances Kv7/M channel activity. Epilepsia. 2016;57:e87–93.

  34. 34.

    Miura Y, Hongu T, Yamauchi Y, Funakoshi Y, Katagiri N, Ohbayashi N, et al. ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6 in mouse hippocampal neurons. Biochem J. 2016;473:2591–602.

  35. 35.

    Donaldson JG, Jackson CL. ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol. 2011;12:362–75.

  36. 36.

    Paleotti O, Macia E, Luton F, Klein S, Partisani M, Chardin P, et al. The small G-protein Arf6GTPrecruits the AP-2 adaptor complex to membranes. J Biol Chem. 2005;280:21661–6.

  37. 37.

    Kyung JW, Cho IH, Lee S, Song WK, Ryan TA, Hoppa MB, et al. Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons. Sci Rep. 2017;7:41620.

  38. 38.

    Krey JF, Dumont RA, Wilmarth PA, David LL, Johnson KR, Barr-Gillespie PG. ELMOD1 stimulates ARF6-GTP hydrolysis to stabilize apical structures in developing vestibular hair cells. J Neurosci. 2018;38:843–57.

  39. 39.

    Finelli MJ, Aprile D, Castroflorio E, Jeans A, Moschetta M, Chessum L, et al. The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons. Hum Mol Genet. 2018;28:584–97.

  40. 40.

    Tona R, Chen W, Nakano Y, Reyes LD, Petralia RS, Wang YX, et al. The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy. Hum Mol Genet. 2019. [Epub ahead of print].

  41. 41.

    Tagliatti E, Fadda M, Falace A, Benfenati F, Fassio A. Arf6 regulates the cycling and the readily releasable pool of synaptic vesicles at hippocampal synapse. eLife. 2016;5:1–18.

  42. 42.

    Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:45e–45.

  43. 43.

    Valente P, Lignani G, Medrihan L, Bosco F, Contestabile A, Lippiello P, et al. Cell adhesion molecule L1 contributes to neuronal excitability regulating the function of voltage-gated Na + channels. J Cell Sci. 2016;129:1878–91.

  44. 44.

    Bean BP. The action potential in mammalian central neurons. Nat Rev Neurosci. 2007;8:451–65.

  45. 45.

    Shu Y, Duque A, Yu Y, Haider B, McCormick DA. Properties of action-potential initiation in neocortical pyramidal cells: evidence from whole cell axon recordings. J Neurophysiol. 2007;97:746–60.

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Acknowledgements

We would like to thank Dr. Anna Corradi for valuable discussion, Dr. Margherita Mancardi for clinical description of the patients, Sara Pepe for help with imaging analysis, Monica Traverso for genotyping and Silvia Casagrande for assistance in primary cell and plasmid preparation. This work is supported by the University of Genoa (FRA grant to A.F.), Compagnia di San Paolo (Grant 2015.0546 to F.B.) and CARIPLO Foundation Milano (Grant 2013 0879 to F.B.). The EU FP7 Integrating Project “Desire” (Grant no. 602531), to FB, FZ and AF is also acknowledged. D.A. was supported in 2018 by a Fondazione CARIGE fellowship (Genova).

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Correspondence to Anna Fassio.

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