Abstract

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal–lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15–Vps34 complex and the Nischarin–Pak1 signaling hub in the development of the telencephalon.

  • Subscribe to Nature Neuroscience for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Ayala, R., Shu, T. & Tsai, L. H. Trekking across the brain: the journey of neuronal migration. Cell 128, 29–43 (2007).

  2. 2.

    Noctor, S. C., Flint, A. C., Weissman, T. A., Dammerman, R. S. & Kriegstein, A. R. Neurons derived from radial glial cells establish radial units in neocortex. Nature 409, 714–720 (2001).

  3. 3.

    Heng, J. I., Chariot, A. & Nguyen, L. Molecular layers underlying cytoskeletal remodelling during cortical development. Trends Neurosci. 33, 38–47 (2010).

  4. 4.

    Gleeson, J. G. & Walsh, C. A. Neuronal migration disorders: from genetic diseases to developmental mechanisms. Trends Neurosci. 23, 352–359 (2000).

  5. 5.

    D’Arcangelo, G. et al. A protein related to extracellular matrix proteins deleted in the mouse mutant reeler. Nature 374, 719–723 (1995).

  6. 6.

    Sheldon, M. et al. Scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature 389, 730–733 (1997).

  7. 7.

    Keays, D. A. et al. Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans. Cell 128, 45–57 (2007).

  8. 8.

    Chae, T. et al. Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron 18, 29–42 (1997).

  9. 9.

    Dhavan, R. & Tsai, L. H. A decade of CDK5. Nat. Rev. Mol. Cell Biol. 2, 749–759 (2001).

  10. 10.

    Keays, D. A., Clark, T. G. & Flint, J. Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens. Mamm. Genome 17, 230–238 (2006).

  11. 11.

    Corbo, J. C. et al. Doublecortin is required in mice for lamination of the hippocampus but not the neocortex. J. Neurosci. 22, 7548–7557 (2002).

  12. 12.

    Hirotsune, S. et al. Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality. Nat. Genet. 19, 333–339 (1998).

  13. 13.

    Stack, J. H., Herman, P. K., Schu, P. V. & Emr, S. D. A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 12, 2195–2204 (1993).

  14. 14.

    Stein, M. P., Feng, Y., Cooper, K. L., Welford, A. M. & Wandinger-Ness, A. Human VPS34 and p150 are Rab7 interacting partners. Traffic 4, 754–771 (2003).

  15. 15.

    Sun, Q., Westphal, W., Wong, K. N., Tan, I. & Zhong, Q. Rubicon controls endosome maturation as a Rab7 effector. Proc. Natl. Acad. Sci. USA 107, 19338–19343 (2010).

  16. 16.

    Ruas, M. et al. TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2. Mol. Cell Biol. 34, 3981–3992 (2014).

  17. 17.

    Tanida, I., Ueno, T. & Kominami, E. LC3 and autophagy. Methods Mol. Biol. 445, 77–88 (2008).

  18. 18.

    Hara, T. et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441, 885–889 (2006).

  19. 19.

    Schwämmle, V., León, I. R. & Jensen, O. N. Assessment and improvement of statistical tools for comparative proteomics analysis of sparse data sets with few experimental replicates. J. Proteome Res. 12, 3874–3883 (2013).

  20. 20.

    Lim, K. P. & Hong, W. Human Nischarin/imidazoline receptor antisera-selected protein is targeted to the endosomes by a combined action of a PX domain and a coiled-coil region. J. Biol. Chem. 279, 54770–54782 (2004).

  21. 21.

    Alahari, S. K., Reddig, P. J. & Juliano, R. L. The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK. EMBO J. 23, 2777–2788 (2004).

  22. 22.

    Seress, L., Gulyás, A. I. & Freund, T. F. Pyramidal neurons are immunoreactive for calbindin D28k in the CA1 subfield of the human hippocampus. Neurosci. Lett. 138, 257–260 (1992).

  23. 23.

    Nemazanyy, I. et al. Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease. EMBO Mol. Med. 5, 870–890 (2013).

  24. 24.

    Gorski, J. A. et al. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J. Neurosci. 22, 6309–6314 (2002).

  25. 25.

    Goebbels, S. et al. Genetic targeting of principal neurons in neocortex and hippocampus of NEX-Cre mice. Genesis 44, 611–621 (2006).

  26. 26.

    Akizu, N. et al. Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction. Nat. Genet. 47, 528–534 (2015).

  27. 27.

    Sobreira, N., Schiettecatte, F., Valle, D. & Hamosh, A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene. Hum. Mutat. 36, 928–930 (2015).

  28. 28.

    Rostislavleva, K. et al. Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes. Science 350, aac7365 (2015).

  29. 29.

    Vadlamudi, R. K. et al. p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B. Mol. Cell Biol. 25, 3726–3736 (2005).

  30. 30.

    Arber, S. et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393, 805–809 (1998).

  31. 31.

    Ding, Y., Milosavljevic, T. & Alahari, S. K. Nischarin inhibits LIM kinase to regulate cofilin phosphorylation and cell invasion. Mol. Cell Biol. 28, 3742–3756 (2008).

  32. 32.

    Chai, X., Förster, E., Zhao, S., Bock, H. H. & Frotscher, M. Reelin stabilizes the actin cytoskeleton of neuronal processes by inducing n-cofilin phosphorylation at serine3. J. Neurosci. 29, 288–299 (2009).

  33. 33.

    Schmid, R. S. et al. alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development. Development 131, 6023–6031 (2004).

  34. 34.

    Waugh, M. G. PIPs in neurological diseases. Biochim. Biophys. Acta. 1851, 1066–1082 (2015).

  35. 35.

    Rivière, J. B. et al. De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes. Nat. Genet. 44, 934–940 (2012).

  36. 36.

    Mirzaa, G. M. et al. Characterisation of mutations of the phosphoinositide-3-kinase regulatory subunit, PIK3R2, in perisylvian polymicrogyria: a next-generation sequencing study. Lancet Neurol. 14, 1182–1195 (2015).

  37. 37.

    Stopkova, P. et al. Identification of PIK3C3 promoter variant associated with bipolar disorder and schizophrenia. Biol. Psychiatry 55, 981–988 (2004).

  38. 38.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 25, 1754–1760 (2009).

  39. 39.

    Bejarano, E. et al. Connexins modulate autophagosome biogenesis. Nat. Cell Biol. 16, 401–414 (2014).

  40. 40.

    Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).

  41. 41.

    Sun, T. et al. Acetylation of Beclin 1 inhibits autophagosome maturation and promotes tumour growth. Nat. Commun. 6, 7215 (2015).

  42. 42.

    Feng, W. et al. Dissection of autophagy in human platelets. Autophagy 10, 642–651 (2014).

  43. 43.

    Ma, Y. et al. Toll-like receptor (TLR) 2 and TLR4 differentially regulate doxorubicin induced cardiomyopathy in mice. PLoS ONE 7, e40763 (2012).

  44. 44.

    Lee, H. et al. Pathological roles of the VEGF/SphK pathway in Niemann-Pick type C neurons. Nat. Commun. 5, 5514 (2014).

  45. 45.

    Willy, J. A. et al. Function of inhibitor of Bruton’s tyrosine kinase isoform α (IBTKα) in nonalcoholic steatohepatitis links autophagy and the unfolded protein response. J. Biol. Chem. 292, 14050–14065 (2017).

  46. 46.

    Jana, N. R., Tanaka, M., Wang, Gh & Nukina, N. Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity. Hum. Mol. Genet. 9, 2009–2018 (2000).

  47. 47.

    Santoh, M. et al. Acetaminophen induces accumulation of functional rat CYP3A via polyubiquitination dysfunction. Sci. Rep. 6, 21373 (2016).

  48. 48.

    Rinaldi, C. et al. Insulinlike growth factor (IGF)-1 administration ameliorates disease manifestations in a mouse model of spinal and bulbar muscular atrophy. Mol. Med. 18, 1261–1268 (2012).

  49. 49.

    Walls, K. C. et al. Lysosome dysfunction triggers Atg7-dependent neural apoptosis. J. Biol. Chem. 285, 10497–10507 (2010).

  50. 50.

    Bertrand, T. et al. Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR. Cell Death Dis. 6, e1924 (2015).

  51. 51.

    Li, S., Leshchyns’ka, I., Chernyshova, Y., Schachner, M. & Sytnyk, V. The neural cell adhesion molecule (NCAM) associates with and signals through p21-activated kinase 1 (Pak1). J. Neurosci. 33, 790–803 (2013).

  52. 52.

    Breuss, M. et al. Mutations in the β-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities. Cell Rep. 2, 1554–1562 (2012).

  53. 53.

    Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, H0034 (2002).

  54. 54.

    Boda, E., Pini, A., Hoxha, E., Parolisi, R. & Tempia, F. Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain. J. Mol. Neurosci. 37, 238–253 (2009).

  55. 55.

    Pichler, P. et al. Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap. Anal. Chem. 82, 6549–6558 (2010).

  56. 56.

    Kita, Y., Kawakami, K., Takahashi, Y. & Murakami, F. Development of cerebellar neurons and glias revealed by in utero electroporation: Golgi-like labeling of cerebellar neurons and glias. PLoS ONE 8, e70091 (2013).

  57. 57.

    Chu, Y. Y. et al. Astrocytic CCAAT/enhancer binding protein delta regulates neuronal viability and spatial learning ability via miR-135a. Mol. Neurobiol. 53, 4173–4188 (2016).

  58. 58.

    Navarro-Quiroga, I., Hernandez-Valdes, M., Lin, S. L. & Naegele, J. R. Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex. J. Comp. Neurol. 497, 833–845 (2006).

  59. 59.

    Vasistha, N. A. et al. Cortical and clonal contribution of Tbr2 expressing progenitors in the developing mouse brain. Cereb. Cortex 25, 3290–3302 (2015).

  60. 60.

    Boekhoorn, K. et al. Doublecortin (DCX) and doublecortin-like (DCL) are differentially expressed in the early but not late stages of murine neocortical development. J. Comp. Neurol. 507, 1639–1652 (2008).

  61. 61.

    Hendzel, M. J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997).

  62. 62.

    Shah, B. et al. C3G/Rapgef1 is required in multipolar neurons for the transition to a bipolar morphology during cortical development. PLoS ONE 11, e0154174 (2016).

  63. 63.

    Mullen, R. J., Buck, C. R. & Smith, A. M. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116, 201–211 (1992).

  64. 64.

    Nóbrega, C. et al. Silencing mutant ataxin-3 rescues motor deficits and neuropathology in Machado-Joseph disease transgenic mice. PLoS ONE 8, e52396 (2013).

  65. 65.

    Keays, D. A. et al. The role of Tuba1a in adult hippocampal neurogenesis and the formation of the dentate gyrus. Dev. Neurosci. 32, 268–277 (2010).

  66. 66.

    Castellano, B. et al. A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures. J. Histochem. Cytochem. 39, 561–568 (1991).

  67. 67.

    Gonchar, Y., Wang, Q. & Burkhalter, A. Multiple distinct subtypes of GABAergic neurons in mouse visual cortex identified by triple immunostaining. Front. Neuroanat. 1, 3 (2008).

  68. 68.

    Gandal, M. J. et al. GABAB-mediated rescue of altered excitatory-inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction. Transl. Psychiatry 2, e142 (2012).

  69. 69.

    Pillai, A. G. et al. Dendritic morphology of hippocampal and amygdalar neurons in adolescent mice is resilient to genetic differences in stress reactivity. PLoS ONE 7, e38971 (2012).

  70. 70.

    Bas-Orth, C., Tan, Y. W., Oliveira, A. M., Bengtson, C. P. & Bading, H. The calmodulin-binding transcription activator CAMTA1 is required for long-term memory formation in mice. Learn. Mem. 23, 313–321 (2016).

  71. 71.

    Oliver, P. L., Keays, D. A. & Davies, K. E. Behavioural characterisation of the robotic mouse mutant. Behav. Brain Res. 181, 239–247 (2007).

  72. 72.

    Deacon, R. M. & Rawlins, J. N. T-maze alternation in the rodent. Nat. Protoc. 1, 7–12 (2006).

  73. 73.

    Marco-Sola, S., Sammeth, M., Guigó, R. & Ribeca, P. The GEM mapper: fast, accurate and versatile alignment by filtration. Nat. Methods 9, 1185–1188 (2012).

  74. 74.

    Derrien, T. et al. Fast computation and applications of genome mappability. PLoS ONE 7, e30377 (2012).

  75. 75.

    Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6, 80–92 (2012).

Download references

Acknowledgements

We thank the family for the donation of the genetic and biological material. We also acknowledge the input of P. Potter and S. Wells from the mutagenesis program at MRC Harwell and the MRC funding that underpinned it (MC U142684172). We are indebted to R. Williams for modeling the VPS15 human mutation. We also thank the transgenic, bio-optics, proteomic and graphics services groups at the IMP/IMBA. We thank The National Center for Medical Genomics (LM2015091) for providing allelic frequencies in ethnically matched populations (project CZ.02.1.01/0.0/0.0/16_013/0001634). We thank Boehringer Ingelheim and the FWF for funding this research (D.A.K., I914, P24267). The human studies were funded by the European Community’s 7th Framework Program (FP7/2007-2013). S.K., A.P. and V.S. were supported by institutional programs of Charles University in Prague (UNCE 204011, PROGRES-Q26/LF1 and SVV 260367/2017). We acknowledge grants 15-28208A and RVO-VFN 64165 from the Ministry of Health of the Czech Republic and the project LQ1604 NPU II from the Ministry of Education.

Author information

Affiliations

  1. Institute of Molecular Pathology (IMP), Vienna Biocentre (VBC), Vienna, Austria

    • Thomas Gstrein
    • , Ines Leca
    • , Martin Breuss
    • , Sandra Pilat-Carotta
    • , Andi H. Hansen
    • , Ratna Tripathy
    • , Anna K. Traunbauer
    • , Tobias Hochstoeger
    • , Gavril Rosoklija
    • , Lukas Landler
    • , Gerhard Dürnberger
    • , Johannes Zuber
    • , Karl Mechtler
    •  & David A. Keays
  2. Wellcome Trust Center for Human Genetics (WTCHG), Oxford, UK

    • Andrew Edwards
    •  & Jonathan Flint
  3. Institute of Inherited Metabolic Disorders, Charles University, Prague, Czech Republic

    • Anna Přistoupilová
    • , Viktor Stránecký
    •  & Stanislav Kmoch
  4. CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

    • Anna Přistoupilová
    • , Marta Gut
    • , Sergi Beltran
    •  & Ivo Gut
  5. Universitat Pompeu Fabra (UPF), Barcelona, Spain

    • Anna Přistoupilová
    • , Marta Gut
    • , Sergi Beltran
    •  & Ivo Gut
  6. Institute for Molecular Biotechnology (IMBA), Vienna, Austria

    • Marco Repic
  7. Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK

    • Thomas M. Keane
    •  & David J. Adams
  8. Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University, Prague, Czech Republic

    • Tomas Honzik
  9. Institute of Human Genetics, University of California, San Francisco, San Francisco, CA, USA

    • Elliott Sherr

Authors

  1. Search for Thomas Gstrein in:

  2. Search for Andrew Edwards in:

  3. Search for Anna Přistoupilová in:

  4. Search for Ines Leca in:

  5. Search for Martin Breuss in:

  6. Search for Sandra Pilat-Carotta in:

  7. Search for Andi H. Hansen in:

  8. Search for Ratna Tripathy in:

  9. Search for Anna K. Traunbauer in:

  10. Search for Tobias Hochstoeger in:

  11. Search for Gavril Rosoklija in:

  12. Search for Marco Repic in:

  13. Search for Lukas Landler in:

  14. Search for Viktor Stránecký in:

  15. Search for Gerhard Dürnberger in:

  16. Search for Thomas M. Keane in:

  17. Search for Johannes Zuber in:

  18. Search for David J. Adams in:

  19. Search for Jonathan Flint in:

  20. Search for Tomas Honzik in:

  21. Search for Marta Gut in:

  22. Search for Sergi Beltran in:

  23. Search for Karl Mechtler in:

  24. Search for Elliott Sherr in:

  25. Search for Stanislav Kmoch in:

  26. Search for Ivo Gut in:

  27. Search for David A. Keays in:

Contributions

A.E., J.F. and D.A.K. performed the phenotypic screen and genetic mapping. T.M.K. and D.J.A. did the whole-exome sequencing on the Marble mouse. T.G., G.R. and T.Hochstoeger analyzed the expression of Vps15. T.G., I.L., M.B., S.P.-C., A.H.H., R.T. and L.L. were responsible for the characterization of the Marble and KO mouse lines. K.M. and G.D. undertook the mass spectroscopy experiments. M.R. and T.G. performed the in utero electroporation analysis. A.P., T.Honzik, M.G., S.B., E.S., S.K., V.S. and I.G. collated the clinical data and performed whole-exome sequencing and analysis of the L1224R patient. T.G. performed the functional experiments. A.K.T., T.G. and J.Z. designed and performed the lentiviral rescue experiment. T.G. and D.A.K. wrote the manuscript and all authors commented on it.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David A. Keays.

Integrated Supplementary Information

Supplementary information