Research ArticleCell biologyNeuroscience Open Access | 10.1172/JCI175435

Gianmarco Pallavicini,1,2 Amanda Moccia,3 Giorgia Iegiani,1,2 Roberta Parolisi,1,2 Emily R. Peirent,4 Gaia Elena Berto,1,2 Martina Lorenzati,1,2 Rami Y. Tshuva,5 Alessia Ferraro,1,2 Fiorella Balzac,6 Emilia Turco,6 Shachi U. Salvi,3 Hedvig F. Myklebust,3 Sophia Wang,3 Julia Eisenberg,3,7 Maushmi Chitale,3 Navjit S. Girgla,3 Enrica Boda,1,2 Orly Reiner,5 Annalisa Buffo,1,2 Ferdinando Di Cunto,1,2 and Stephanie L. Bielas3,4,7

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Pallavicini, G. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Moccia, A. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Iegiani, G. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Parolisi, R. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Peirent, E. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Berto, G. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Lorenzati, M. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Tshuva, R. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Ferraro, A. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Balzac, F. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Turco, E. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Salvi, S. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Myklebust, H. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Wang, S. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Eisenberg, J. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Chitale, M. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Girgla, N. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Boda, E. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Reiner, O. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Buffo, A. in: JCI | PubMed | Google Scholar

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Di Cunto, F. in: JCI | PubMed | Google Scholar |

1Neuroscience Institute Cavalieri Ottolenghi, Turin, Italy.

2Department of Neuroscience “Rita Levi Montalcini,” University of Turin, Turin, Italy.

3Department of Human Genetics and

4Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.

5Departments of Molecular Genetics and Molecular Neuroscience, Weizmann Institute of Science, Rehovot, Israel.

6Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

7Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA.

Address correspondence to: Ferdinando Di Cunto, Neuroscience Institute Cavalieri Ottolenghi Regione Gonzole, 10, 10043 Orbassano (TO), Italy. Phone: 39.011.6706616; Email: ferdinando.dicunto@unito.it. Or to: Stephanie L. Bielas, Department of Human Genetics, University of Michigan Medical School, 3703 Med Sci II, 1241 E. Catherine St., Ann Arbor, Michigan, 48109-5618, USA. Phone: 1.734.647.8890; Email: sbielas@med.umich.edu.

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Find articles by Bielas, S. in: JCI | PubMed | Google Scholar |

Authorship note: GP, AM, and GI are co–first authors and contributed equally to this work.

Published September 24, 2024 - More info

Published in Volume 134, Issue 21 on November 1, 2024
J Clin Invest. 2024;134(21):e175435. https://doi.org/10.1172/JCI175435.
© 2024 Pallavicini et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published September 24, 2024 - Version history
Received: September 20, 2023; Accepted: September 6, 2024 View PDF Abstract

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/threonine kinase (CIT) missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Graphical Abstract Introduction

The mammalian cerebral cortex forms through a species-specific sequence of neural precursor cell proliferation and differentiation events (1, 2). Neuroepithelial stem cells expand through a series of symmetric proliferative divisions and then give rise to multipotent apical neural radial glia progenitor cells (aNPCs). In the ventricular zone, aNPCs divide symmetrically to produce additional multipotent progenitors and asymmetrically to produce basal neural radial glia progenitor cells (bNPCs) and intermediate progenitor cells, which continue to proliferate in the outer-subventricular zone or terminally differentiate into excitatory neuron subtypes (3). Expansion of multipotent aNPCs by symmetric proliferation and generation of progeny through asymmetric cell division are carefully regulated species-specific features of corticogenesis that contribute to the final size and folding of the cerebral cortex (1, 2, 4). Given the consequential role of aNPC symmetrical division in brain size, the apical-basal polarity of aNPCs is tightly controlled. aNPCs span the breadth of neuroepithelium with apical end-feet secured at the ventricular border by adhesion junctions that contain overlapping fate and polarity factors. The precise distribution of fate determinants between aNPC daughter progeny determines cell fate. During neurogenesis, cleavage furrow constriction of mitotic aNPCs in neuroepithelium is polarized; contractile ring constriction occurs from the basal-to-apical membrane to localize abscission at the apical border of the cell, adjacent to the ventricle (4). The position of the mitotic spindle relative to the apical border of aNPCs, shifting between perpendicular to oblique or even parallel, is also critical in the transition from symmetric to asymmetric divisions (5). The importance of this biology for corticogenesis is accentuated by primary hereditary microcephaly (MCPH) and microlissencephaly that result from errors in this developmental process (6, 7).

MCPH-associated proteins participate in many biological processes that affect aNPC proliferation and differentiation (7–11). Disentangling the complex physiological relationships between MCPH genes is complicated by the limited knowledge about their functional interactions and the difficulty of modeling human microcephaly in rodents, which do not recapitulate all details of human cortical development. Indeed, inactivation of MCPH genes in mice frequently produces significantly milder phenotypes than those observed in affected individuals (12, 13). We previously identified pathogenic biallelic variants in citron rho-interacting serine/threonine kinase (CIT) (OMIM ID: 605629) as the genetic basis for MCPH17 (14, 15). CIT represents a growing list of human genetic findings linking defects in cytokinesis machinery to clinical features of severe microcephaly. An overarching feature of these findings is their disproportionate effect on brain development despite widespread expression across tissues, highlighting a unique vulnerability of the brain to cytokinesis defects. CIT encodes a Ser/Thr protein kinase (CIT-K) that localizes to the contractile ring and midbody during cytokinesis. CIT-K is functionally critical for cytokinesis completion, midbody positioning, abscission, and maintenance of genomic integrity (16–20). CIT-K is characterized by an N-terminal kinase domain and C-terminal protein interaction domains. The C-terminal domains permit integration into the contractile ring and midbody, which is required for scaffolding functions. Biallelic CIT missense variants that disrupt kinase function (kinase-inactive [KI]) and loss-of-function (LOF) frameshift (FS) variants that destroy both kinase and scaffolding functions are the genetic basis of microcephaly and microlissencephaly, respectively (14, 15, 21, 22). The increasing severity of these clinical phenotypes indicate that both kinase and scaffolding functions are crucial for brain development.

In this report, we describe complementary mouse and human CIT KI models generated to discriminate the developmental mechanisms of CIT-K kinase function from the more severe CIT LOF corticogenesis phenotypes. Cit LOF rodent models displayed severe microcephaly as a result of altered neurogenesis, binucleation, and TP53-dependent apoptosis (18, 23, 24). CitKI/KI mice do not phenocopy MCPH17 microcephaly, although they display aNPC molecular phenotypes consistent with Cit LOF models. Similar to the described mouse models, we observed progressively more severe molecular pathology between CITKI/KI and CITFS/FS human 3D forebrain organoids. However, both genotypes showed striking alterations in the polarized organization and structural integrity of neural rosette neuroepithelium, revealing a function of CIT-K kinase activity in aNPC cytokinesis that is critical for early brain development. Our results underscore the relevance of species-specific sensitivity to CIT-K cytokinesis functions and further highlight human 3D forebrain organoids as a fundamental model to better understand human microcephaly.

Results

Brain size is unchanged in the CitKI/KI mouse model. To investigate the role of CIT-K catalytic activity in brain development, we generated a new mutant mouse model. Two nucleotides were edited using traditional mouse embryonic stem cell–based (ESC-based) knockin technologies (25) to generate a Cit (c.376_377AA>GC; p.K126A) KI mouse line. This Cit missense substitution is similar to the CIT missense variants described in individuals with MCPH17 microcephaly (Supplemental Figure 1, A–E; supplemental material available online with this article; https://doi.org/10.1172/JCI175435DS1) (15). The K126A amino acid substitution was previously shown to fully abolish CIT-K kinase activity (Supplemental Figure 1B) (26). Homozygous CitKI/KI pups were born at the expected Mendelian ratios. CIT-K had the highest expression in proliferating neural tissue. Western blot analysis of P4 cerebellum and cortex showed a 40% reduction in CIT-K expression in the cerebellum of CitKI/KI mice and normal expression of CIT-N, a shorter brain-specific isoform lacking the amino terminal kinase domain (Supplemental Figure 1, F and G) (27,