Neuropediatrics 2013; 44 - PS16_1251
DOI: 10.1055/s-0033-1337810

Effects of CDK5RAP2 downregulation in murine embryonic stem cells correlate with cellular phenotype in MCPH3 patients

N Krämer 1, L Issa 1, G Neubert 1, A Seiler 2, D Morris-Rosendahl 3, O Ninnemann 4, A Kaindl 1
  • 1Institut für Zell- und Neurobiologie, Charité Berlin, Klinik für Pädiatrie m. S. Neurologie, Charité Berlin, Berlin, Germany
  • 2Bundesinstitut für Risikobewertung, Berlin, Germany
  • 3Institut für Humangenetik, Universitätsklinik Freiburg, Freiburg, Germany
  • 4Institut für Zell-und Neurobiologie, Charité Berlin, Berlin, Germany

Aims: Primary autosomal recessive microcephaly (MCPH) is a rare, genetically heterogeneous neurodevelopmental disease, characterized by an isolated defect of the brain. Patients with MCPH develop severe microcephaly in utero with pronounced reduction in brain volume, particularly of the neocortex. Homozygous mutations of the CDK5RAP2 gene cause MCPH type 3 (MCPH3). CDK5RAP2 localizes to the centrosome and the pericentriolar matrix and plays a role in various cellular processes such as centrosome functions, spindle formation, DNA repair and apoptosis. A current hypothesis for the microcephaly phenotype pathomechanism implicates a premature shift from symmetric to asymmetric cell divisions and thus premature neurogenesis with a subsequent depletion of the progenitor pool. Still, the exact underlying pathomechanism remains to be definitely established. The aim was to analyze the pathomechanism underlying MCPH in an ES cell-based in vitro model and in immortalized lymphocytes (LCLs) from human MCPH3 patients.

Methods: The effects of CDK5RAP2 deregulation were analyzed on LCLs from human MCPH3 patients and on an in vitro model, which we established through lentiviral CDK5RAP2-shRNAi infection of murine embryonic stem cells (mESC) resulting in a stable downregulation of CDK5RAP2 in these cells.

Results: In LCLs from MCPH3 patients, the loss of CDK5RAP2 function results in mitotic spindle defects and centrosomal disorganization. In undifferentiated mESC, downregulation of CDK5RAP2 resulted in the same defects as well as in a slight reduction of proliferation. Induction of neural differentiation in these cells led to a severe phenotype with an excessive loss of cells due to an increased proliferation defect.

Conclusion: Taken all together, our results support the hypothesis that microcephaly in MCPH3 results not only from cell division defects in progenitor cells, possibly on the basis of a disruption of centrosome integrity/function and spindle defects, but also from an accumulating proliferation defect of differentiating stem cells as well as from cell death of differentiating and early postmitotic cells.