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DOI: 10.1055/s-0032-1307168
Microcephaly and Effects of Cdk5rap2 Downregulation in Murine Embryonic Stem Cells
Aims: In 2005 homozygous mutations in the Cycline dependent kinase 5 regulatory subunit associated protein 2 gene CDK5RAP2 were discovered to cause primary autosomal recessive microcephaly type 3 (MCPH3). MCPH is a rare, genetically heterogeneous disease, characterized by an isolated developmental defect of the brain, specifically targeting the cerebral cortex. Patients with MCPH develop a severe microcephaly in utero, affecting particularly the cerebral cortex. CDK5RAP2 is a centrosomal protein, playing a role in various cellular processes such as centrosome functions, spindle formation and dynamics, kinetochore attachment to spindles, spindle checkpoint control, DNA repair and apoptosis. These processes are important for a normal cortical development. While their deregulation contributes to the human MCPH phenotype, the exact pathomechanism is still unknown. The aim was to analyze the pathomechanism underlying MCPH in an in vitro model.
Methods: To analyze the effects of Cdk5rap2 deregulation, we established an in vitro model through lentiviral Cdk5rap2-shRNAi infection of murine embryonic stem cells (mESC). These cells with a stable downregulation of Cdk5rap2 were used to study the effects on proliferation, differentiation and apoptosis in mESC.
Results: In undifferentiated mESC, downregulation of Cdk5rap2 resulted only in a slight reduction of proliferation and the stem cell character was maintained. After neural differentiation induction, a severe phenotype appeared with a groß loss of cells, which will be further analyzed (reduced proliferation, increased cell death). While in undifferentiated mESC Cdk5rap2 is localized at the centrosomes and thereby associated with the nucleus, the differentiation induction leads to a significant change of Cdk5rap2 localization with an assembly of Cdk5rap2 signals in the center of cell clusters, far away from the nuclei.
Conclusion: The results indicate a potentially important role of Cdk5rap2 in regulating the transition between symmetric and asymmetric proliferation and in polarization of differentiating cells.
Microcephaly - MCPH - Cdk5rap2 - stem cells