The Function Of Akt3 In Mouse Cortical Development

The mammalian neocortex is a highly organized 6-layered structure, which is esse ntial for its structural integrity and normal function. The development of cerebral corte x includes proliferation of progenitor cells, neuronal migration, and ultimately their dif ferentiation and establishment of connection. Many genetic factors influence cortical de velopment and cause brain malformation, such as mental retardation, epilepsy, lissencep haly, microcephaly and macrocephaly. At the early stage of human brain development,hyperactivity of PI3K-Akt3 signaling pathway induces hemimegalencephaly or megale ncephaly. However, the pathogenic mechanism is still largely unclear. Akt3 is predomi nantly expressed in the nervous system. Akt3 knockout mice display impaired brain de velopment, but the brain structures show no anatomical malformations. Akt3 overexpre ssion and mutation that induces hyperactivity this kinase were detected in many types of cancers and in some brain malformations. To reveal the function of Akt3 in brain development, we used cell culture and transfection, plasmid construction, point mutant,in utero electroporation and immunofluorescence to study the function of Akt3 in bra in development. The major results of this study includes:1. We successfully cloned the Akt3 gene and constructed the Akt3 overexpression pl asmid. Using lipofectin, we transfected CHO cells with Akt3 and found that overexpre ssion of Akt3 promoted the formation of lamellipodia.2. Using in utero electroporation, we overexpressed Akt3-wt in the developing mous e cortical neurons at E15.5 and found that overexpression of Akt3-wt significantly inhi bited neuronal migration, promoted the branching of leading process, enlarged the som a size and nuclear size of the transfected neurons, induced the formation of basal den drite and promoted axonal elongation.3. Using point mutation, we constructed Akt3-E17 K, an active form of Akt3. We tra nsfected the Akt3-E17 K plasmid into the mouse cortical neurons and found that Akt3-E17 K not only inhibited neuronal migration, but also promoted the aggregation of the transfected neurons. Likes the Akt3-wt, Akt3-E17 K enlarged the soma size, promoted leading process branching, basal dendrite formation and axonal outgrowth of the trans fected neurons. These results indicate that hyperactivity of Akt3 could promote assembly of neuronal cytoskeleton.4. To elucidate the function of the phosphorylation site of Akt3 in cortical developm ent, we constructed the Akt3-T305 A expression plasmid. Using in utero electroporation,we found that Akt3-T305 A suppressed the activity of Akt3, and alleviated the effects of Akt3 on neuronal migration, leading process branching, soma size regulation, dend rite formation and axon growth.5. We used the online software to analyze the structure of Akt3 and found that the D271 might be the active site of this kinase. To elucidate whether D271 is essential f or its activity, we constructed the Akt3-D271 A and transfected into the developing mo use brain. The results revealed that Akt3-D271 A completely eliminated the inhibition e ffect of Akt3 on neuronal migration, as well as the effects on axon growth, soma size and neuronal morphology. These results suggest that D271 is the active site of Akt3 and plays an important role in cortical development.