Figure 5.

Abi-1 protein levels influence dendritogenesis and synaptic maturation. (A, B) The transfection of HeLa cells and hippocampal neurons with the Abi-1-RNAi construct reduces the level of endogenous Abi-1 protein to barely detectable levels. (C, D) In neurons transfected at day 3, the downregulation of Abi-1 leads to a highly branched MAP2-positive dendritic tree. The TNBPs is significantly elevated in RNAi-transfected neurons (E–G). This phenotype is also observed after transfection on day 7, showing especially high numbers of secondary and tertiary dendrites. Overexpression of Abi-1 protein results in a simplified dendritic tree, with a reduction of the TNBPs compared with controls. Significances compared with the empty vector controls are indicated above the bars. Note that RNAi-resistant wild-type Abi-1 cotransfected with RNAi (RNAi_Abi-1+Mut Abi-1) reduces TNBPs significantly, whereas the RNAi-resistant Abi-1-(53Y-A) construct is not able to compensate (or overcompensate) the loss of endogenous Abi-1 (RNAi_Abi-1+Mut-Abi-1-(53Y-A)) (H–J). Analysis of synaptic contacts reveals a significant reduction of Bassoon-positive boutons in Abi-1-RNAi-transfected neurons. Moreover, RNAi-transfected neurons show a high percentage of immature synaptic contacts on thin filopodia-like protrusions with apical Bassoon-positive presynaptic terminals (arrow heads). In contrast, the overexpression of Abi-1-GFP results in a significant increase of mature synaptic contacts at the dendritic shaft. Note that RNAi-resistant Abi-1 construct cotransfected with RNAi (RNAi_Abi-1+Mut Abi-1) also significantly increases the synapse number as well as maturity. In contrast, the RNAi-resistant Abi-1-(53Y-A) construct alone or cotransfected with RNAi_Abi-1 is not able to increase synapse number or change synapse morphology. Significances compared with the empty vector controls pSuper and pSuper+pEGFP are indicated above the bars. Significances: ^**0.001%; ^***0.0001; ns, not significant.