Technical Reference #3159

3159.

Reelin Signals through Apolipoprotein E Receptor 2 and Cdc42 to Increase Growth Cone Motility and Filopodia Formation Jost Leemhuis; Elisabeth Bouche´; Michael Frotscher; Frank Henle; Lutz Hein; Joachim Herz; Dieter K. Meyer; Marina Pichler; Gu¨nter Roth; Carsten Schwan; and Hans H. Bock, Albert-Ludwigs-University, Journal of Neuroscienc, 30(3159), (2010)
Link To Paper

Abstract:
Lipoprotein receptor signaling regulates the positioning and differentiation of postmitotic neurons during development and modulates neuronal plasticity in the mature brain. Depending on the contextual situation the lipoprotein receptor ligand Reelin can have opposing effects on cortical neurons. We show that Reelin increases growth cone motility and filopodia formation and identify the underlying signaling cascade. Reelin activates the Rho GTPase Cdc42 known for its role in neuronal morphogenesis and directed migration in an apolipoprotein E receptor 2- Disabled-1- and phosphatidylinositol 3-kinase-dependent manner. We demonstrate that neuronal vesicle trafficking a Cdc42-controlled process is increased after Reelin treatment and further provide evidence that the peptidergic VIP/ PACAP38 system and Reelin can functionally interact to promote axonal branching. In conclusion Reelin-induced activation of Cdc42 contributes to the regulation of the cytoskeleton of individual responsive neurons and converges with other signaling cascades to orchestrate Rho GTPase activity and promote neuronal development. Our data link the observation that defects in Rho GTPases and Reelin signaling are responsible for developmental defects leading to neurological and psychiatric disorders.

Materials & Methods:
Time-lapse light microscopy. Cortical neurons were plated on poly-Llysine- coated glass-bottom microwell dishes (MatTek). For time-lapse light microscopy neurons were incubated in a custom-made chamber at 37°C in a humidified atmosphere (6.5% CO2 9%O2) staged on a Zeiss Axiovert 200 microscope system equipped with a digital camera (Coolsnap HQ RoperScientific). Differential interference contrast (DIC)- images were acquired every 30 s with a Zeiss DIC 40 A:1.4 oil-immersion objective for up to 8 h. To minimize phototoxicity an automatic shutter was used (50 ms/frame). The MetaMorph 7.1 software (Universal Imaging) was used to acquire and process the resulting stacks of images. Only stage II and stage II neurons at day in vitro (DIV) 1 were selected for analysis. To quantify motility stacks (time-lapse movies) were automatically thresholded to binarize the information of the images. The distal 15 m neurites were defined and marked. The numbers of pixel changes compared with the respective previous image of a stack were determined by using the “region measurements” function of the MetaMorph software. To compare motility of the same growth cone of the “control period” with the “treatment period” pixel changes of one or two 15 min intervals of the control period and within the first 45 min of the treatment period were determined. Afterward the SDs of pixel changes of the 15 min intervals were divided by the average of the total pixel area to normalize the motility of growing structures. When GFP-labeled neurons were used the grayscale image of the fluorescence signal was also binarized by thresholding. To analyze the motility compared with fluorescence resonance energy transfer (FRET) activity the yellow fluorescent protein (YFP) image was thresholded to generate a binary mask with a value of zero outside the cell and a value of one inside the cell. The aspects of a resulting binarized image of a DIC FRET or GFP image were different as the degree of coverage of the thresholded to original image was different but this fact did not influence quantification as relative pixel differences were analyzed. All of these three thresholding methods obtained similar results. To express motility of the treatment period in a relative context to the control period the average of the control period was set as 1. For each treatment group the neurites from at least n 6 different neurons were analyzed. With this method only motility changes without any information about the origin of motility within one neuron can be described.

Microscopic Technique
Time-lapse light microscopy

Cell Type(s)
Cortical neurons