Di Re, Hsu et al Disrupted interactions of proteins at the axon initial segment induced by AKT/GSK3 kinase signaling

Published: 6 April 2021| Version 1 | DOI: 10.17632/jgcsykf8xv.1
Contributors:
Jessica Di Re,
Fernanda Laezza,
Wei-Chun Jim Hsu

Description

We used confocal imaging to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate select AIS proteins. Image-based features representing cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrinG, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed. Analysis revealed bIV spectrin as a converging downstream target of the AKT/GSK3 pathway with the AKT inhibitor triciribine increasing bIV Spectrin localization to the AIS and altering its polar distribution within neurons.

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Hippocampal neurons (DIV 14) were fixed in fresh 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 15 min. Following permeabilization with 0.25% Triton X-100 and blocking with 10% BSA for 30 min at 37 °C, neurons were incubated overnight at room temperature with primary antibodies. Neurons were then washed three times in PBS and incubated for 45 min at 37 °C with appropriate secondary antibodies. Coverslips were then washed six times with PBS and mounted on glass slides with Prolong Gold anti-fade reagent. Confocal images were acquired with a Zeiss LSM-510 Meta confocal microscope with a 63X oil immersion objective (1.4 NA). Multi-track acquisition was done with excitation lines at 488 nm for Alexa 488, 543 nm for Alexa 568 and 633 nm for Alexa 647. Respective emission filters were band-pass 505–530 nm, band-pass 560–615 nm and low-pass 650 nm. Z-stacks were collected at z-steps of 1 μm with a frame size of 512 × 512, pixel time of 2.51 μs, pixel size 0.28 × 0.28 μm or 0.39 × 0.39 μm and a 4-frame Kallman averaging. Acquisition parameters, including photomultiplier gain and offset, were kept constant throughout each set of experiments. We derived our image-based features from fluorescent intensity profiles of specific analytes, namely PanNav, bIV spectrin, ankyrinG, and neurofascin, measured along the AIS and along representative dendrites of neurons the images. For each selected neuron, we computed fluorescent intensities as follows. (1) We manually selected a rectangular region-of-interest (ROI) around the AIS and a dendrite in the fluorescent image using a width of 3 or 4 pixels corresponding to a width 1.12 or 1.18 mm. (2) Background subtraction was performed by selecting and area away from the neurites of each image and subtracting the average value from each point of the ROI. (3) For the AIS, in order to compare areas of accumulated protein of interest, we used a method adapted from Guo et al. 2017. Briefly, we created an average of 3-point along the AIS and used this average to find the peak of fluorescent intensity for each AIS. From this peak, we determined the point at which the signal decreased below 15% of the peak after background subtraction and used these points to define the beginning and end of the AIS, creating an analyte specific length from every protein examined.