Abstract
Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.
Highlights
Several have biologic targets with previously reported involvement in axon degeneration, our findings reveal that inhibitors of IB kinase (IKK)2 and glycogen synthase kinase 3 (GSK3) suppress axotomy-induced axon degeneration
The extent of axon fragmentation can be quantified from brightfield or phase-contrast images of axons using a previously described image analysis algorithm that distinguishes fragmented from intact axonal segments [10]
In this study we present an image-based screening method to identify new members of the axon degeneration signaling cascade
Summary
DRG Neuron Culture—Pregnant female mice at 12 days postcoitus (Charles River Laboratories) were anesthetized prior to cervical dislocation. Image Acquisition and Data Analysis—At three time points following axotomy (0, 6, and 24 h), axons distal to the site of transection (Fig. 1B, panel a) were visualized by automated brightfield microscopy using an InCell Analyzer 1000 fitted with a ϫ20 objective (GE Healthcare). In addition to the images of injured axons acquired at three time points, two images of uninjured axons proximal to the site of injury in each well was acquired at 24 h post-axotomy to monitor toxicity of each compound (Fig. 1B, panel b). All wells in which the degeneration index at 0 h post-axotomy was above 0.3 (30 wells; 2% of those screened), usually resulting from rapid drug toxicity, were excluded from further analysis. Starting with a maximum concentration 5-fold above the initial screening dose, compounds were 2-fold serially diluted in DMSO (total of 8 concentrations), prior to addition to DRG cultures. Degeneration indices were plotted as a function of compound concentration and sigmoid curves with Equation 3, b yϭaϪ
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