Abstract
After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.
Highlights
In both chronic neuropathological conditions and following acute injury, Dual Leucine-zipper Kinase (DLK) signals via its downstream target c-Jun N-terminal Kinase (JNK) to activate pro-degenerative transcription and subsequent neuronal death[1,2,3,4,5,6,7]
Subcellular localization changes of this type are often used as readouts in High Content Screening (HCS)[16,17], an approach that might be well suited to identify compounds that inhibit Dual leucine-zipper kinase (DLK) palmitoylation
Because a non-neuronal cell line might be more amenable to HCS approaches than primary neurons, we assessed whether DLK localization is palmitoylation-dependent in HEK293T cells
Summary
In both chronic neuropathological conditions and following acute injury, Dual Leucine-zipper Kinase (DLK) signals via its downstream target c-Jun N-terminal Kinase (JNK) to activate pro-degenerative transcription and subsequent neuronal death[1,2,3,4,5,6,7]. Consistent with the importance of palmitoylation for DLK-JNK signaling, genetically mutating DLK’s palmitoylation site prevented JNK phosphorylation in non-neuronal cells, and blocked JNK-dependent responses to axonal injury in cultured neurons[11]. These findings suggested to us that compounds that prevent DLK palmitoylation might be as neuroprotective as inhibitors of DLK’s kinase activity. Pursuing this therapeutic strategy would require development of an effective screening method to identify such compounds. Our screening assay has the potential to identify novel modulators of DLK palmitoylation, which may have considerable therapeutic potential
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