Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons

Abstract

Summary Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors (JUN), activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11)) as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Intrinsic mechanisms of neuronal axon regeneration

“…Studies in retinal ganglion cells (RGCs) suggest that interactions are one mechanism of specifying the cellular response following DLK activation 65 . Whereas DLK signalling in peripheral neurons is necessary for the activation of several pro-regenerative pathways 63 , in RGCs it promotes cell death 66 .…”

“…Whereas DLK signalling in peripheral neurons is necessary for the activation of several pro-regenerative pathways 63 , in RGCs it promotes cell death 66 . In the latter context, DLK interacts with leucine zipper-bearing kinase (LZK; also known as MAP3K13), another MAPKKK, to activate a set of transcription factors that leads to RGC death after optic nerve injury 65 .…”

“…In the context of cellular reprogramming, the accessibility of the chromatin to a given transcription factor dictates the effectiveness of the conversion 227 . Differences in chromatin accessibility may also lead to different outcomes, as genomic regions that are differentially bound by given transcription factors or differences in signalling pathways may lead to different cellular responses, such as the cell death and regeneration that are promoted by transcription factor SOX11 (REF 143 ) and dual leucine zipper bearing kinase (DLK) 65 , respectively. As such, therapies that address both cellular competency and the manipulation of gene expression may be necessary.…”

An axonal stress response pathway: degenerative and regenerative signaling by DLK

“…Endogenous DLK associates with vesicles [3], and live imaging studies of GFP-DLK transgenes suggest these vesicles are transported both anterogradely and retrogradely in axons [3,32]. DLK function is required cell autonomously for nuclear responses induced by axonal injury, including the activation of specific transcription factors [2,4,5,7,11•,14,33••]. These include phosphorylated STAT3, which is thought to be retrogradely transported in peripheral nerves from axons to the nucleus [5], and also transcriptional reporters for JNK signaling [3].…”

“…In mammals, DLK (MAP3K12) has a sister kinase, MAP3K13 (LZK), which has some partially overlapping biochemical activities and roles [9,11•]. Worms ( Caenorhabditis elegans ) and flies ( Drosophila melanogaster ) each have a single orthologue of equivalent homology to both DLK and LZK, named DLK-1 and Wallenda.…”