ABSTRACT
JNKs are implicated in a range of brain pathologies and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools, and c-Jun/ATF2 targets of JNK in death of central nervous system neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs, and pharmacological inhibitors. Combined small interfering RNA knockdown of all JNKs 1, 2, and 3, provides substantial neuroprotection. In contrast, knockdown or knock-out of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely pharmacological. Complete knockdown of c-Jun and ATF2 using small interfering RNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect, whereas cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective.
Subject(s)
Apoptosis , Cell Nucleus/enzymology , Mitogen-Activated Protein Kinase 10/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Neurons/enzymology , Activating Transcription Factor 2/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Apoptosis/drug effects , Brain Diseases/drug therapy , Brain Diseases/enzymology , Cytoplasm/enzymology , Gene Silencing , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 10/antagonists & inhibitors , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Mitogen-Activated Protein Kinase 9/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-jun/metabolism , RNA, Small Interfering/pharmacologyABSTRACT
Glycogen synthase kinase 3 (GSK-3) is implicated in neuronal death through a causal role, and precise mechanisms have not been unambiguously defined. We show that short hairpin RNA (shRNA) knockdown of GSK-3beta, but not GSK-3alpha, protects cerebellar granule neurons from trophic-deprivation-induced death. Using compartment-targeted inhibitors of the Wnt-regulated GSK-3 pool, NLS-FRAT1, NES-FRAT1, and axin-GSK-3-interacting domain (axin-GID), we locate proapoptotic GSK-3 action to the cytosol and regulation of Bim protein turnover despite constitutive cycling of GSK-3 between the cytosol and nucleus, revealed by leptomycin B. We examine the importance of Ser21/9 (GSK-3alpha/beta) phosphorylation on proapoptotic GSK-3 function. Neurons isolated from GSK-3alpha/beta(S21A/S9A) knock-in mice survive normally and are fully sensitive to trophic-deprivation-induced death. Nonetheless, inhibition of GSK-3 catalytic activity with lithium or SB216763 protects GSK-3alpha/beta(S21A/S9A) neurons from death. This indicates that dephosphorylation of GSK-3beta/Ser9 and GSK-3alpha/Ser21 is insufficient for GSK-3 proapoptotic function and that another level of regulation is required. Gel filtration reveals a stress-induced loss of neuronal GSK-3beta from a high-molecular-mass complex with a concomitant decrease in axin-bound GSK-3beta. These data imply that Wnt-regulated GSK-3beta plays a nonredundant role in trophic-deprivation-induced death of neurons.