u-0126 and Wallerian-Degeneration

Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of 'slow Wallerian degeneration' (Wld (S)), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld(S). Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld(S)-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld(S) expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld(S) or NMNAT2.

Topics: Animals; Axons; Butadienes; Cell Line; Enzyme Stability; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Kinase 5; MAP Kinase Signaling System; Nicotinamide-Nucleotide Adenylyltransferase; Nitriles; Phenotype; Protein Kinase Inhibitors; Rats; Superior Cervical Ganglion; Wallerian Degeneration

Treatment of transected distal axons of rat sympathetic neurons in compartmented cultures with MG132 (5 microM) and other inhibitors of proteasome activity, preserved axonal mitochondrial function, assessed by Mitotracker-Orange and MTT staining, for at least 24 h. MG132 similarly protected axons from undergoing branch elimination (pruning) in response to local NGF deprivation. Axons protected by MG132 displayed persistent phosphorylation of Erk1/2, and pharmacological inhibition of MEK activity with U0126 (50 microM) restored rapid axonal degeneration. Therefore, the proteasome does not appear to be necessary as a general effector of protein degradation during axonal degeneration. Rather, the proteasome functions in the regulation of signaling pathways that control axonal survival and degeneration. Specifically, the down-regulation of the MEK/Erk pathway by the proteasome plays roles in Wallerian degeneration of severed axons and axonal pruning in response to local NGF deprivation. Identification of the pathways that regulate axonal survival and degeneration will provide possible target sites for pharmacological treatments of neurodegenerative diseases and traumatic injury.

Topics: Animals; Animals, Newborn; Axons; Butadienes; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Inhibitors; Leupeptins; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Nerve Growth Factor; Nerve Regeneration; Nitriles; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Superior Cervical Ganglion; Sympathetic Nervous System; Wallerian Degeneration