Neuronal stress response following proteasomal inhibition, and its prevention by astrocytic thiol supply

Abstract

Introduction: The underlying mechanisms of neurodegenerative diseases such as Parkinson’s disease (PD) are not completely understood. One key event in PD, amongst others, is the disturbance of the ubiquitin proteasome system (UPS). The resulting misfolded and aggregated proteins have been discussed to induce cell death. Various attempts in the past to treat these diseases and to slow down the progressive neuronal loss failed at different clinical stages. Most of these attempts followed the concept of blocking specific stress pathways (e.g. caspase activation). An alternative therapeutic approach would be to identify and harness endogenous cellular defense mechanisms and thereby to prevent the activation of deleterious cascades.Methods: In this study we used human dopaminergic neurons (LUHMES cells) and treated them with the proteasome inhibitor MG-132 to model neurodegeneration after disturbance of the UPS in vitro. Cell death, as well as several biochemical and signaling changes were determined in neuronal cultures and astrocyte-neuron co-cultures.Results: In a first step, we monitored the stress responses orchestrating the neurodegeneration in LUHMES cells following proteasomal inhibition. Neurons, exposed to MG-132 (nanomolar concentrations), underwent rapid apoptotic cell death. Prior to caspase activation, we observed an increase in AKT and p38 phosphorylation. Moreover, the ATF4 stress response was induced, and this lead to an increase in GSH synthesis capacity. In a second step, we identified cysteine supplementation as a protective intervention. This intervention prevented ATF4 activation, AKT and p38 phosphorylation and cell death, while other amino acids showed no effect. Since glia cells are known to support neurons with thiols, we wondered, whether they might have protective properties in a co-culture model. When astrocytes were added to neurons, the latter were protected against MG-132. Under these conditions, the neuronal GSH increase was not observed and ATF4 activation was not detected. The major part of the protective effect of astrocytes was mediated by thiol supply to neurons and this indicates that elevation of cysteine levels in brain might have a protective effect against disturbances of the UPS system in neurons.Conclusion: The ATF4-mediated induction of DDIT4, NOXA and PUMA, and the mechanism of thiol-transfer from astrocytes to neurons may provide new pharmacological targets to prevent neurodegeneration.