Astrocytic thiol supply protects neurons from proteotoxic and oxidative stress

Abstract

Neurodegenerative disorders are marked by a progressive neuronal loss, often induced by proteotoxic and oxidative stress. Since many approaches to specifically block neuronal cell death pathways have failed in clinical trials, the modification of astrocytic support for stressed neurons should be pursued as an alternative strategy. Following this concept, we investigated astrocytic support during various neuronal stress conditions. We developed an in vitro co-culture model of human neurons (LUHMES) with astrocytes, and compared this to neuronal mono-cultures. Neurons in mono-culture were highly sensitive to proteotoxic (protein aggregates; ATF- 4 induction) and oxidative (GSH depletion; NRF-2 induction) stress, triggered with the frequently used proteasome inhibitor MG-132 or the mitochondrial toxicant 1-methyl-4-phenylpyridinium (MPP+). The presence of astrocytes protected the neurons, and drastically reduced neurodegeneration. The transcription factor ATF-4 was identified as a coordinator of the neuronal stress response, and its upregulation was attenuated in the presence of astrocytes, while NRF-1/NFE2L1, the transcription factor counter-regulating neuronal proteotoxic stress, was upregulated. We identified thiols, secreted by astrocytes, to be the triggers for the stress response adaption. Addition of GSH, transfer of astrocyte-conditioned medium and also the presence of astrocytes in direct co-cultures resulted in increased intracellular GSH levels of neurons. Additionally, cysteine depletion in neurons, occurring upon proteasomal inhibition, was reversed upon co-treatment with GSH. The improved thiol supply did not only attenuate oxidative stress and modify several other stress response pathways, it also increased the ability of neurons to degrade aggregated poly-ubiquitinated proteins. Thiol supply by astrocytes therefore results in an attenuation of cell death-related stress response with an enhanced recovery from proteotoxic stress and eventually in decreased neurodegeneration. Having established the important role of astrocytes during neuronal stress response, we further refined the experimental model. Neurons and astrocytes were cultured in form of 3D organoids, into which iPSC-derived microglia are incorporated as a third relevant cell type. We currently investigate the effect of quiescent vs activated microglia on astrocytic thiol supply and neuronal stress response.