Basal activation of astrocytic Nrf2 in neuronal culture media: Challenges and implications for neuron-astrocyte modelling

As a gatekeeper of antioxidant and anti-inflammatory cell protection, the transcription factor Nrf2 is a promising therapeutic target for several neurodegenerative diseases, leading to the development of Nrf2 activators targeting Keap1-dependent and independent regulatory mechanisms. Astrocytes play a crucial role in regulating neuronal physiology in health and disease, including Nrf2 neuroprotective responses. As neurons require specific conditions for their differentiation and maintenance, most 2D and 3D co-culture systems use medias that are compatible with neuronal differentiation and function, but also ensure astrocyte survival. Few studies, however, assess the molecular adaptations of astrocytes to changes from astrocyte maintenance medias alone, and their subsequent effects on neurons which may represent technical rather than physiological responses. Our findings show that while Nrf2 can be effectively activated by the Keap1-Nrf2 protein-protein interaction disruptor 18e, and classical Nrf2 activators dimethylfumarate and CDDO-Me, in human primary cortical astrocyte monocultures, their efficacy is lost in LUHMES neuron-astrocyte co-cultures. Further investigation revealed that the Advanced DMEM/F12-based LUHMES differentiation media maximally induced basal Nrf2 activity in astrocytes alone, compared to astrocyte maintenance media, thus preventing pharmacological activation. Although Neurobasal slightly activated basal Nrf2, this was not significant and did not prevent further activation by dimethylfumarate, suggesting that this media has less impact on astrocytic Nrf2 activity relative to Advanced DMEM/F12. As Nrf2 is a key regulator of oxidative damage and neuroinflammation, modelling these common features of neurodegenerative diseases may be confounded by environments that maximally activate basal Nrf2. Our findings thus suggest caution in media selection for neuron-astrocyte co-culture in disease modelling and therapeutic Nrf2 activator discovery, and suggest use of Neurobasal over Advanced DMEM/F12 medias for this purpose.