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

As a gate-keeper of anti-oxidant, anti-inflammatory and xenobiotic cell protection mechanisms, the transcription factor Nrf2 has been implicated as a promising therapeutic target for several neurodegenerative diseases, leading to the development of Nrf2 activators targeting Keap1-dependent and independent regulatory mechanisms. This study aimed to evaluate the efficacy of a Keap1-Nrf2 protein-protein interaction disruptor, 18e, in comparison with classical electrophilic Nrf2 activators, CDDO-Me and Dimethylfumarate (DMF), with a view to measuring their effects on neuronal protection using LUHMES neuron-astrocyte co-cultures. 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 containing high glucose and a variety of growth factors, allowing astrocytes to survive without the media negatively impacting neuronal function. Few studies, however, assess the molecular adaptations of astrocytes in response to changes from astrocyte maintenance medias alone, and the potential consequences for neuronal function, which may represent technical rather than physiological changes. Our findings show that while Nrf2 can be effectively activated by 18e, DMF and CDDO-Me in human primary cortical astrocyte monocultures, their efficacy is lost in the LUHMES-astrocyte co-culture, as measured by NQO1 enzymatic activity. Further investigation revealed that the Advanced DMEM/F12-based LUHMES differentiation media maximally induced basal Nrf2 activity in astrocytes alone, in comparison to complete astrocyte maintenance media. Analysis of media components revealed that this was not due tetracycline or high glucose, and was unlikely to be due to REDOX-inducing phenol-red, the concentration of which is comparable across all medias used in our study. Although Neurobasal slightly activated basal Nrf2 compared to astrocyte media, trends toward further activation were observed in the presence of 18e and DMF, suggesting that this media impacts astrocytic Nrf2 responses less than Advanced DMEM/F12. Numerous studies model oxidative stress and neuroinflammation, key features of neurological diseases, using neuronal systems. As Nrf2 is a key regulator of cellular damage, the effects of these stressors could be confounded by cellular environments that maximally activate basal Nrf2, as observed in our experiments. Hence, this study highlights the need for caution in media selection for neuron-astrocyte co-culture modelling, not only for researchers investigating Nrf2 therapeutics, but also for other mechanisms by which astrocytes influence neuronal function in health and disease.