Experiments using neuron and glia co-culture in vitro suggest that activation of innate immunity in the CNS can trigger neuronal death (Lehnardt et al., 2003). Since NSC-derived neurons always co-exist with astrocytes, it is possible that astrocytes contributed to the neurotoxic effect of the microglia CM. To explore this possibility, we performed sequential CM experiments employing isolated primary microglia and astrocytes and using Neuro2A cells as a read-out for neurotoxicity. Primary murine astrocytes and microglia were infected with shCtrl- or shNurr1-lentivirus used for the injection into the SN. Cells were then stimulated with LPS and CM was harvested as described in Figure 2G. CM of microglia infected with shNurr1 induced significant cell death in Neuro2A cultures, whereas CM of astrocytes infected with shNurr1 had much less effect on the death of Neuro2A cells. Intriguingly, sequential conditioning of media from microglia to astrocytes indicated that astrocytes significantly amplified the production of neurotoxic factors when exposed to microglia-conditioned media (Fig. 2J lane 2 to lane 6 and 7). This effect was further increased when expression of Nurr1 was reduced in astrocytes (Fig.
