Abstract
The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy.
Highlights
Animals have developed adaptive, protective defence programs mediated by de novo gene expression to protect against oxidative stress
Astrocytes are the only contributor to Nrf2 re sponses in mixed cultures, because Nrf2 expression is repressed in neurons to the extent that they are unable to mediate Nrf2-dependent gene expression [12,16]
We performed a systematic analysis of induction of Nrf2 target genes, defined in a comprehensive study [18] as those whose promoters contain a ChIP-seq peak and are either downregulated in Nrf2-deficient MEFs (Nrf2+/+ vs. Nrf2− /− ) or up-regulated in Kelch-like ECH-associated protein 1 (Keap1)-deficient MEFs (Keap1+/+ vs. Keap1− /− ). 41 Nrf2 target genes as defined by this criteria were induced >2-fold in astrocytes (Table S1), but none of the 41 were induced in neurons (Fig. 1b and c, Table S1)
Summary
Protective defence programs mediated by de novo gene expression to protect against oxidative stress. Nrf activation in astrocytes, either via small molecular Keap inhibitors, or astrocyte-specific overexpression protects the astrocytes themselves, and protects nearby neurons in vitro by a mechanism at least in part due to the production and release of the antioxidant glutathione [10,14,15] This non-cell-autonomous neuroprotective ef fect is observed in mice over-expressing Nrf subjected to models of ALS, PD, stroke and hypo-perfusion [6,8]. A key issue for the therapeutic tar geting of Nrf in astrocytes (e.g. via small molecules) in neuro logical/neurodegenerative diseases associated with oxidative stress is whether such stress has already activated Nrf via Keap inhibition, occluding the effect of any therapeutic intervention. We found that activation of Nrf2-dependent gene expression in astro cytes by mild oxidative stress occurs via a non-canonical Keap1-inde pendent pathway in contrast to pharmacological Keap inhibitors, rendering such compounds effective even under conditions of oxidative stress
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