Cellular redox regulation by the neuronal glutamate and cysteine transporter, EAAT3

• Published in: The FASEB journal 2022-05, Vol.36 (S1), p.n/a
• Main Authors: Radhakrishnan, Saranya, Amara, Susan G.
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.L7661

Glutamate transporters are essential for clearing glutamate from the extracellular space and thus limit the actions of glutamate within the brain. The neuronal glutamate transporter EAAT3 has also been shown to transport cysteine, which serves as a rate‐limiting substrate for the synthesis of neuronal glutathione, an essential regulator of oxidative stress in cells. Hence, EAAT3 can contribute to regulating cellular redox by acting as an essential cysteine transporter in neurons. EAAT3 mutations are also associated with several neuropsychiatric disorders such as schizophrenia and obsessive‐compulsive disorders. The activity of plasma membrane transporters can be altered by changes in trafficking, expression levels and recycling of the transporters. EAAT3 can rapidly cycle to and from the cell surface and through intracellular compartments within a few minutes and this can be regulated through several different intracellular signaling pathways. Previous studies have demonstrated that activation of protein kinase C (PKC) can regulate EAAT3 trafficking and increase the localization of EAAT3 at the membrane surface. There is evidence for some redox‐triggered mechanisms that can directly or indirectly influence signaling by altering the catalytic properties or influencing subcellular compartmentation of different PKC isoforms. This suggests a mechanism by which EAAT3 trafficking could be modulated by PKC as a response to cellular redox stress to increase cysteine import and facilitate the synthesis of the antioxidant glutathione. To study this, we monitored if oxidizing agents such as hydrogen peroxide can stimulate a redox response in the cell and lead to increased membrane localization of EAAT3. We found that application of hydrogen peroxide leads to an increase in EAAT3 membrane localization in Neuro2A cells expressing EAAT3. We used TIRF and biotinylation assays to confirm that EAAT3 localization can be altered by PKC activation and further examined whether this surface expression can be additionally modulated during redox stress. While studying downstream redox changes upon cysteine and antioxidant N‐acetyl cysteine treatments, we noticed a transient oxidative response in the mitochondria resulting from the generation of sulfane sulfur species and hydrogen sulfide in the mitochondria. To understand these downstream effects of cysteine and N‐acetyl cysteine on cytosolic and mitochondrial stress, we monitored the redox changes in the cytosol and mitochondria