Autophagy induced by STING, an unnoticed and primordial function of cGAS

Cyclic GMP-AMP synthase (cGAS), combined with dsDNA, produces cyclic GMP-AMP (cGAMP) to stimulate STING and activates the kinases TBK1 and IKK to induce interferon and other cytokines, which play an important role in host defense. In addition to the traditional interferon inflammatory signaling pathway of cGAS, recently, Chen et al.¹ discovered a new mechanism in which cGAS-cGAMP-STING activated autophagy through WIPI2 and ATG5. Autophagy is a process induced under various cellular stress conditions, such as oxygen deficit, nutrient deprivation, lack of growth factors, and infection, aiming to maintain the basic survival of cells or promote apoptosis of senescent cells.^2,3 The activation of the autophagy-related (ATG)1/Unc-51-like kinase (ULK) complex is vital for autophagy induction. The ATG1/ULK1 complex phosphorylates or recruits its downstream proteins, contributing to autophagosome formation.³ Under the regulation of related genes, “cargo” is transferred to the lysosome and digested by various enzymes. In addition, cyclic GMP-AMP synthase (cGAS), which detects exogenous or self-damaged dsDNA in the cytoplasm after binding to dsDNA, produces cGAMP to activate the adaptor protein stimulator of interferon genes (STING), triggering the downstream pathway and secretion of interferon or other cytokines.⁴

Chen et al. discovered that STING, after treatment with cGAMP, not only induces the secretion of interferon or other cytokines but also initiates autophagy.¹ The researchers found that STING from the sea anemone Nematostella vectensis (NvSTING) facilitates autophagy rather than inducing the production of interferon upon interacting with cGAMP.¹ cGAMP from human or N. vectensis was shown to be highly conservative, and NvSTING does not contain the C-terminal domain, which is required for the activation of the interferon pathway.¹ Moreover, truncated human STING(1–340) without the C-terminal domain still maintains NvSTING function after treatment with cGAMP. A mutation within amino acid residues 330–334 in the full-length STING resulted in a failure to induce autophagy and phosphorylation of TBK1 and IRF3 after treatment with cGAMP. In this report, the authors hypothesized that autophagy induced by STING is independent of TBK1-IRF3 activation and is a primordial function of the cGAS pathway.¹

The formation of autophagosomes is an inevitable step in autophagy. Protein LC3 is a key marker of the autophagosome and is transferred to the growing autophagosome by LC3 lipidation.⁵ Therefore, it is important to determine which type of lipid or membrane source serves as a substrate for LC3 lipidation. Proteins are transported to their destinations and initiated in the endoplasmic reticulum (ER). They move to the Golgi apparatus, from where they are sent to various destinations or to the lysosome for degradation.⁵ From the ER to the Golgi, they pass through the ER–Golgi intermediate compartment (ERGIC).^5,6 STING first colocalizes with ERGIC-53, which is a marker of the ERGIC, before interacting with LC3–GFP-positive autophagosomes.¹ ERGIC was shown to serve as a membrane source for LC3 lipidation.⁵ Further evidence suggested that ERGIC from cGAS-activated ATG5^−/− HEK293T-STING cells incubated with cytosol from inactivated wild-type cells leads to LC3 conversion.¹ However, ERGIC from cGAS-stimulated cells treated with brefeldin A (an ERGIC inhibitor) failed to stimulate LC3 conversion.¹ This observation indicates that ERGIC serves as a membrane source for LC3 lipidation (Fig. 1). In addition, the transport from the ER to ERGIC is dependent on the COP-II complex, including SEC24C and ARF1 GTPases.⁵ SEC24C gene knockdown in HEK293T-STING cells leads to LC3 conversion, while phosphorylation of TBK1 and IRF3 is impaired. cGAMP treatment enhanced the binding between ARF1 GTPase and its effector protein, GGA3.¹

Fig. 1.

Autophagy induced via cGAS-STING and its signaling pathway. a Exogenously or endogenously damaged dsDNA stimulates cGAS to produce cGAMP. cGAMP binds to STING, and the cGAMP-STING complex recruits SAR1 and SEC24C to form the COP-ll complex. The COP-ll complex translocates from the ER to ERGIC. b The ERGIC serves as a membrane source for LC3 lipidation, which is dependent on WIPI2. LC3-positive membranes enclose dsDNA, bacteria or viruses and form autophagosomes. c The autophagosome transferred to the lysosome is digested by various enzymes

To investigate the mechanisms of autophagy induced by STING, autophagy-related gene analysis was performed. ATG5 but not ATG9 deficiency was found to impair LC3 lipidation. In addition, mTOR is a well-studied autophagic regulatory pathway. Although HT-DNA or cGAMP treatment induced more LC3 conversion than an mTOR inhibitor, there was no significant increase in mTOR or 4E-BP1 dephosphorylation compared with the positive control. Compared to canonical autophagy, ULK1, ULK2, and beclin1 showed less influence on autophagy induced by STING, while WIPI2-deficient cells showed reduced LC3 lipidation after treatment with cGAMP. In essence, Chen et al. found that autophagy induced by STING is dependent on WIPI2 and ATG5, which operate distinctly from conventional autophagy.

As mentioned previously, autophagy induction via STING trafficking is a primordial function in the cGAS pathway. Whether it eliminates the “threat” within the cytoplasm is not yet clear. As expected, the LC3 puncta accumulate around the exogenous DNA and enclose it. This process is accelerated after treatment with cGAMP, and endogenous damaged DNA was shown to be decreased significantly in the cytoplasm compared to the control. In terms of antiviral functions, both HIV-1 and HSV-1 virus titers were decreased in cGAMP-stimulated HEK293T cells that express full-length STING or STING(1–340). Notably, STING(1–340) did not induce interferon or other cytokines. Knocking out ATG5 but not TBK1 would largely abrogate this effect, revealing that autophagic events induced by STING play an antiviral role during viral infections.

The misfolding or irreversible aggregation of cytoplasmic proteins is a characteristic of many neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD).⁷ In these diseases, the aggregated proteins are unable to be degraded and become toxic. Unlike other cells, the homeostasis of highly differentiated neurons is based on autophagy because misfolded or damaged organelles fail to be eliminated after cell division.⁸ Obviously, if neurons cannot eliminate this threat, cell apoptosis is inevitable. Autophagy, a cellular process for clearing damaged proteins and organelles, plays an important role in these neurodegenerative diseases.⁹ Rapamycin, a mTOR inhibitor, provides neuroprotection in several experimental models of neurodegenerative diseases.¹⁰ Other studies have suggested that ATG5 expression promotes cerebral cortical cell development and differentiation, while ATG5 deficiency abrogates this effect.¹¹ Overexpression of ATG5 in mice enhanced autophagy, and the ATG5 transgenic mice showed better tolerance to weight loss and aging and a decrease in insulin sensitivity.^11,12 When neurons are damaged, the promotion of autophagy is dependent on ATG5, which may contribute to restoring homeostasis. Hence, autophagy induced by STING may lead to neuroprotective effects. Neurodegenerative diseases affect the quality of human life, especially during old age, and most drugs developed for neurodegenerative diseases are unsuccessful. The findings reported here may provide a new direction for the development of drugs for neurodegenerative diseases. At the same time, as a supplement to the cGAS-cGAMP signaling pathway, this finding lays the foundation for future studies explaining autophagy-related mechanisms in the fields of immunity, inflammation, antitumor studies, and neurodegenerative diseases.

Competing interests

The authors declare no competing interests.