In amyotrophic lateral sclerosis, abnormally accumulated TDP-43 can damage mitochondria and promote the release of mitochondrial DNA into the cytoplasm. After recognizing this abnormal DNA, cGAS generates 2′,3′-cGAMP and activates STING, subsequently initiating TBK1–IRF3 and NF-κB signaling and converting mitochondrial damage into persistent neuroinflammation. The cGAS–STING pathway thus represents an important molecular axis connecting TDP-43 pathology with motor neuron injury.

Keywords: amyotrophic lateral sclerosis; TDP-43; mitochondrial DNA; cGAS; STING; TBK1; IRF3; neuroinflammation

1 Amyotrophic Lateral Sclerosis and TDP-43 Pathology

1.1 Why Do Motor Neurons Undergo Progressive Degeneration?

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized primarily by the progressive loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. Following motor neuron injury, neural impulses can no longer be transmitted normally to skeletal muscles, and patients gradually develop muscle weakness, muscle atrophy, dysphagia, and respiratory failure.

ALS is not caused by a single molecular abnormality. Disrupted proteostasis, abnormal RNA metabolism, impaired axonal transport, glutamate excitotoxicity, mitochondrial dysfunction, and neuroinflammation may coexist and amplify one another during disease progression.

1.2 Normal Functions and Pathological Transformation of TDP-43

(1) Normal TDP-43 Is Located Mainly in the Nucleus

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein that is normally distributed mainly in the nucleus and participates in pre-mRNA splicing, regulation of RNA stability, RNA transport, and transcriptional control. TDP-43 can shuttle dynamically between the nucleus and cytoplasm, and its localization is tightly regulated.

(2) Nuclear Depletion and Cytoplasmic Aggregation Occur Simultaneously

In most ALS cases, TDP-43 is mislocalized from the nucleus to the cytoplasm, where it forms phosphorylated, ubiquitinated, and aberrantly cleaved protein aggregates. This change produces two consequences simultaneously: loss of the normal RNA-regulatory functions of nuclear TDP-43 and toxic gain of function by abnormal cytoplasmic TDP-43.

(3) Mitochondria Are an Important Site of TDP-43 Toxicity

A proportion of abnormal TDP-43 can enter mitochondria and disrupt the respiratory chain, membrane potential, ATP production, and reactive oxygen species homeostasis. When the homeostasis of the mitochondrial inner and outer membranes is disrupted, mitochondrial DNA that is normally confined within mitochondria may enter the cytoplasm and be recognized by the innate immune system.

2 How the cGAS–STING Pathway Recognizes Abnormal DNA

2.1 cGAS Is a Cytoplasmic DNA Sensor

Cyclic GMP-AMP synthase (cGAS) is a cytoplasmic DNA sensor. Free double-stranded DNA is almost absent from the cytoplasm of normal cells; viral DNA, bacterial DNA, damaged nuclear DNA, or mitochondrial DNA may all be recognized by cGAS after entering the cytoplasm.

cGAS responds primarily to the spatial localization, double-stranded structure, and length of DNA rather than to a specific nucleotide sequence. This recognition mechanism facilitates the rapid detection of pathogens, but it also means that cellular DNA appearing in the wrong compartment can trigger inflammation.

2.2 How cGAS Generates 2′,3′-cGAMP

Binding of double-stranded DNA to cGAS promotes cGAS dimerization and higher-order assembly, shifting its catalytic domain into an active state. Activated cGAS uses ATP and GTP as substrates to synthesize 2′,3′-cyclic GMP-AMP (2′,3′-cGAMP), which contains both 2′–5′ and 3′–5′ phosphodiester bonds.

2′,3′-cGAMP is not a conventional metabolic intermediate but a second messenger responsible for transmitting cytoplasmic DNA signals. It converts the spatial information that abnormal DNA has appeared in the cytoplasm into a chemical signal that can be recognized by a downstream membrane protein.

2.3 How STING Transduces the Signal

(1) Ligand Binding and Conformational Change

Stimulator of interferon genes (STING) is a transmembrane adaptor protein located in the endoplasmic reticulum membrane. Binding of 2′,3′-cGAMP to the cytoplasmic domain of STING promotes conformational closure of the STING dimer and the formation of higher-order complexes.

(2) Translocation from the Endoplasmic Reticulum to the Golgi Apparatus

Activated STING leaves the endoplasmic reticulum and is transported through the ER–Golgi intermediate compartment to the Golgi apparatus. At the Golgi apparatus, STING undergoes palmitoylation and aggregation, which are important steps in the formation of a downstream signaling platform. STING that fails to undergo proper trafficking or palmitoylation is generally unable to sustain TBK1 activation.

(3) Activation of TBK1–IRF3 and NF-κB

STING at the Golgi apparatus recruits TANK-binding kinase 1 (TBK1). TBK1 phosphorylates the carboxyl terminus of STING and interferon regulatory factor 3 (IRF3), promoting IRF3 dimerization and nuclear translocation and inducing the expression of IFN-β and interferon-stimulated genes. STING can also activate NF-κB-related signaling and promote the expression of TNF, IL-6, and other inflammatory mediators.

2.4 How STING Signaling Is Terminated

Persistently activated STING is transported from the Golgi apparatus to endosomes and lysosomes, where degradation limits signaling intensity. STING trafficking is both necessary for signal initiation and an important component of signal termination. If trafficking, degradation, or negative-feedback mechanisms are impaired, a transient DNA response may develop into persistent inflammation.

3 How TDP-43 Activates the cGAS–STING Pathway

3.1 Abnormal TDP-43 Disrupts Mitochondrial Homeostasis

TDP-43 that enters mitochondria can affect mitochondrial RNA metabolism, respiratory chain function, and oxidative phosphorylation, resulting in reduced ATP production, elevated reactive oxygen species, and abnormal membrane potential. When mitochondria remain under sustained stress, their membrane structure and ionic homeostasis gradually become dysregulated, creating conditions that permit leakage of mitochondrial contents.

3.2 mPTP-Mediated Release of Mitochondrial DNA

Persistent opening of the mitochondrial permeability transition pore (mPTP) can increase the permeability of the mitochondrial inner membrane, causing matrix swelling, collapse of the membrane potential, and impaired energy metabolism. Under TDP-43-associated stress, mPTP participates in the release of mitochondrial DNA into the cytoplasm.

mtDNA is normally confined to the mitochondrial matrix and nucleoid structures and does not directly contact cytoplasmic cGAS. After entering the cytoplasm, the identity of mtDNA remains unchanged, but its spatial localization becomes abnormal. cGAS recognizes this compartmental mislocalization as a danger signal and initiates 2′,3′-cGAMP synthesis.

3.3 The TDP-43–mtDNA–cGAS–STING Signaling Axis

TDP-43-associated signaling can be summarized as follows: abnormal TDP-43 accumulation → entry of TDP-43 into mitochondria → impairment of mitochondrial function and membrane homeostasis → mPTP-mediated mtDNA release → cGAS-mediated synthesis of 2′,3′-cGAMP → STING activation → enhancement of TBK1–IRF3 and NF-κB signaling. This signaling axis connects protein aggregation, organelle damage, and innate immune responses, extending TDP-43 pathology from the intracellular environment to the inflammatory signaling network.

4 How cGAS–STING Amplifies Neuroinflammation

4.1 Type I Interferon Response

Following STING activation, TBK1 mediates IRF3 phosphorylation and promotes IFN-β expression. Secreted IFN-β can bind type I interferon receptors on the cell surface, activate JAK–STAT signaling, and induce interferon-stimulated genes. A short-term interferon response contributes to antimicrobial defense, but when it persists in the absence of pathogens, it may alter neuronal metabolism, synaptic function, and cell survival.

4.2 NF-κB-Associated Inflammatory Responses

STING can also promote the expression of TNF, IL-6, and multiple chemokines through the NF-κB pathway. These inflammatory mediators can activate microglia and astrocytes and enhance local immune-cell recruitment and inflammatory signal propagation. Prolonged exposure of motor neurons to inflammatory mediators, oxidative stress, and an excitotoxic environment facilitates the accumulation of cellular damage.

4.3 Mitochondrial Damage and Inflammation Form a Positive-Feedback Loop

Inflammatory factors and reactive oxygen species can further damage mitochondria and promote the entry of additional mtDNA into the cytoplasm. Newly released cytoplasmic mtDNA continues to activate cGAS–STING, forming a positive-feedback loop of “mitochondrial damage–DNA leakage–innate immune activation–secondary mitochondrial damage.”

This mechanism indicates that neuroinflammation in ALS is not merely a passive response following neuronal death but may actively contribute to disease progression. TDP-43 pathology provides the upstream injury signal, whereas cGAS–STING amplifies this signal and transmits it to a broader cellular environment.

5 Intervention Levels for Targeting the cGAS–STING Pathway

5.1 Inhibition of cGAS: Blocking the Initiation of Abnormal DNA Signaling

cGAS inhibitors can reduce 2′,3′-cGAMP production and prevent cytoplasmic mtDNA signals from being transmitted to STING. This strategy acts upstream in the pathway and is suitable for DNA-dependent STING activation, but it cannot block signals generated by exogenous cyclic dinucleotides or other factors that directly activate STING.

5.2 Inhibition of STING: Blocking the Common Signaling Hub

STING is located downstream of cGAS and serves as a key hub connecting 2′,3′-cGAMP with TBK1–IRF3 signaling. Inhibition of STING ligand binding, endoplasmic reticulum-to-Golgi trafficking, oligomerization, or palmitoylation may reduce type I interferon and NF-κB-associated inflammatory outputs.

STING integrates multiple types of cytoplasmic DNA signals, and its inhibition generally has a broader scope than selective cGAS blockade. At the same time, STING participates in antiviral immunity and immune surveillance, and long-term systemic inhibition may weaken host defense.

5.3 Restriction of mtDNA Release: Controlling the Upstream Danger Signal

Stabilizing mitochondrial membranes, reducing abnormal mPTP opening, or improving mitochondrial proteostasis can reduce the entry of mtDNA into the cytoplasm. These strategies do not directly inhibit core innate immune proteins but instead reduce the endogenous DNA source that triggers the pathway.

Mitochondrial damage in ALS involves multiple interconnected processes, and restricting mtDNA release alone may not correct all abnormalities in RNA metabolism, axonal transport, and proteostasis caused by TDP-43.

5.4 Inhibition of TBK1 and Downstream Inflammatory Outputs

TBK1 inhibition can reduce IRF3 phosphorylation and type I interferon expression, but TBK1 also participates in autophagy, selective protein degradation, and regulation of cell survival. The effects of multitarget TBK1 inhibitors cannot be attributed entirely to the cGAS–STING pathway, and their actions on IKKε, PDK1, and other kinases must be considered during interpretation.

5.5 cGAS–STING Is Not Simply a Pathogenic Switch

cGAS–STING has protective functions during acute infection and the early stages of tissue injury, whereas persistent or excessive activation is more likely to cause chronic inflammation. ALS has a prolonged disease course and involves complex cell types, and the role of the pathway may vary according to disease stage, tissue region, and immune status. The aim of intervention is not to completely eliminate innate immunity but to restrict aberrant signaling persistently driven by self-derived mtDNA.

6 Products Related to the cGAS–STING Pathway

6.1 Products for Modulating the cGAS–STING Pathway

6.2 Products for Target Expression and Signal Analysis

6.3 Products for Mitochondrial Damage and Downstream Inflammatory Outputs

TDP-43 can activate the cGAS–STING pathway through mPTP-mediated mtDNA release, converting mitochondrial damage into TBK1–IRF3, NF-κB, and type I interferon responses. This mechanism provides a new molecular framework for understanding neuroinflammation in ALS and a potential intervention strategy for limiting the inflammatory amplification caused by TDP-43 pathology.

References

[1] Barber GN, et al. STING: infection, inflammation and cancer. Nat Rev Immunol. 2015;15(12):760–770.

[2] Motwani M, et al. DNA sensing by the cGAS-STING pathway in health and disease. Nat Rev Genet. 2019;20(11):657–674.

[3] Yu CH, et al. TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS. Cell. 2020;S0092-8674(20)31161-2.

[4] Wang R, et al. Activation of interferon signaling pathways in spinal cord astrocytes from an ALS mouse model. Glia. 2011;59(6):946–958.

For more related articles, please see below:

[1] mtDNA Stress and cGAS–STING/AIM2 Innate Immune Activation Mediated by CRAT Deficiency