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

Intracellular DNA sensing is not always triggered directly by exogenous pathogens. In many noninfectious stress settings, its upstream origin is first a disruption of mitochondrial metabolic homeostasis. Taking CRAT deficiency as the point of entry, acetyl-group buffering failure, mtDNA stress, cGAS–STING activation, and AIM2 inflammasome amplification can be integrated into one continuous mechanistic chain. This framework helps explain, from the level of metabolic fundamentals, how sterile innate immune activation is established.

Keywords: CRAT; mtDNA stress; cGAS–STING; AIM2; acetylcarnitine; mitochondrial stress; type I interferon; inflammasome

1. Significance of CRAT Deficiency as a Metabolic Starting Point

1.1 Metabolic positioning of CRAT

(1) An acetyl-group buffering node

CRAT (carnitine acetyltransferase) catalyzes the reversible conversion between acetyl-CoA and acetylcarnitine. The significance of this reaction lies not merely in acetylcarnitine production itself, but more importantly in buffering excess mitochondrial acetyl groups, maintaining free CoA availability, and coordinating carbon-flux linkage among fatty acid oxidation, pyruvate oxidation, and entry into the tricarboxylic acid cycle. Accordingly, CRAT is positioned at the interface between mitochondrial substrate-processing capacity and acetyl-group redistribution capacity, and is one of the key nodes required for maintenance of metabolic flexibility.

(2) Interface properties within the carnitine network

Under high metabolic load, CRAT is not an isolated carnitine-metabolism enzyme, but rather an interface layer linking fatty acid oxidation, acetyl-group export, CoA cycling, and organic-acid metabolism. Changes in its activity affect not only acetylcarnitine formation, but also the mitochondrial capacity to buffer acute metabolic perturbation. Consequently, the consequences triggered by CRAT deficiency are usually manifested as network-level imbalance rather than as an abnormality of a single step.

1.2 Metabolic reprogramming after CRAT deficiency

(1) Reduced buffering capacity for acetyl-CoA

After CRAT deficiency, the capacity to convert acetyl-CoA toward acetylcarnitine declines, and the flexibility with which mitochondria regulate acetyl-group burden correspondingly decreases. Under these conditions, the cell is not simply facing reduction of a single metabolite, but rather insufficient handling and temporary storage capacity for acetyl groups. The result is generally manifested as relative accumulation of acetyl-CoA, restricted regeneration of free CoA, and poorer coupling among multiple oxidative metabolic pathways.

(2) Impaired substrate-switching capacity

Under normal conditions, cells can switch relatively flexibly between fatty acid oxidation and glucose oxidation according to nutrient supply and energy demand. Under CRAT deficiency, however, this switching capacity declines, and mitochondria more readily enter a state in which substrate input remains continuous while processing flexibility is reduced. At this stage, the earliest change is usually not immune output itself, but mitochondrial metabolic homeostasis.

(3) Shifts in cholesterol- and bile-acid-related metabolism

CRAT deficiency may also be accompanied by changes in the direction of cholesterol catabolism and in the distribution of related metabolic intermediates. The pathological significance of this is not that a single intermediate rises in isolation, but that these metabolic changes further increase local mitochondrial stress and drive the environment surrounding mtDNA from stability toward fragility, thereby creating conditions for subsequent DNA sensing.

1.3 Why CRAT deficiency is particularly suitable as the starting point of this mechanistic axis

(1) It shifts metabolic abnormality forward as the primary pathological layer

If one begins with cGAS–STING or AIM2 themselves, what is usually observed is already a downstream immune phenotype. By contrast, taking CRAT deficiency as the starting point makes it easier to see how abnormal metabolic homeostasis occurs first and gradually generates abnormal DNA signals that can be read by the immune system.

(2) It highlights the immunological significance of carnitine-related metabolism

One of the most important insights provided by this mechanistic axis is that carnitine-related metabolism does not merely serve energy supply. It may exert profound effects on acetyl-group buffering, mitochondrial stability, and the threshold for cytosolic DNA sensing. Accordingly, metabolic abnormality is no longer just a background condition for immune responses, but becomes their true upstream driving layer.

2. Formation of mtDNA Stress and Its Intermediate Role

2.1 The actual meaning of mtDNA stress

(1) Not simply a change in mtDNA abundance

mtDNA stress is not equivalent to an increase in mitochondrial DNA copy number, nor is it merely the broad notion of "mitochondrial DNA damage." More precisely, it refers to abnormalities in mtDNA packaging, stability, localization, and compartmental isolation, such that mtDNA becomes more prone to exposure, mislocalization, or leakage into the cytosol.

(2) The key issue is spatial disorganization

What DNA-sensing innate immunity truly responds to is not total DNA abundance, but whether DNA appears in the wrong compartment. Once mtDNA escapes its normal mitochondrial confinement and enters the cytosol, it is converted from an internal mitochondrial genetic component into a potential danger-associated signal. Therefore, the essence of mtDNA stress is spatial disorganization rather than simple quantitative fluctuation.

2.2 Why metabolic pressure readily evolves into mtDNA stress

(1) Imbalance of the mitochondrial redox environment

The acetyl-group handling defect caused by CRAT deficiency can gradually increase intramitochondrial metabolic stress, thereby disturbing electron transport, reactive oxygen species homeostasis, and the local redox environment. For mtDNA, these changes first disrupt the surrounding microenvironment, making it more vulnerable to oxidative damage and loosening of packaging structure.

(2) Disturbance of membrane structure and crista organization

When metabolic stress persists, mitochondrial membrane permeability, crista structure, and coordination between the inner and outer membranes also gradually destabilize. The consequence is not necessarily immediate catastrophic rupture, but more commonly a progressive decline in the ability of mitochondria to maintain compartmental control over DNA. At that stage, the probability of abnormal mtDNA exposure or mislocalization increases markedly.

(3) Insufficient quality control

If damaged mitochondria cannot be isolated, repaired, or cleared in time, mtDNA stress is more likely to persist. In other words, mtDNA stress is not usually a transient event, but rather the combined result of metabolic abnormality, structural fragilization, and defective quality control.

2.3 Position of mtDNA stress within the mechanistic chain

(1) It is the intermediate layer connecting metabolic abnormality to immune abnormality

Within this mechanistic axis, CRAT deficiency does not directly activate cGAS–STING. The actual conversion step is accomplished by mtDNA stress. Only when mtDNA loses its original positional constraints and enters the cytosol do DNA-sensing pathways gain access to an actual ligand source.

(2) It determines whether the downstream immune response can become sustained

If mitochondria undergo only transient metabolic fluctuation without generating stable mtDNA stress, DNA-sensing responses are often difficult to maintain. If mtDNA stress persists over the long term, cGAS–STING is more likely to remain activated and to provide a sustained basis for subsequent AIM2 amplification.

Table 1. Upstream changes from CRAT deficiency to mtDNA stress

Level	Key Change	Main Consequence
Acetyl-group metabolism	CRAT deficiency	Reduced acetyl-group buffering capacity
Mitochondrial metabolism	Disturbed balance between acetyl-CoA and CoA	Reduced substrate-switching capacity
Mitochondrial homeostasis	Increased redox and membrane-structural stress	Destabilization of the local mtDNA environment
DNA stress	Formation of mtDNA stress	Provision of substrate for activation of DNA-sensing pathways

3. The Signal-Translation Layer of cGAS–STING

3.1 cGAS–STING recognizes DNA in an abnormal compartment

(1) Activation logic depends on DNA location rather than source

cGAS is a cytosolic double-stranded DNA sensor. As long as DNA abnormally appears in the cytosol, whether it originates from viruses, bacteria, the nucleus, or mitochondria, it may be recognized. Under mitochondrial stress, once mtDNA leaks into the cytosol, it can function as an effective ligand for cGAS.

(2) STING undertakes signal translation and amplification

After cGAS binds DNA, it catalyzes the production of cGAMP, which then activates STING and initiates the TBK1–IRF3 axis and related downstream programs. The core function of this step is to translate the structural abnormality of DNA mislocalization into an amplifiable immune signal rather than merely completing ligand recognition.

3.2 Establishment of the type I interferon program

(1) First-stage output is dominated by transcriptional reprogramming

Within this mechanistic axis, the first outcome established after cGAS–STING activation is not the inflammasome, but rather type I interferon and related transcriptional programs. The main consequences generally include:

① increased type I interferon expression;

② upregulation of interferon-stimulated genes;

③ establishment of an inflammation-related transcriptional background;

④ increased sensitivity of cells to subsequent DNA stimulation.

Accordingly, cGAS–STING is closer to a state-establishing layer than to a terminal execution layer.

(2) Formation of a pre-inflammatory environment

Establishment of an interferon background has dual significance. On the one hand, it indicates that mtDNA abnormality has been effectively translated into an immune signal. On the other hand, it means that the cell has entered a presensitized state in which inflammatory amplification can more readily occur. This state is not yet equivalent to full inflammasome activation, but it creates the necessary condition for subsequent AIM2 upregulation.

3.3 Hierarchical position of cGAS–STING in the main mechanistic axis

(1) A middle-stage translation node

In the axis connecting CRAT deficiency, mtDNA stress, and innate immune activation, cGAS–STING is not the most upstream event. The true starting point is disruption of metabolism and mitochondrial homeostasis, whereas cGAS–STING functions as the middle-stage translation layer, converting mtDNA abnormality into a systemic immune transcriptional response.

(2) It determines whether pathology progresses downstream

If cGAS–STING activation is only transient and low-level, pathology may remain at the stress-transcription layer. If its activation is sustained, however, cells are placed for a prolonged period under an interferon-related background, making subsequent inflammasome programs more likely to be driven upward. Therefore, cGAS–STING is both an intermediate pathway and a key amplification node determining whether pathology progresses toward the execution layer.

4. Secondary Amplification by the AIM2 Inflammasome

4.1 Functional positioning of AIM2

(1) Inflammasome-type DNA sensing

AIM2 can also recognize cytosolic DNA, but its downstream output is not primarily interferon, but rather inflammasome assembly, caspase-1 activation, and maturation and release of IL-1beta and IL-18. AIM2 is therefore closer to the inflammatory execution end than to the transcriptional preparation end.

(2) Sequential relationship with cGAS–STING

In the present mechanistic axis, AIM2 is not a completely parallel, independent pathway with cGAS–STING. Rather, after cGAS–STING establishes a type I interferon background, AIM2 enters a state of higher expression and higher sensitivity. AIM2 is therefore more appropriately understood as a secondary amplification layer rather than an initial recognition layer.

4.2 Why AIM2 rises under this background

(1) Priming effect of the interferon background

A sustained interferon environment can lower the response threshold of cells to DNA stimulation, increase AIM2 expression, and raise the tendency toward inflammasome assembly. Under these conditions, mtDNA abnormality that would previously have been sufficient only to trigger a transcriptional response can more readily push the system further into inflammatory execution.

(2) Initiation of the inflammatory execution layer

Once AIM2 enters a state of high expression and high sensitivity, cellular immune output shifts from a transcriptional response dominated by interferon to a more destructive inflammasome-layer response. The result is generally manifested as:

① activation of caspase-1;

② maturation and release of IL-1beta and IL-18;

③ enhancement of local inflammatory execution programs;

④ amplification of cellular injury and tissue-level inflammation.

4.3 Functional differences between the two amplification stages

(1) First stage: transcriptional priming

The first stage, dominated by cGAS–STING, is mainly responsible for recognizing DNA abnormality and establishing an interferon background. Its defining features are state establishment and increased sensitivity.

(2) Second stage: inflammatory execution

The second stage, dominated by AIM2, converts the earlier primed state into an inflammasome-execution response and determines whether pathology escalates from reversible stress to injurious inflammation.

Table 2. Continuous output from mtDNA stress to cGAS–STING/AIM2

Stage	Key Molecule	Main Output	Biological Significance
First stage	cGAS–STING	Type I interferon, interferon-stimulated genes	Establishment of a pre-inflammatory background
Second stage	AIM2 inflammasome	Caspase-1 activation, IL-1beta/IL-18 maturation	Amplification of inflammatory execution and tissue injury

5. Disease Relevance and Pathological Extrapolation

5.1 Cardiovascular injury and inflammatory remodeling of the myocardium

(1) A continuous pathological chain in the myocardial context

Within this mechanistic axis, the most direct disease relevance is first manifested in cells with high mitochondrial burden, especially cardiomyocytes. The acetyl-group buffering defect caused by CRAT deficiency not only increases metabolic stress, but can further drive mtDNA stress, cGAS–STING-dependent type I interferon responses, and subsequently enhanced AIM2 inflammasome activation. In this way, metabolic imbalance, mitochondrial DNA stress, and myocardial inflammatory amplification are integrated into the same pathological axis.

(2) Mechanistic significance in cardiovascular disease

In the context of cardiovascular disease, the importance of this axis lies not merely in indicating the presence of inflammation, but in explaining why tissues under high metabolic load are more likely to convert mitochondrial stress into sustained immune signaling. Once cGAS–STING remains continuously activated and further drives inflammasome programs, the consequences are often not limited to transcriptional reprogramming, but may also promote cardiomyocyte injury, expansion of local sterile inflammation, and worsening of cardiac remodeling.

5.2 Metabolic inflammation and organ injury

(1) Shared logic in metabolic disease

From a broader disease perspective, the mtDNA stress–cGAS–STING layer is not restricted to myocardium. In obesity, insulin resistance, fatty liver disease, and other metabolic disorders, there is likewise a clear mechanistic relationship among disruption of mitochondrial homeostasis, enhanced cytosolic DNA sensing, and chronic low-grade inflammation. Their common logic is that metabolic overload first causes disruption of mitochondrial DNA homeostasis, and DNA-sensing pathways then convert that metabolic stress into a sustained inflammatory background.

(2) AIM2 drives metabolic inflammation from the stress layer into the injury layer

If the pathological process remains confined to the cGAS–STING stage, the main manifestation usually remains upregulation of interferon- and inflammation-related gene expression. Once the AIM2 inflammasome is further brought into a highly sensitive state, however, disease consequences are more likely to shift from metabolic stress toward tissue-injurious inflammation. Thus, in metabolic inflammation, AIM2 involvement generally means that pathology has progressed from simple homeostatic imbalance to the stage of inflammatory execution and cellular injury.

5.3 Bidirectional effects in the tumor microenvironment

(1) The immune-recognition initiation layer

In the tumor context, mtDNA leakage and cGAS–STING activation do not necessarily function only pathologically. At certain stages, mtDNA released from tumor cells or damaged cells may enhance innate immune recognition and increase local inflammatory tone, thereby helping initiate antitumor immunity. In other words, this axis can serve an immune-activating role under specific spatiotemporal conditions.

(2) The chronicized inflammatory maintenance layer

If mtDNA–cGAS–STING signaling persists chronically at a low level, however, the result is not necessarily effective antitumor immunity. Sustained stimulation may instead lead to establishment of a chronic inflammatory background associated with immune suppression, adaptive microenvironmental change, and remodeling of tumor tissue. If AIM2 is simultaneously elevated in this context, the inflammatory execution layer may be further intensified, making the local ecosystem even more complex. Therefore, in tumors, this axis is clearly biphasic: it can either enhance immune recognition or, once chronicized, participate in maintenance of pathological inflammation.

5.4 Chronic amplification in aging and neurodegenerative disease

(1) An explanatory framework for chronic low-grade inflammation

Aging and neurodegenerative diseases are not always accompanied by overt inflammatory bursts. More commonly, they involve long-term low-grade inflammation and mitochondrial stress. In such settings, mtDNA stress is especially explanatory because it can convert progressively accumulated disruption of mitochondrial homeostasis into chronic DNA-sensing stimulation. cGAS–STING therefore functions not only as an acute danger-signaling pathway, but also as an important maintenance layer for chronic sterile inflammation.

(2) Pathological escalation after AIM2 involvement

If, in aging or neurodegenerative settings, an interferon-related environment persists and drives AIM2 upregulation, inflammasome programs become more likely to be secondarily amplified. At that point, the disease phenotype may progress from mitochondrial functional decline toward neuroinflammation, cellular injury, and degenerative tissue changes. Accordingly, the significance of AIM2 in this mechanistic axis is not merely that of another DNA receptor, but that of a key control layer determining whether chronic stress progresses into the injury-execution stage.

6. Related Research Products

Table 3. Key reagents in research on the cGAS–STING–AIM2 axis

Name	CAS No.	Experimental Stage	Key Use	Use Notes
Carnitine acetyltransferase from pigeon breast muscle	9029-90-7	Validation of upstream metabolic node	Used to construct in vitro CRAT-related acetyl-transfer reaction systems and validate the upstream effects of acetyl-CoA/acetylcarnitine conversion on mitochondrial metabolic homeostasis and the downstream immune axis	More suitable as a tool for CRAT enzymatic-function validation rather than as a downstream immune readout tool
L-Carnitine hydrochloride	6645-46-1	Upstream metabolic intervention	Used to establish carnitine-supply and CRAT-related acetyl-group buffering backgrounds and evaluate the effects of changes in the carnitine pool on mitochondrial homeostasis and DNA-sensing activation	More suitable for fundamental metabolic supplementation and control-group design, and not directly equivalent to restoration of CRAT function
Acetyl-L-carnitine	3040-38-8	Upstream metabolic intervention	Used to assess the effects of acetyl-group export and the acetylcarnitine buffering layer on mitochondrial stress and downstream innate immune signaling	Suitable as a compensatory intervention tool for acetyl-group buffering under CRAT-deficient conditions
O-Acetyl-L-carnitine hydrochloride	5080-50-2	Upstream metabolic intervention	Used to study the regulatory effects of acetylated carnitine forms on mitochondrial metabolic stress and the immune-activation chain	Suitable for parallel comparison with L-carnitine and acetyl-L-carnitine
Palmitoyl-L-carnitine	1935-18-8	Lipotoxicity/mitochondrial stress model	Used to establish a background of long-chain acylcarnitine accumulation and simulate increased metabolic load and enhanced mitochondrial input stress	More suitable for modeling the upstream scenario of "excessive metabolic input with limited mitochondrial processing capacity"
Palmitic acid	57-10-3	Lipid-overload model	Used to construct lipid-overload and mitochondrial-stress models as a preceding stimulus for mtDNA stress and innate immune activation	Commonly used together with carnitine/acetylcarnitine systems to analyze amplification of metabolic stress
C-176 (STING inhibitor)	314054-00-7	STING inhibition validation	Used to block STING and determine whether the interferon program and subsequent inflammatory amplification depend on the STING node	Suitable for causal blockade experiments
STING agonist-1	702662-50-8	Pharmacological STING activation	Used to strengthen STING activation and amplify type I interferon responses in order to validate the middle-stage signal-translation role of this axis	Suitable for cell-based functional validation
H-151	941987-60-6	STING inhibition validation	Used as a STING antagonist to determine whether the interferon program and subsequent inflammatory amplification depend on the STING node	Suitable for cross-validation with C-176
cGAMP disodium	2407516-83-8	Validation of STING-pathway activation	Used as a direct STING-activating tool to validate downstream responsiveness of the cGAS–STING axis	Suitable as a positive control for STING activation
NLRP3/AIM2-IN-2	791840-86-3	Validation of inflammasome branches	Used to distinguish the contributions of the STING-upstream transcriptional program and the AIM2/NLRP3 inflammasome execution layer	Suitable for the later part of the mechanistic axis to validate whether the inflammatory execution layer is truly elevated
NLRP3/AIM2-IN-3	1787787-60-3	Validation of inflammasome branches	Used to inhibit AIM2/NLRP3-related inflammasome activation and assess the contribution of the late-stage execution layer to the overall phenotype	More suitable as a tool for validation of downstream inflammatory amplification

Table 4. Functional tool products in research on the cGAS–STING–AIM2 axis

Catalog No.	Name	Grade and Purity	Experimental Stage	Research Direction / Intended Use
Ab129563	Recombinant STING Antibody	ExactAb™, Validated, recombinant, 0.4 mg/mL	STING protein readout	Suitable for detecting changes in STING expression and localization in WB, IF, IHC, and related experiments, and for validating whether the STING axis is activated after mtDNA stress
EJ1514083	Human Stimulator of interferon genes (STING) ELISA Kit	BioReagent	STING expression detection	Suitable for quantitative analysis of changes in STING protein levels and for comparing the extent of STING-pathway activation under different treatment conditions
M1492773	Mitochondrial Reactive Oxygen Species (ROS) Production Rate Assay Kit (Fluorometric Method)	BioReagent	Mitochondrial stress readout	Used to detect increased mitochondrial ROS under CRAT deficiency or metabolic-stress conditions, serving as an upstream indicator of mitochondrial pressure in mtDNA stress
F486463	Fluorometric Intracellular Ros Kit	Sufficient for 200 fluorescence assays (red)	Oxidative stress readout	Suitable for evaluating overall cellular oxidative-stress levels and for assisting in determining whether mitochondrial stress has expanded to whole-cell stress
M1505999	Mitochondrial Membrane Potential Assay Kit (Rhodamine 123)	BioReagent, for cell culture, sterile	Mitochondrial homeostasis readout	Used to monitor changes in mitochondrial membrane potential and evaluate mitochondrial functional destabilization under CRAT deficiency or metabolic-load conditions
M273063	Mitochondrial Membrane Potential Detection Kit (JC-1)	—	Mitochondrial homeostasis readout	Suitable for comparing the degree of mitochondrial depolarization among different treatment groups and for evaluating functional status before and after mtDNA stress formation
M1509036	Mitochondrial Membrane Potential Assay Kit (JC-10)	BioReagent	Mitochondrial homeostasis readout	Suitable for detection of membrane-potential changes in cell models and can serve as an alternative or parallel validation tool to the JC-1 system
A1515819	ATP Content Assay Kit (AHM, Micro Method)	BioReagent	Energy metabolism readout	Used to detect changes in cellular ATP levels and to assist in determining whether CRAT deficiency has already caused impaired mitochondrial energy output
E1501756	Enhanced ATP Assay Kit	BioReagent	Energy metabolism readout	Suitable for highly sensitive detection of changes in cellular ATP and for analyzing the relationship between mitochondrial stress and energy-metabolism disorder
D1372306	Magnetic Universal Genomic DNA Kit	BioReagent, for DNA and RNA applications	DNA extraction/qPCR	Suitable for extraction of cellular DNA combined with qPCR analysis of mtDNA abundance or cytosolic DNA-related readouts, and for validation of mtDNA stress
M665559	Blood/Cell/Tissue Genomic DNA Extraction Kit (Spin Column)	BioReagent	DNA extraction/qPCR	Suitable for routine DNA extraction from cells or tissue samples and for mtDNA quantification, copy-number analysis, and detection related to DNA leakage
A1468337	AIM2 Human Pre-designed siRNA Set A	—	AIM2 genetic intervention	Suitable for downregulation of AIM2 expression and for validating the necessity of AIM2 in downstream inflammatory amplification within the main mechanistic axis
P743707	pLenti-AIM2-sgRNA	—	AIM2 node validation	Suitable for protein-level controls or pathway validation under AIM2-deficient backgrounds
P743708	pLenti-AIM2-sgRNA	—	AIM2 node validation	Suitable for RNA-level controls and validation of downstream transcriptional readouts under AIM2-deficient backgrounds
EJ1515164	Human Absent In Melanoma 2 (AIM2) ELISA Kit	BioReagent	AIM2 expression detection	Suitable for quantitative analysis of changes in AIM2 protein levels and for comparing whether AIM2 expression increases before and after establishment of the interferon background

Overall, this mechanistic axis can be summarized as follows: CRAT deficiency first disrupts acetyl-group buffering and mitochondrial homeostasis, thereby inducing mtDNA stress; mislocalized mtDNA activates cGAS–STING and establishes a type I interferon background; AIM2 is then pushed into a highly sensitive state, and inflammasome output is further amplified. In this way, CRAT deficiency, mtDNA stress, cGAS–STING, and AIM2 together constitute a continuous metabolism-mitochondria-innate immunity mechanistic axis.