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ISGylation of STING contributes to neuroinflammation and neurodegeneration via activating c-Fos in Parkinson's disease models

This study demonstrates that ISGylation of STING drives neuroinflammation and dopaminergic neuronal death in Parkinson's disease models by activating c-Fos through a positive feedback loop, suggesting that targeting this ISG15-STING axis offers a novel therapeutic strategy.

Original authors: Xuan Liu, Shuzhen Xu, Pinyuan Huang, Xueer Jiang, Aiwen Dong, Tingjue Wu, Tianwen Huang, Qinyong Ye, En Huang

Published 2026-06-25

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CC BY 4.0

Original authors: Xuan Liu, Shuzhen Xu, Pinyuan Huang, Xueer Jiang, Aiwen Dong, Tingjue Wu, Tianwen Huang, Qinyong Ye, En Huang

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Big Picture: A "False Alarm" That Won't Turn Off

Imagine your brain is a bustling city. Normally, the city's security system (the immune system) stays quiet until a real intruder (like a virus) shows up. When it does, the security guards sound an alarm, fight the intruder, and then turn the alarm off once the danger is gone.

In Parkinson's disease, however, this alarm system gets stuck in the "ON" position. It keeps screaming "DANGER!" even when there is no intruder. This constant noise causes chaos, damaging the city's most important workers: the dopamine-producing neurons.

This study discovered a specific mechanism that keeps this alarm stuck on. It involves a protein called STING (the alarm siren) and a molecular "sticky note" called ISG15.

The Main Characters

STING (The Alarm Siren): A protein that usually detects threats and starts an immune response.
ISG15 (The Sticky Note): A small protein that acts like a tag. When it attaches to another protein, it's called "ISGylation."
c-Fos (The Panic Button): A gene that gets turned on when the cell is stressed. It tells the cell to start dying or causing inflammation.
The Dopamine Neurons: The hardworking cells in the brain that control movement. In Parkinson's, these are the ones getting destroyed.

What the Scientists Found

The researchers used two types of "simulated" Parkinson's disease models:

In a Petri Dish: They treated brain cells with a toxin (MPP+) or infected them with a virus carrying a Parkinson's-related protein (Alpha-synuclein A53T).
In Mice: They injected mice with toxins or viruses to create Parkinson's symptoms.

Here is the chain reaction they discovered:

1. The Sticky Note Makes the Siren Louder

In healthy cells, STING is quiet. But in Parkinson's models, the "sticky note" (ISG15) attaches itself to STING at a specific spot (called K289).

The Analogy: Imagine STING is a microphone. Normally, it has a volume knob set to "Low." The sticky note (ISGylation) acts like a duct tape that jams the knob all the way to "Max Volume."
The Result: The study showed that when they removed the sticky note (by using a mutation called K289A or blocking ISG15), the microphone volume went back down. The alarm wasn't screaming as loud.

2. The Panic Button Gets Pressed

When the STING alarm is stuck on "Max Volume," it triggers a gene called c-Fos.

The Analogy: c-Fos is like a panic button in the control room. When the alarm is too loud, it slams the panic button, which tells the cell, "We are under attack! Shut down and cause a riot!"
The Result: The study found that when STING was modified by the sticky note, c-Fos levels skyrocketed. This led to the death of the neurons and a massive inflammatory response (neuroinflammation).

3. The Vicious Cycle

The study found a "feedback loop."

The Analogy: It's like a microphone picking up its own speaker sound. The loud STING alarm tells the cell to make more sticky notes (ISG15). These new sticky notes attach to more STING, making the alarm even louder, which creates even more sticky notes.
The Result: This loop keeps the inflammation going and destroys more brain cells, making the disease worse.

The "Fix" in the Experiment

The researchers tried to break this cycle in three ways, and it worked:

The Mutation (K289A): They changed the STING protein so the sticky note couldn't attach to it.
The Blocker (shISG15): They reduced the amount of sticky notes available in the cell.
The Silencer (C-176): They used a drug to stop the STING alarm from working in the first place.

The Outcome: In both the petri dishes and the mice, all three methods did the same thing:

They stopped the "sticky note" from attaching.
They lowered the volume of the alarm.
They stopped the panic button (c-Fos) from being pressed.
Crucially: The mice moved better, their brains had less inflammation, and more of their dopamine neurons survived.

The Conclusion

The paper concludes that in Parkinson's disease, the "sticky note" (ISGylation) attaches to the alarm siren (STING), making it hyper-active. This hyper-activity triggers a panic gene (c-Fos) that kills brain cells and causes inflammation.

By stopping the sticky note from attaching to the alarm, the researchers were able to calm the system down, save the brain cells, and improve movement in the mice. They suggest that targeting this specific "sticky note" process could be a new way to treat Parkinson's, though this study only proved it works in models, not yet in humans.

Technical Summary: ISGylation of STING Contributes to Neuroinflammation and Neurodegeneration via Activating c-Fos in Parkinson's Disease Models

Problem Statement
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons (DN) in the substantia nigra pars compacta (SNc) and the formation of Lewy bodies. While the aggregation of misfolded $\alpha$ -synuclein and mitochondrial dysfunction are well-established factors, the role of chronic neuroinflammation and the innate immune response remains a critical area of investigation. Specifically, Type-I interferons (IFNs) and their downstream interferon-stimulated genes (ISGs) have been implicated in neurodegeneration, yet the precise mechanisms by which ISG15-mediated protein modification (ISGylation) influences PD pathogenesis are not fully understood. This study addresses the gap in knowledge regarding whether the IFN-ISG15 pathway contributes to PD by ISGylating the DNA sensor STING (Stimulator of Interferon Genes), thereby driving neuroinflammation and neuronal death.

Methodology
The study employed a comprehensive approach combining in vitro and in vivo models to investigate the STING-ISG15-c-Fos axis:

In Vitro Models: Human neuroblastoma SH-SY5Y cells were treated with MPP+ (a neurotoxin) or infected with lentiviral vectors expressing human $\alpha$ -synuclein A53T (PLV-SynA53T) to mimic PD pathology.
- Genetic Manipulation: Cells were infected with lentiviruses encoding wild-type STING (STING-WT), a non-ISGylation mutant (STING-K289A), or shRNA targeting ISG15 (shISG15).
- Pharmacological Intervention: The STING inhibitor C-176 was used to block STING activity.
- Assays: Western blotting, Co-Immunoprecipitation (Co-IP), and RNA-sequencing (transcriptomics) were used to analyze protein interactions, ISGylation status, and gene expression. Cell viability was assessed via CCK-8 assays.
In Vivo Models: Two distinct PD mouse models were utilized:
1. MPTP Model: C57BL/6J mice received intraperitoneal MPTP injections.
2. AAV Model: Mice received bilateral stereotaxic injections of AAV2/1-SynA53T into the SNc.
- Interventions: Mice were pre-treated with lentiviral vectors (STING-WT, STING-K289A, shISG15) or the STING inhibitor C-176 prior to or during PD induction.
- Behavioral Testing: Open field tests, Rotarod tests, and Pole tests were conducted to evaluate motor function and coordination.
- Histology and Molecular Analysis: Immunohistochemistry (IHC) and immunofluorescence (IF) were performed on SNc sections to quantify dopaminergic neurons (TH-positive), glial activation (GFAP, Iba1), and protein co-localization (STING, c-Fos). Western blotting was used to quantify ISG15, ISG-STING, c-Fos, NF- $\kappa$ B, and TNF- $\alpha$ levels.

Key Contributions and Results

Induction of ISG15 and ISG-STING in PD Models:
The study demonstrated that both ISG15 expression and the ISGylation of STING (ISG-STING) were significantly upregulated in in vitro (MPP+ and SynA53T) and in vivo (MPTP and AAV-SynA53T) PD models. Co-IP confirmed that ISG15 covalently links to STING, forming a complex with a molecular weight of approximately 52 kDa (sum of ISG15 ~15 kDa and STING ~37 kDa).
Identification of K289 as the ISGylation Site:
Transcriptomic analysis of STING-WT overexpression revealed the upregulation of immediate early genes (IEGs), most notably c-Fos. The study identified lysine 289 (K289) as the critical site for ISGylation. The STING-K289A mutant, which cannot be ISGylated, failed to induce high levels of c-Fos, NF- $\kappa$ B, or TNF- $\alpha$ , and did not promote cell death to the same extent as STING-WT.
The STING-c-Fos Axis Drives Neuroinflammation and Cell Death:
- Mechanism: ISGylation of STING acts as a "gain-of-function" modification, augmenting STING activity. This leads to the upregulation of c-Fos, a component of the AP-1 transcription factor complex.
- Downstream Effects: The activation of the STING-c-Fos axis resulted in increased expression of inflammatory mediators (NF- $\kappa$ B, TNF- $\alpha$ ) and subsequent dopaminergic neuronal death.
- Positive Feedback Loop: The study proposes a positive feedback mechanism where STING activation induces downstream IFN and ISG15, which in turn further ISGylates STING, potentiating the neurodegenerative process.
Therapeutic Interventions Reversed Pathology:
- Genetic Knockdown/Modification: In both MPTP and AAV-SynA53T mice, the expression of STING-K289A or knockdown of ISG15 (shISG15) significantly reduced ISG-STING levels, c-Fos expression, and neuroinflammation.
- Pharmacological Blockade: Treatment with the STING inhibitor C-176 yielded similar protective effects.
- Functional Recovery: These interventions restored dopaminergic neuron counts (TH-positive cells), reduced glial activation (GFAP and Iba1), and significantly improved motor behaviors (distance traveled, rotation retention time, and pole climbing time) compared to control groups.
Cellular Localization:
Immunofluorescence analysis revealed that in PD models, STING and c-Fos are predominantly induced and co-localized within neurons (NeuN-positive), rather than solely in glial cells, suggesting a cell-autonomous role of neuronal STING in driving PD pathology.

Significance and Claims
The paper claims to establish a novel mechanistic link between the innate immune ISG15 pathway and Parkinson's disease pathogenesis. Specifically, it posits that:

ISGylation of STING at residue K289 is a critical driver of neuroinflammation and dopaminergic neuronal loss in PD.
This modification enhances STING function (gain-of-function), leading to the activation of c-Fos and a subsequent positive feedback loop involving IFN and ISG15 that exacerbates neurodegeneration.
Targeting the ISG15-mediated ISGylation process or the interaction between ISG15 and STING represents a potential novel therapeutic strategy for PD.

The study concludes that disrupting the ISG-STING-c-Fos axis can ameliorate motor deficits and neuronal death in PD models, offering a new perspective on treating neurodegenerative diseases by modulating specific post-translational modifications of immune sensors.