Introduction

Alzheimer’s disease affects over 50 million people worldwide, with projections reaching 150 million by 2050. Current FDA-approved antiamyloid antibody treatments offer only modest effects in slowing disease progression during mild dementia stages, highlighting the need for therapeutic approaches that address the complex neuroinflammatorymechanisms underlying Alzheimer’s pathogenesis.

Microglia, the brain’s resident immune cells, play a central role in AD progression. These cells respond to neurodegeneration by transitioning from a homeostatic state to what we term “neurodegenerative microglia” (MGnD) or “disease-associated microglia” (DAM). This activation state presents a therapeutic paradox: while MGnD can enhance clearance of amyloid-beta plaques, these cells also produce inflammatory mediators that maycontribute to neuronal damage and disease progression. Recent genome-wide association studies have confirmedthat microglia-specific genes correlate strongly with late-onset AD risk, establishing these cells as primary therapeutic targets.

The challenge lies in modulating microglia to maximize their beneficial functions while minimizing harmfulinflammation. Recent research has identified an intermediate activation state termed “pre-MGnD” that may represent an optimal therapeutic target. This state, regulated by interferon-gamma (IFN-γ) signaling, appears to restrict amyloidpathology and reduce dystrophic neurites without triggering the full inflammatory cascade associated with classical MGnD activation.

Xenon gas presents a novel approach to achieving this microglial modulation. As an inhaled anesthetic agent with clinical use and the ability to cross the blood–brain barrier, xenon is well positioned to directly influence central nervous system immune cells. The present study investigates whether xenon inhalation can shift microglia toward the beneficialpre-MGnD state, offering a potential disease-modifying treatment that addresses amyloid pathology through immune system modulation rather than direct protein targeting.

Xenon Creates Protective Intermediate Microglial States

The Discovery: Four Distinct Pre-MGnD States

 To understand how xenon affects microglia, we used single-cell RNA sequencing to examine individual brain immune cells from Alzheimer’s model mice treated with weekly xenon inhalations. This detailed analysis revealed something surprising: microglia don’t simply exist in “on” or “off” states.Instead, there are multiple intermediate activation states called “pre-MGnD” that represent critical transition points in disease progression.

Pre-MGnD cells represent an intermediate activation state where microglia have begun responding to pathology but have not yet committed to the chronic inflammatory phenotype characteristic of advanced disease. This transitional period represents a critical intervention window where cellular fate can be directed toward either protective orpathogenic outcomes.

The four distinct pre-MGnD subsets were identified, each with different functions:

• Pre-MGnD-cytokine (e.g., Ccl2, Il1a, Nfkbia): Inflammatory subset expressing genes like IL-1, TNF, and chemokines
• Pre-MGnD-interferon (e.g., Isg15, Stat1, Ifit3): Interferon-responsive subset involved in regulated immune responses and antigen presentation
• Pre-MGnD-HSP: Stress response subset expressing heat shock proteins that help manage cellular stress
• Pre-MGnD-ribosome: (e.g., Rps/Rpl genes) protein synthesis subset supporting cellular functions

Xenon predominantly changed the mix of these intermediate states: the cytokine pre-MGnD population decreased, while interferon, HSP, and ribosome pre-MGnD populations increased. This represents selective immunomodulation rather than broad immune suppression. Xenon creates what we describe as a “regulated intermediate state” thatbalances protective functions with reduced inflammatory damage.

Molecular Mechanisms and Functional Outcomes

At the molecular level, xenon created a distinct gene expression signature in microglia. The treatment suppressedinflammatory pathways associated with neuronal damage, including:

• Proinflammatory cytokines (IL-1, TNF, IL-6)
• Chemokines that recruit additional immune cells
• NF-κB signaling, a master regulator of inflammation (Core NF-κB regulators such as Sgk1 and Nfkbiz were downregulated)

Simultaneously, xenon enhanced protective pathways:

• Interferon-gamma signaling components
• Heat shock proteins (cellular stress-response proteins)
• Ribosomal genes supporting protein synthesis
• Antigen presentation molecules including MHC-II (proteins that present antigens to other immune cells)

We validated increased STAT1 (a key interferon signaling transcription factor) and MHC-II protein expression in brain tissue. Notably, a subpopulation of microglia enriched for MHC-II antigen-presenting genes appeared specifically after xenon exposure.

This isn’t broad immune suppression. Xenon selectively reduces harmful inflammatory programs while preserving theinterferon responses and phagocytic machinery that microglia need to clear toxic proteins. The result is what we call a “regulated intermediate state.”

IFN-γ Signaling: The Mechanistic Switch That Translates to Human Microglia

We identified interferon-gamma (IFN-γ) signaling as the specific molecular pathway responsible for xenon’s effects. IFN-γ is a cytokine (a signaling molecule) typically associated with immune activation, and its signaling is required for most xenon-induced microglial gene changes. Blocking IFN-γ worsens amyloid pathology outcomes in the xenon context.

To prove this pathway was essential, we ran experiments with mice depleted of the IFN-γ receptor in microglia(Cx3cr1-CreERT2:Ifngr1-flox mice). They treated animals with xenon and examined what changed. The results were striking: approximately 70% of the gene

expression changes normally induced by xenon disappeared. Without functional IFN-γ receptors on microglia, xenon lost its ability to suppress proinflammatory regulators and dampen NF-κB and cytokine signaling pathways.

To confirm these findings in the context of actual pathology, we treated APP/PS1 Alzheimer’s model mice with IFN-γneutralizing antibodies before xenon exposure (weekly for 2 weeks). Blocking IFN-γ didn’t just eliminate geneexpression changes but actually reversed the therapeutic benefits. Microglia adopted a more proinflammatory signature with increased expression of inflammatory genes (Nfkbia, Il1r1, Cd14). More importantly, pathology worsened: amyloid plaque deposition increased, dystrophic neurites increased, and the numbers of beneficial Clec7a+ microglia decreased.

These experiments establish that IFN-γ signaling is mechanistically required for xenon’s effects, and disrupting IFN-γ signaling doesn’t just change gene expression, it eliminates therapeutic benefit and worsens disease outcomes.

Understanding the Paradox

IFN-γ is well-known as a proinflammatory cytokine, so its role in reducing inflammation may seem paradoxical. However, we hypothesize that IFN-γ signaling is highly context-dependent. Under certain conditions, IFN-γ can actually suppress TNF and IL-1 production through synergistic induction of the anti-inflammatory cytokine IL-10 andthrough blocking NF-κB activation. This dual nature allows IFN-γ to promote controlled immune activation (antigen presentation, phagocytosis) while simultaneously dampening the most damaging inflammatory cascades. Thiscontext-dependent activity aligns perfectly with the “regulated intermediate state” profile xenon creates in microglia.

Translation to Human Microglia

A critical question for any preclinical finding is whether the mechanism operates in human cells. We examined xenon’seffects in mice transplanted with human iPSC-derived microglia that exhibit gene expression profiles and functional properties similar to primary human microglia from brain tissue.

Xenon produced remarkably similar effects: reduced inflammatory gene expression and increased interferon-responsive genes. STAT1 protein (the key transcription factor downstream of IFN-γ receptor activation) was significantly elevated in xenon-treated human microglia, confirming the IFN-γ to STAT1 signaling axis operates in human cells.

The functional outcomes translated as well: human microglia in xenon-treated mice contributed to reduced amyloid burden and fewer dystrophic neurites, even when treatment began after pathology was established.

Conclusion

What this study ultimately shows is xenon’s ability to perform state-specific immune tuning. Xenon shifts microglia toward protective intermediate states that maintain their ability to clear pathological proteins while reducinginflammatory damage. This addresses a critical limitation of previous anti-inflammatory strategies that failed by suppressing both beneficial and harmful immune activities indiscriminately. By shifting microglia toward protective intermediate states rather than simply suppressing their activity, xenon offers proof of concept for a more sophisticated approach to neuroinflammation in Alzheimer’s disease.