New Insights into Neuroinflammation: From "Activation" to "Cellular State Remodeling"
Historically, the brain was considered an "immune privileged" organ isolated from peripheral
immune surveillance. However, advances in
neuroimmunology have fundamentally reshaped this perspective, revealing extensive interactions
between the CNS and peripheral immunity.
Immune cells are now recognized as critical regulators of brain development, homeostasis, and
tissue repair. In addition, the meninges,
choroid plexus, perivascular spaces and lymphatic drainage system collectively establish a
highly connected immune microenvironment within
the brain[2].
Under pathological conditions, these regulatory barriers undergo dynamic remodeling. In
neurodegenerative disorders such
as
Alzheimer's disease (AD),
Parkinson's disease (PD),
Huntington's disease (HD), and
amyotrophic lateral sclerosis (ALS), bidirectional communication between neural and immune
systems becomes increasingly dysregulated
[3]. Blood-brain barrier
(BBB) disruption facilitates infiltration of peripheral immune cells, thereby
exacerbating inflammatory signaling and accelerating neurodegeneration. Peripheral immune
populations, including CD8
+ and CD4
+ T cells, together
with inflammatory mediators such as APOE4, B2M and TREM2, are strongly associated with disease
progression and BBB dysfunction
[3].
This evolving view of "immune ecology" suggests that neuroinflammation is a highly coordinated,
multicellular, and temporally regulated process involving interactions among innate immunity,
adaptive immunity, glial cells, and neurons.
The Refinement of Microglial Cell States
Microglia, the resident immune cells of the brain, have emerged as central regulators of
neurodegenerative disease progression and promising therapeutic targets. These cells originate
from erythromyeloid progenitors that migrate from the yolk sac into the developing CNS,
alongside macrophage populations located CNS border interfaces.
Microglia exhibit both protective and pathogenic functions within the brain. Under physiological
conditions, they remove neuronal debris and
pathological protein aggregates while maintaining tissue homeostasis. They also regulate
neuroinflammatory responses through secretion of
cytokines (e.g.,
IL-1β and
TNF)
and immune mediators such as
IL-4 and
arginase-1 (Arg1). Traditionally, microglial activation was
classified into two polarized states: pro-inflammatory "M1" phenotype and anti-inflammatory
"M2" phenotype
[4].
Recent advances in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics have
substantially expanded our understanding of microglial
heterogeneity, shifting the field from the classical M1/M2 framework toward a dynamic cell-state
lineage model. In 2017, Keren-Shaul et al. first
identified disease-related microglia (DAM) in AD mouse models using single-cell sequencing. This
microglial state was subsequently validated
in ALS, PD,
multiple sclerosis (MS), and aging models, indicating
conserved inflammatory
programs across neurodegenerative diseases
[4].
DAM activation occurs through a two-step transition process. Initially, homeostatic genes are
downregulated while DAM-related genes,
including Apoe, become progressively induced. Full DAM activation subsequently requires
Trem2-dependent signaling, enabling enhanced phagocytic
and inflammatory activity. Furthermore, DAM populations can be subdivided into pro-inflammatory
and anti-inflammatory subtypes according to
their transcriptional profiles[4].
Figure 1. A two-step transition for Disease-associated microglia (DAM) activation[4].
Aging and neurodegeneration further drive the emergence of additional microglial states,
including activated response microglia (ARM),
microglial neurodegenerative phenotypes (MGnD), lipid droplet-accumulating microglia (LDAM),
dark microglia (DM) and interferon-responsive
microglia (IRM). Among these, IRM populations are strongly associated with aging-related
inflammatory signaling and
neurodegenerative pathology[5].
Communication Between Astrocytes and Microglia
Astrocytes and microglia are the two major non-neuronal cell populations within the CNS and
together form a highly interconnected inflammatory network.
Astrocytes maintain synaptic homeostasis, regulate neurotransmitter recycling, support ion
balance, and contribute to
synapse formation and elimination[6].
Similar to microglia, astrocytes can transition between protective and pathogenic reactive
states. Pro-inflammatory A1 astrocytes release
cytokines such as IL-1β, TNF-α, and complement proteins that contribute to neuronal injury,
whereas anti-inflammatory A2 astrocytes support
neuronal survival and tissue repair through secretion of neurotrophic factors. Astrocyte
dysfunction is widely observed across
neurodegenerative diseases and frequently amplifies neuroinflammatory signaling[7].
Microglia do not function in isolation; their bidirectional communication with astrocytes
represents a core component of the neuroinflammatory
network. Microglia directly communicate with astrocytes through the release of Wnts, promoting
astrocytes process redistribution and
facilitating microglia-mediated synapse elimination. In addition, CX3CL1-CX3CR1 signaling plays
an important role in the microglial
regulation of synapses[6].
Figure 2. Bidirectional molecular crosstalk between microglia and astrocytes under
neuroinflammatory conditions[7].
Microglia and astrocytes are essential for maintaining CNS homeostasis and responding to
injuries and diseases. Under pathogenic stimulation,
activated microglia release pro-inflammatory mediators, including IL-1α, TNF-α, and complement
component C1q, which induce astrocytes to
adopt the reactive A1 state. A1 astrocytes further secrete inflammatory cytokines, sustaining
microglial activation and forming a harmful
positive feedback loop that amplifies neuroinflammation and accelerates neurodegeneration. In
parallel, enhanced synaptic pruning and
complement-mediated microglial phagocytosis contribute to synaptic and neuronal loss. Persistent
inflammation can also disrupt the BBB,
allowing peripheral immune cells to infiltrate the CNS And further exacerbate inflammation
damage[8][9].
Neuroinflammation is therefore no longer viewed simply as the presence or absence of
inflammation, but rather a complex regulatory network
involving specific cell types, activation states, temporal stages, and intercellular
communication pathways. This conceptual shift highlights
the importance of single-cell and spatiotemporal approaches in neuroinflammation research.
The NLRP3-STING axis: A hub for Neuroinflammatory Signaling
NLRP3 inflammasome: Amplifier of Inflammation
The
NLRP3 inflammasome is a crucial multiprotein complex involved in innate immunity, and
its dysregulation is closely associated with
neuroinflammatory diseases such as MS. Post-translational modifications (PTMs) including
ubiquitination, phosphorylation, and acetylation
finely regulate inflammasome activation. Dysregulation of these processes drives
inflammatory cytokine release, demyelination, and
neurodegeneration
[10].
Upon activation, NLRP3 recruits the connexin apoptosis-associated spot-like protein (ASC)
and the effector protein pro-caspase-1 to form the
inflammatory complex. Activated NLRP3 inflammasome then mediates the self-cleavage of
pro-caspase-1, producing enzymatically
active caspase-1[11]. Since its discovery in 2002, multiple
activation mechanisms for NLRP3 have been identified,
including canonical, non-canonical, and alternative pathways.
Figure 3. Molecular mechanisms underlying priming and activation of the NLRP3
inflammasome[10].
Classical NLRP3 activation consists of two phases: priming and activation. During priming,
toll-like receptors (TLRs) and inflammatory
cytokine receptors recognize PAMPs or cytokines such as TNF, leading to increased expression
of NLRP3, pro-IL-1β and pro-IL-18.
This process is further regulated by PTMs, which license inflammasome components for
subsequent activation[10].
Activation signals promote NLRP3 inflammasome assembly through pathways including ion flux,
mitochondrial dysfunction, lysosomal damage,
and immune metabolic reprogramming. PTMs such as ubiquitination, phosphorylation,
acetylation, and SUMOylation finely regulate NLRP3
activation by modulating its stability, complex interaction, and subcellular
localization[10].
Mechanistically, the balance between ubiquitination and deubiquitination controls NLRP3
degradation and inflammasome assembly, while
phosphorylation and acetylation regulate its ATPase activity and interactions with ASC and
NEK7, thereby determining inflammasome
activation efficiency. Together, these PTMs constitute a central regulatory network
governing NLRP3 inflammasome activation[10].
cGAS-STING pathway: DNA Sensing and Inflammation Initiation
The cGAS-STING signaling pathway is a core component of innate immune sensing that detects
abnormal cytoplasmic double-stranded DNA (dsDNA).
This pathway is composed of the cyclic GMP-AMP synthase (cGAS), the interferon
gene-stimulating protein (STING), and its downstream
signaling adaptor molecules.
Under physiological conditions, DNA is confined to the nucleus and mitochondria, while cGAS
remains inactive. Upon pathogen invasion or
cellular stress, cytosolic dsDNA binds to cGAS in a sequence-independent manner, triggering
the synthesis of cyclic guanosine
monophosphate-adenosine monophosphate (cGAMP). cGAMP subsequently activates STING, promoting
its translocation from the
endoplasmic reticulum to the Golgi apparatus and recruitment of
TBK1 and IKK. This leads to
activation of IRF3 and
NF-κB signaling,
inducing the production of type I interferons and pro-inflammatory cytokines, thereby
initiating innate immune responses
[11].
Figure 4. Overview of the cGAS-STING signaling pathway[11].
The cGAS–STING pathway is a major driver of age-related inflammation neurodegeneration. In
ageing brains, particularly in microglia, increased cytosolic
mitochondrial DNA (mtDNA) activates cGAS-STING signaling, leading to a type I interferon
(IFN-I) signature closely associated with ageing and
neurodegenerative diseases. Beyond promoting neuroinflammation, cGAS–STING activation also
regulates non-immune pathways shared across ageing
and multiple neurodegenerative disorders[12].
The STING-NLRP3 Axis: A positive Feedback Inflammatory Network
Recent studies indicate that cGAS-STING and NLRP3 signaling are not independent pathways,
but rather form a tightly coupled inflammatory network.
Increasing evidence suggests that NLRP3 functions downstream of the cGAS-STING signaling
pathway[13]. Mechanistically, the release of mtDNA activates
cGAS–STING signaling, which promotes NF-κB nuclear translocation and transcriptional
upregulation of NLRP3. Mun Y et al. demonstrated that rotenone
treatment activated the cGAS–STING axis in PMA-differentiated THP-1 macrophages, as
indicated by increased cGAS expression, phosphorylation of STING
and TBK1, and enhanced NF-κB nuclear translocation[14]. This activation subsequently
promoted NLRP3 inflammasome priming and IL-1β secretion.
Pharmacological inhibition of STING with H-151 significantly suppressed NLRP3 expression,
NF-κB activation, and IL-1β release. Likewise,
cyclosporin A attenuated mitochondrial reactive oxygen species (ROS) production, cytosolic
oxidized mtDNA accumulation, and downstream
cGAS–STING activation, thereby suppressing inflammasome activation. Collectively, these
findings indicate that rotenone-induced NLRP3
inflammasome activation is mediated through mitochondrial ROS-dependent mtDNA release and
subsequent activation of the cGAS–STING–NF-κB signaling axis.
Figure 5. Rotenone activates the NLRP3 inflammasome via mPTP opening and mtDNA-mediated
cGAS–STING signaling[14].
Mitochondria: A Central Hub for Inflammatory Signaling
Mitochondrial damage occupies a central integrating role in the STING-NLRP3 axis, extending
far beyond the traditional concept of mitochondria
as cellular "energy factories". In addition to their essential functions in cellular
metabolism, mitochondria are critical regulators of
innate immune responses triggered by pathogenic infection or cellular stress. Under harmful
stimuli, mitochondrial stress promotes the
release of mtDNA into the cytoplasm. Cytosolic mtDNA acts as a damage-associated molecular
pattern (DAMP), activating DNA-sensing
pathways and such as cGAS-STING signaling and subsequently inducing the production of IFN-I
and inflammatory factors such as TNF-α
and
IL-6. In parallel, mtDNA and mitochondrial ROS serve as
major DAMPs that promote inflammasome activation and intersect with
multiple pathways of regulated cell death (RCD)
[15].
The Influence of the NLRP3-STING Axis in Neurodegenerative Diseases
The NLRP3-STING pathway plays a crucial role in the progression of neurodegenerative
diseases, although the mechanisms underlying its activation may differ across disease
contexts. Accordingly, targeting the NLRP3-STING signaling axis has emerged as a promising
therapeutic strategy for neurodegenerative diseases.
Alzheimer's Disease
Aberrant activation of the cGAS-STING pathway plays a critical role in AD pathogenesis,
which is characterized by progressive cognitive
decline, amyloid-β (Aβ) deposition and tau pathology. Under physiological conditions,
microglia clear Aβ and pathological tau; however,
chronic activation of inflammatory pathways, particularly the NLRP3 inflammasome, impairs
microglial phagocytosis and promotes sustained
neuroinflammation. Inhibition of cGAS signaling alleviates cognitive deficits, reduces Aβ
accumulation, and suppresses
interferon-stimulated gene expression, highlighting the importance of the cGAS-STING axis in
neuroimmune regulation. In addition,
tau aggregates can activate innate immune responses through cGAS-STING-related mechanisms,
and AD mouse models exhibit enhanced expression
of cGAS-STING pathway components alongside neurodegenerative microglial signatures[16].
Parkinson's Disease
PD is characterized by Lewy pathology, including Lewy bodies and neurites, accompanied by
progressive dopaminergic neuronal loss in the substantia
nigra and other vulnerable brain regions. Persistent neuroinflammation, marked by microglial
activation and elevated pro-inflammatory
cytokines, is a key contributor to dopaminergic neurodegeneration. Emerging evidence
indicates that defects in
PINK1/Parkin-dependent
mitophagy result in the accumulation of damaged mitochondria, which may contribute to PD
pathogenesis. Studies in Parkin-deficient mice
further demonstrated that impaired mitophagy leads to mtDNA leakag and subsequent activation
of the cGAS-STING pathway, resulting in
increased production of pro-inflammatory cytokines and IFN-I. Under physiological
conditions, the PINK1-Parkin pathway maintains
mitochondrial quality control by eliminating damaged mitochondria, thereby preventing
excessive cGAS-STING activation and neuroinflammatory
responses
[16].
Multiple Sclerosis
MS is a progressive neurodegenerative disorder of CNS characterized by immune-mediated
damage to oligodendrocytes and their myelin sheaths, leading to
chronic neuroinflammation and axonal degeneration. inflammatory responsesand subsequent
neuronal fiber deterioration. Emerging evidence suggests that
modulation of the cGAS-STING axis and subsequent IFN-I production may represent a potential
therapeutic strategy for MS. For example, the serine protease
inhibitor Bowman-Birk inhibitor has been shown to alleviate autoimmune neuroinflammation
through STING-dependent induction of IFN-β. In addition,
IFN-γ induces neuronal STING expression, which is retained in the endoplasmic reticulum (ER)
through interaction with STIM1[16].
Amyotrophic Lateral Sclerosis
ALS is a progressive neurodegenerative disorder characterized by selective degeneration of
upper and lower motor neurons in the brain
and spinal cord. A major pathological hallmark of ALS is the cytoplasmic accumulation of
TDP-43, which is associated with enhanced
neuroinflammatory signaling, including activation of NF-κB and IFN-I pathways. Studies have
demonstrated that mitochondrial accumulation
of TDP-43 promotes mtDNA leakage, leading to activation of the cGAS-STING signaling pathway
and subsequent pro-inflammatory responses.
Consistently, increased cGAMP levels have been detected in spinal cord tissues from ALS
patients, further supporting the involvement of
cGAS–STING signaling in ALS pathogenesis[16].
Figure 6. The interaction mechanism between STING and various NDs[16].
From Mechanistic Insights to Therapeutic Opportunities
Multidimensional Detection of Inflammasome Activation
Accurate evaluation of the NLRP3-STING activation requires integration of multiple
biomarkers and technical platforms.
Detection of Inflammasome Activation
Detection of inflammasome activation is primarily based on assessing inflammasome complex
assembly, Caspase-1 activation,
downstream cytokine release and pyroptotic cell death[17].
1. ASC Speck Formation (Microscopy & Flow Cytometry)
Upon inflammasome activation, the adaptor protein ASC aggregates from a diffuse cytoplasmic
distribution into large micrometer-sized structures known as ASC specks, which serve as a
hallmark of inflammasome assembly.
2. Caspase-1 Activation Assays (Western Blotting)
Caspase-1 is the major effector protease of the inflammasome and detection of its cleavage
or activation is widely used as a direct indicator of inflammasome activity.
3. Cytokine Release Measurements (ELISA)
Activated Caspase-1 cleaves pro-IL-1β and pro-IL-18 into their mature secreted forms;
therefore, measurement of IL-1β and IL-18 release is commonly used to evaluate inflammasome
activation.
4. Pyroptosis Detection (LDH Release Assay)
Inflammasome activation often induces pyroptosis, a highly inflammatory form of programmed
cell death characterized by membrane rupture and lactate dehydrogenase (LDH) release.
5. Gene Expression (qPCR)
Many inflammasome components, such as NLRP3, require a priming step that upregulates their
transcription prior to activation, making qPCR a useful method for assessing inflammasome
priming status.
Integration of Spatial and Single-Cell Technologies
Spatial transcriptomics and single-cell multi-omics have revealed substantial cellular and
spatial heterogeneity in neuroinflammation.
Plaque-associated microglia exhibit graded DAM activation surrounding Aβ deposits, whereas
astrocytes display region-specific reactive
states. In addition, integrated multi-omic approaches such as CITE-seq and ASAP-seq enable
simultaneous transcriptomic and proteomic
profiling, facilitating precise characterization of microglial subpopulations, including DAM
and interferon-responsive states defined by Interferon-stimulated gene (ISG) signatures.
Therapeutic Strategies Targeting the NLRP3-STING Axis
NLRP3 Inhibitors
Targeting NLRP3 inflammasome activation has emerged as a promising therapeutic strategy for
neurodegenerative diseases. Several NLRP3
inhibitors, including
JC124,
MCC950,
OLT1177, and
ZYIL1, have demonstrated
anti-neuroinflammatory and neuroprotective effects in
preclinical studies
[18].
OLT1177, an orally available β-sulfonyl nitrile compound, reduces microglial activation,,
inflammatory cytokine release, and cortical plaque burden, while exhibiting favorable safety
and tolerability profiles in clinical studies. JC124, a sulfonamide analogue derived from
the glyburide, attenuates neuroinflammation, microglial activation, astrogliosis, and Aβ
plaque accumulation, while also improving synaptic plasticity, hippocampal neurogenesis, and
cognitive function.
MCC950 is one of the most extensively studied NLRP3 inhibitors and suppresses
NLRP3-dependent neuroinflammation through modulation of autophagy and inhibition of NF-κB
signaling. In models of AD and PD, MCC950 reduces Aβ plaque formation, attenuates
pathological α-synuclein (pathαSYN)-associated neuroinflammation, prevents dopaminergic
neuronal loss, and reduces CD4+ and CD8+ T cell infiltration.
Similarly, the orally administered inhibitor ZYIL1 (
Usnoflast)
suppresses LPS/ATP-induced IL-1β and
IL-18 production and has
demonstrated favorable tolerability in early clinical studies.
STING Modulator
Increasing evidence suggests that modulation of the cGAS-STING pathway represents a
promising therapeutic strategy for neurodegenerative diseases.
Several compounds, including
H-151,
RU.521, and
C-176, have shown beneficial effects
in AD models.
For example, H-151 inhibits STING palmitoylation, whereas RU.521 targets the catalytic site
of cGAS; both agents significantly reduce
Aβ pathology in 5xFAD mice. C-176, an irreversible STING inhibitor that covalently binds the
Cys91 residue of STING, markedly suppresses
Aβ25-35-induced neuroinflammation in microglia, with enhanced
effects observed when combined with RU.521. In addition, several non-specific
modulators of the cGAS-STING pathway, including
nicotinamide
riboside (NR),
NAD+,
melatonin, and
dapsone, have also
demonstrated
therapeutic potential in ameliorating AD-related pathology
[19].
Figure 7. Overview of the inhibitors targeting the cGAS-STING signal pathway[19].
The Key Challenges in Clinical Translation
Despite the therapeutic potential of targeting the NLRP3-STING axis, several challenges
remain for clinical translation.
Target Complexity and Insufficient Mechanistic Understanding
The molecular interplay between NLRP3 and STING in neuroinflammation remains incompletely
understood, and their activation patterns may differ across neurological disorders. In
diseases such as AD, PD, and ischemic stroke, activation of the NLRP3-STING axis may involve
distinct cell types (e.g., microglia, neurons) and divergent signaling pathways,
underscoring the need for a more precise understanding of its disease-specific roles.
Drug Delivery and Blood-Brain Barrier (BBB) Penetration
Effective neuroinflammatory therapy requires sufficient drug penetration across the BBB.
Although many NLRP3 and STING inhibitors show potent in vitro activity, limited brain
permeability often prevents effective therapeutic concentrations from being achieved in
vivo, thereby restricting clinical efficacy. Consequently, the development of
brain-penetrant compounds and advanced delivery systems remains a major challenge.
Safety and Immune Homeostasis Concerns
NLRP3 and STING are essential components of innate immunity. Long-term inhibition may impair
host defense and immune surveillance. In the CNS, excessive suppression of inflammatory
signaling may also disrupt microglial homeostatic and potentially exacerbate
neurodegeneration. Therefore, achieving a balance between anti-inflammatory efficacy and
preservation of immune function is critical.
Patient Heterogeneity and Stratification Difficulties
Neurodegenerative and neuroinflammatory disorders exhibit substantial heterogeneity in
genetic background, disease stage, and inflammatory status. The lack of robust patient
stratification strategies hinders the identification of subpopulations most likely benefit
from NLRP3-STING-targeted therapies, thereby limiting the efficiency and reliability of
clinical trials.
Summary
Research on neuroinflammation is shifting from the concept of a "single inflammatory response"
toward that of a "complex immune network". The NLRP3 inflammasome and the cGAS–STING pathway
constitute a tightly interconnected inflammatory axis centered on mitochondrial dysfunction,
whose positive feedback loop actively contributes to neuronal dysfunction and neurodegeneration.
This evolving understanding highlights the importance of integrating single-cell technologies,
spatiotemporal dynamic analysis, and functional validation to elucidate the heterogeneity and
dynamics of neuroinflammatory networks. Precision therapies targeting the NLRP3–STING axis in a
disease stage- and cell state-specific manner may represent a promising direction for
next-generation neurodegenerative diseases treatment.
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References
[1]
Lawrence GMEP, et al. Immunity. 2022 Aug 9;55(8):1331-1333.
[2]
Castellani G, et al. Science. 2023 Apr 7;380(6640):eabo7649.
[3]
Zhang S, et al. Mol Neurodegener. 2025 Feb 21;20(1):22.
[4]
Cheng YH, et al. PLoS Biol. 2025 Oct 21;23(10):e3003426.
[5]
Wei Y, et al. Immun Ageing. 2022 Oct 8;19(1):44.
[6]
Faust TE, et al. Cell. 2025;188(19):5212-5230.e21.
[7]
Bhusal A, et al. Curr Neuropharmacol. 2023;21(10):2020-2029.
[8]
Gao C., et al. Sig Transduct Target Ther 8, 359 (2023).
[9]
Sweeney M., et al. Nat Rev Neurol 14, 133-150 (2018).
[10]
Shin H.J., et al. Exp Mol Med 58, 650-663 (2026).
[11]
Zhang Z, et al. Nat Rev Immunol 25, 425-444 (2025).
[12]
Gulen M.F., et al. Nature 620, 374-380 (2023).
[13]
Chen KQ, et al. Front Immunol. 2025 May 20;16:1594133.
[14]
Mun Y, et al. Antioxidants (Basel). 2025 Oct 24;14(11):1276.
[15]
Marchi S., et al. Nat Rev Immunol 23, 159-173 (2023).
[16]
Zhang H, et al. Front. Aging Neurosci. 17:1659216.
[17]
Tweedell RE, et al. Nat Protoc. 2020 Oct;15(10):3284-3333.
[18]
Yin L, et al. Drug Discov Today. 2025 Jun;30(6):104375.
[19]
Quan S, et al. Mol Neurodegener. 2025 Mar 4;20(1):25.
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New Insights into Neuroinflammation: From "Activation"
to "Cellular State Remodeling"
The NLRP3-STING axis: A hub for Neuroinflammatory
Signaling
From Mechanistic Insights to Therapeutic Opportunities
