Abstract

   Alzheimer's disease (AD) has traditionally been associated with
   amyloid-β plaques, but growing evidence underscores the role of
   neuroinflammation in disease progression. The autoinflammatory
   hypothesis of AD suggests chronic immune dysfunction contributes to
   neuronal damage, making immune modulation a promising therapeutic
   strategy. Cannabidiol (CBD), a phytocannabinoid with anti-inflammatory
   properties, may offer therapeutic potential. This study investigates
   how CBD independently influences two key neuroinflammatory regulators
   in AD: the indoleamine 2,3-dioxygenase (IDO) pathway and the cyclic
   GMP-AMP synthase (cGAS) pathway. Though mechanistically distinct, both
   shape CNS immune responses. Targeting these immune-metabolic axes
   provides a mechanistic alternative to amyloid- or tau-based approaches
   by addressing upstream drivers of neuroinflammation and immune
   dysregulation. Using the male 5XFAD transgenic AD mouse model, we
   administered CBD via inhalation and assessed IDO and cGAS expression
   using flow cytometry, immunofluorescence (IF), and gene expression
   analysis. We evaluated cytokine levels and used STRING-based
   bioinformatics to identify CBD-target interactions. CBD treatment
   significantly reduced IDO and cGAS expression, correlating with
   decreased proinflammatory cytokines, including TNF-α, IL-1β, and IFN-γ.
   Bioinformatics identified potential interactions between CBD and immune
   targets such as AKT1, TRPV1, and GPR55. These targets were prioritized
   based on their roles in neuroinflammatory signaling and high-confidence
   interactions with CBD. AKT1 regulates inflammatory signaling and cell
   survival, TRPV1 modulates nociception and neuroinflammation, and GPR55
   influences immune cell activation. These findings support CBD as a
   potential monotherapy or adjunctive treatment for AD by targeting
   distinct neuroinflammatory pathways, including IDO and cGAS. Further
   studies are warranted to fully explore its therapeutic potential.

   Keywords: Alzheimer's, cannabidiol, CBD, cGAS, IDO, neuroinflammation

Significance Statement

   This study highlights the therapeutic potential of cannabidiol (CBD) in
   targeting neuroinflammation, a major driver of Alzheimer's disease (AD)
   progression. By modulating the IDO and cGAS pathways—critical
   regulators of CNS immune responses—CBD reduces proinflammatory
   cytokines and ameliorates immune dysfunction. These findings support
   the emerging autoinflammatory hypothesis of AD, which posits that
   chronic inflammation underlies neuronal damage. The IDO/cGAS signaling
   axis, located at the intersection of innate immunity and metabolic
   regulation, remains underexplored in AD and represents a key
   intervention point to disrupt neuroinflammatory loops. This study
   positions CBD as a promising mono- or adjunctive therapy and reinforces
   the need to consider multitargeted strategies that address upstream
   immune mechanisms in neurodegenerative disease.

Introduction

   Alzheimer's disease (AD) remains a major challenge for healthcare, with
   no effective treatment to halt or slow its progression
   ([50]Yiannopoulou et al., 2019; [51]Yiannopoulou and Papageorgiou,
   2020; [52]van der Flier et al., 2023). Current therapies, such as
   amyloid-targeting treatments and cognitive enhancers, only offer
   symptomatic relief, failing to address the root causes of
   neurodegeneration ([53]Yiannopoulou and Papageorgiou, 2020; [54]Zhang
   et al., 2024). This highlights a critical gap in our understanding.

   The complexity of AD, driven by genetic, environmental, and immune
   factors, means no single theory can explain it fully. While amyloid-β
   (Aβ) plaques and tau tangles are central to the traditional AD model,
   they do not account for the disease's full scope ([55]Morris et al.,
   2014; [56]Rischel et al., 2023; [57]Weaver, 2023). For instance,
   individuals with amyloid plaques may not exhibit cognitive decline, and
   some with severe neurodegeneration lack significant amyloid or tau
   pathology ([58]Farrell et al., 2017; [59]Weigand et al., 2020). This
   suggests the amyloid-tau framework is incomplete and may miss key
   aspects of AD's true mechanisms.

   Immune system dysfunction is considered a critical and obligatory step
   in the progression of AD. While Aβ accumulation is a key hallmark of AD
   pathology, recent evidence suggests that immune dysregulation can
   independently drive neurodegeneration. Specifically, the activation of
   immune responses, including microglial and astrocytic activation, has
   been shown to exacerbate neuroinflammation and contribute to neuronal
   damage. In the 5xFAD mouse model, while Aβ deposition occurs early in
   the disease, the immune system's role in driving neurodegenerative
   processes extends beyond Aβ-related events. This aligns with the
   autoinflammatory theory of AD, which posits that immune dysfunction,
   rather than being solely a consequence of Aβ accumulation, is an early
   and central driver of disease progression ([60]Meier-Stephenson et al.,
   2022). Therefore, immune system dysfunction is considered a central
   feature of AD pathology that plays a significant role in disease
   progression, potentially independent of the Aβ-induced toxic effects.

   The autoinflammatory theory of AD (AD^2) suggests the emerging role of
   indoleamine 2,3-dioxygenase (IDO) in AD progression. IDO, an enzyme
   involved in tryptophan metabolism, plays a dichotomic role in
   neuroinflammation. It can either promote immune tolerance and
   anti-inflammatory responses or contribute to neurotoxic inflammation
   ([61]Kwidzinski and Bechmann, 2007; [62]Baban et al., 2011;
   [63]Salminen, 2022). In AD, elevated IDO activity may indicate a
   maladaptive immune response, amplifying neuroinflammation and
   accelerating neurodegeneration ([64]Yu et al., 2015; [65]Savonije et
   al., 2023). This suggests that IDO could be a central mediator of the
   immune dysregulation seen in AD, linking the immune system's response
   to the neuroinflammatory processes driving the disease.

   Cytokines and innate immune sensors independently contribute to the
   complex neuroinflammatory milieu of AD. Interferon-gamma (IFN-γ), a
   Type 2 interferon predominantly secreted by activated T-cells and
   natural killer cells, plays a pivotal role in amplifying inflammation
   by upregulating IDO in the kynurenine pathway that promotes neurotoxic
   tryptophan metabolites. IFN-γ–mediated induction of IDO fosters a
   chronically proinflammatory environment and drives microglial
   activation, which, in turn, enhances cytokine release and sustains
   neuroinflammation ([66]Smith et al., 2012; [67]Yu et al., 2015;
   [68]Ogunmokun et al., 2021; [69]Govindarajulu et al., 2023;
   [70]Savonije et al., 2023; [71]Chen et al., 2024). Independently, the
   cyclic GMP-AMP synthase (cGAS) pathway serves as a cytosolic DNA sensor
   that activates the STING signaling cascade, culminating in robust Type
   1 interferon responses and the expression of inflammatory mediators.
   This cGAS-STING axis has been increasingly implicated in the
   progression of AD through its role in sensing DNA damage and triggering
   maladaptive microglial activation, thereby exacerbating
   neuroinflammation and neuronal dysfunction ([72]Smith et al., 2012;
   [73]Govindarajulu et al., 2023; [74]Chen et al., 2024). While these
   pathways are mechanistically distinct, they intersect functionally
   within the broader context of neuroimmune signaling. For example,
   IFN-γ–driven IDO expression and cGAS-STING–mediated interferon
   production converge on overlapping downstream inflammatory cascades.
   This convergence may establish a synergistic feedback loop that
   intensifies immune activation, sustains glial reactivity, and
   accelerates disease progression. Understanding these parallel and
   intersecting pathways offers mechanistic insight into AD pathogenesis
   and highlights the therapeutic potential of modulating both
   cytokine-dependent and nucleic acid-sensing pathways. Building on this
   concept, recent insights into neurodegeneration increasingly support
   the idea that immune-metabolic dysregulation is not merely a downstream
   effect but a fundamental driver of AD pathogenesis. Specifically, the
   cGAS-STING pathway, a cytosolic sensor of DNA damage, and the
   IDO-kynurenine axis converge to regulate both innate immune activation
   and metabolic reprogramming in the brain. Targeting this dual-pathway
   interface introduces a paradigm shift beyond amyloid- and tau-focused
   models by offering a more integrated approach to modulating chronic
   neuroinflammation at its source. This perspective aligns with the AD^2
   theory and further strengthens the rationale for therapeutic strategies
   aimed at restoring neuroimmune homeostasis through immune-metabolic
   intervention.

   Thus, targeting IDO activity, along with cGAS modulation, could offer a
   novel approach for restoring immune balance in AD. By addressing both
   the immune dysregulation and neuroinflammatory components of the
   disease, therapies aimed at these pathways could provide a promising
   therapeutic strategy within the autoinflammatory framework of AD^2,
   potentially offering disease-modifying effects beyond amyloid- and
   tau-focused treatments.

   Several studies have demonstrated that cannabidiol (CBD) can alleviate
   AD symptoms and improve patient outcomes ([75]Baban et al., 2021;
   [76]Xiong and Lim, 2021; [77]Trojan et al., 2023). CBD, a
   nonpsychoactive phytocannabinoid derived from cannabis, has gained
   attention as a promising therapeutic agent for inflammatory diseases,
   largely due to its anti-inflammatory properties, low toxicity, and
   ability to modulate various proinflammatory signals ([78]Baban et al.,
   2021; [79]Singh et al., 2023). Recent findings suggest that CBD
   significantly increased TREM2 expression in glial cells and reduced
   IL-6 levels in peripheral blood leukocytes, which were associated with
   improved cognitive function in a preclinical AD model ([80]Khodadadi et
   al., 2021). Additionally, CBD not only enhanced astrocytic IL-33
   expression, a cytokine that promotes microglial phagocytosis of Aβ and
   improves contextual memory, but also boosted acetylcholine production,
   further improving cognitive performance ([81]Khodadadi et al., 2021).
   These results support the potential of CBD as a clinically safe and
   effective disease-modifying treatment to slow neurocognitive decline in
   AD.

   CBD's anti-inflammatory and immunomodulatory properties may influence
   key immune pathways such as IDO and cGAS, which are involved in chronic
   inflammation and neurodegeneration. While direct evidence of CBD's
   interaction with these pathways is still limited, it is plausible that
   CBD could modulate both the IDO and cGAS pathways, offering potential
   therapeutic benefits for neuroinflammation in neurodegenerative
   diseases like AD. In this study, we aimed to investigate the
   association between CBD, IDO, and cGAS to uncover new insights into
   CBD's role in modulating immune responses and slowing
   neurodegeneration, ultimately contributing to more effective,
   disease-modifying treatments for AD.

Materials and Methods

Experimental design and treatment protocol

   Adult male 5xFAD mice were purchased from Jackson Laboratory. The 5xFAD
   mice express human amyloid precursor protein and preseniln-1 transgenes
   with five AD-linked mutations, used as a preclinical model of AD
   ([82]Oakley et al., 2006; [83]Schneider et al., 2014). At 9–12 months,
   mice were randomized into two groups (n = 10/group), receiving either
   placebo or inhaled CBD (10 mg/mouse, Thriftmaster Global Bioscience)
   every day for 4 weeks. Each CBD inhaler contained 985 mg of
   broad-spectrum CBD (winterized crude hemp extract) plus 15 mg of
   cosolvent, surfactant, and propellant, with a total volume of 1,000 mg
   (1.78 mg dose per actuation, with 200 ml/min flow rate). For the
   placebo, the 985 mg of broad-spectrum CBD was replaced with 985 mg of
   hemp seed oil. As described previously ([84]Khodadadi et al., 2024),
   inhalers were modified by adding an extra nozzle piece to adjust to the
   mouse model and to further control the intake of CBD. The study design
   included three independent cohorts, ensuring the robustness and
   reproducibility of the results. Inhalation was selected as the route of
   administration based on its favorable pharmacokinetic profile,
   including rapid systemic absorption and avoidance of first-pass hepatic
   metabolism, which can reduce the bioavailability of orally administered
   CBD ([85]Stella et al., 2021; [86]Varadi et al., 2023). This method
   allows for more consistent plasma concentrations and a faster onset of
   action, which is essential for capturing dynamic neuroimmune responses
   within a defined experimental window.

IF staining

   Fresh brain tissues were fixed with 10% neutral buffered formalin. The
   tissues then were processed, embedded, and subsequently cut into 4 μm
   sections for IF assessments as described previously ([87]Khodadadi et
   al., 2021). Briefly, after blocking endogenous peroxidase activity with
   hydrogen peroxide (diluted 1:10 in distilled water for 10 min),
   sections were treated with Proteinase K for 10 min and washed twice in
   PBS. Slides were then labeled with specific, fluorescent-conjugated
   antibodies with fluorescent conjugation against IDO (PE anti-IDO1,
   BioLegend, catalog #654006), transmembrane protein 119 (Alexa Fluor 488
   anti-TMEM119, for microglia, Thermo Fisher Scientific, catalog
   #53-6119-82), glial fibrillary acidic protein (Alexa Fluor 488
   anti-GFAP, for astrocytes, BioLegend, catalog #644704), and cGAS
   (catalog #79978, purified, Cell signaling Technology). All slides were
   counterstained using DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher
   Scientific, catalog #D1306) prior to examination and imaging by Zeiss
   Fluorescence Microscope. The integrated density of the area chosen (in
   pixels) and the mean gray value (the measurement of the brightness)
   were measured using the lasso tool of Adobe Photoshop CS4 extended.

Analytical flow cytometry

   For flow cytometry analysis, single-cell suspension was prepared from
   the brain by sieving the brain tissues through a 100 μm cell strainer
   (BD Biosciences) followed by centrifugation (252 × g, 10 min). All
   cells were then stained with fluorescent antibodies based on routine
   flow cytometry staining protocol as described previously ([88]Khodadadi
   et al., 2021). Briefly, all cells were then stained with fluorescent
   antibodies to phenotype and quantify microglia (TMEM119^+CD45^+/lo) and
   infiltrating macrophages (TMEM119^−CD45^+/hi; Alexa Fluor 488
   anti-TMEM119, Thermo Fisher Scientific, catalog #53-6119-82 and PE
   anti-mouse CD45, BioLegend, catalog #157604). Then cells were fixed and
   permeabilized and stained intracellularly for cytokines including IFN-γ
   (APC anti-mouse IFN-γ, BioLegend, catalog #505809), interleukin-1β
   (PerCP anti-mouse IL-1β, Thermo Fisher Scientific, catalog
   #46-7114-82), tumor necrosis factor alpha (Pacific Blue anti-mouse
   TNFα, BioLegend, catalog #506318), and interleukin-10 (Biotin
   anti-mouse IL-10, BioLegend, catalog #505003). Cells were then run
   through a NovoCyte Quanteun flow cytometer (Agilent Technologies) and
   analyzed by the FlowJo V10 analytical software. To confirm the
   specificity of primary antibody binding and rule out the nonspecific Fc
   receptor binding to cells or other cellular protein interactions,
   negative control experiments were conducted using isotype controls
   matched to each primary antibody's host species, isotype, and
   conjugation format.

Behavioral tests

   Cognitive and exploratory behaviors were evaluated using the open field
   (OF) and novel object recognition (NOR) paradigms, adapted from
   established protocols ([89]Khodadadi et al., 2024). For NOR, mice were
   initially placed in an enclosed arena containing two identical objects
   positioned within a 10 cm circular boundary at a fixed distance apart.
   After a familiarization phase, animals were briefly removed, and upon
   reintroduction, one of the familiar objects was substituted with a
   novel object differing in shape, texture, and appearance. Recognition
   memory was quantified using a discrimination index (DI), calculated as
   (T[n] − T[f]) / (T[n] + T[f]), where T[n] represents time spent
   exploring the novel object and T[f] the familiar object.

   In the OF test, mice were placed individually in a square arena
   (40 × 40 × 40 cm) for 10 min, while the activity was recorded by an
   overhead camera. Locomotor parameters, including total distance
   traveled, average velocity, and time spent in the central zone, were
   quantified using the EthoVision XT software (version 17.5, Noldus
   Information Technology).

Integrated bioinformatic analysis

Network construction: protein–protein interaction

   To investigate the IDO/cGAS genes, we used the interactive database
   platform STRING v.11.0 ([90]https://string-db.org/). STRING is a
   database of known and predicted protein–protein interactions (PPI;
   [91]Szklarczyk et al., 2023). The interactions include direct
   (physical) and indirect (functional) associations; they stem from
   computational prediction, from knowledge transfer between organisms,
   and from interactions aggregated from other (primary) databases.

   Next, the PPI network was constructed. The confidence score cutoff was
   set at 0.4, and other settings were set to default.

Gene ontology functional analysis

   After identification of the IDO/cGAS targets, the Metascape platform
   and the Enrichr database were used to analyze their main biological
   processes, cellular components, and molecular functions to perform
   enrichment analysis ([92]Gable et al., 2022; [93]Szklarczyk et al.,
   2023). The results were visualized using biological online tools.

Statistical analysis

   For statistical analysis, Brown–Forsythe and Welch analysis of variance
   (ANOVA) was used to establish significance (p < 0.05) among groups. For
   tissue quantification statistical analysis, the area of expression was
   compared in both placebo and CBD-treated groups using two-way ANOVA,
   followed by post hoc Sidak testing for multiple comparison (p < 0.05).

Results

Inhaled CBD reduces IDO expression and alters glial activation in mice with
AD

   IF staining was performed to evaluate the effects of inhaled CBD on IDO
   expression in the brains of 5XFAD mice, a well-established model of AD.
   A significant reduction in IDO expression was observed in the
   entorhinal cortex area of both microglia and astrocytes in CBD-treated
   mice compared with untreated controls ([94]Fig. 1). Specifically, in
   Panel A, TMEM119+ microglial cells showed a notable decrease in IDO
   expression following CBD treatment, while panel B demonstrated a
   similar reduction in IDO levels in GFAP+ astrocytes. While TMEM119
   expression is known to decrease in highly activated microglia,
   particularly in later stages of AD, it was used here as a resident
   microglial marker to examine IDO expression within this population.
   Quantitative analysis, shown in Panel C, further confirmed these
   observations, with pixel intensity measurements revealing a significant
   decrease in total IDO expression as well as in both microglia and
   astrocytes in CBD-treated AD mice (***p < 0.001). In addition to these
   reductions in IDO expression, pixel intensity analysis indicated
   changes in glial activation. Specifically, CBD treatment was associated
   with increased microglial activation, as reflected by a higher pixel
   intensity of TMEM119+ staining, suggesting a heightened state of
   microglial response. In contrast, astrocyte activation, measured by
   GFAP intensity, was reduced in CBD-treated animals compared with
   controls, indicating a potential modulatory effect of CBD on astrocyte
   function. While the differences in both microglial and astrocyte
   activation were nonsignificant (ns), the observed trends, higher
   microglial activation and lower astrocyte activation, are significant
   in the context of AD, as increased microglial activation and reduced
   astrocyte activation are considered beneficial for AD progression.
   These changes support the potential therapeutic effects of CBD in
   modulating neuroinflammatory pathways in AD. The reduction in IDO
   expression, particularly in microglia and astrocytes, suggests that CBD
   may modulate key inflammatory pathways involved in AD pathology. IDO, a
   crucial enzyme in tryptophan metabolism, is known to be upregulated in
   neuroinflammatory conditions and has been implicated in
   neurodegenerative diseases like AD. By reducing IDO expression, CBD
   could potentially mitigate neuroinflammation and its contribution to AD
   progression, highlighting its therapeutic potential in targeting the
   immunoinflammatory aspects of the disease.

Figure 1.

   [95]Figure 1.
   [96]Open in a new tab

   Inhalation of CBD reduces IDO expression in 5xFAD mice with AD. IF
   staining (Panel A and B) demonstrated a significant reduction in IDO
   expression in microglia (TMEM119+ cells, Panel A, and astrocytes (GFAP+
   cells, panel B, within the entorhinal cortex of CBD-treated 5xFAD mice
   compared with untreated controls. The images shown are representative
   of three independent experimental cohorts. C, Quantitative analysis of
   IDO expression, based on pixel intensity measurements, confirmed a
   significant decrease in total IDO expression, as well as in both
   microglia and astrocytes, in CBD-treated mice with AD (***p < 0.001).
   Each graph shows all individual data points (n = 10 mice per group),
   representing one of three independent experiments with consistent
   findings across cohorts. Additionally, pixel intensity analysis
   revealed increased microglial activation (higher TMEM119 intensity) and
   reduced astrocyte activation (lower GFAP intensity) in CBD-treated
   animals relative to untreated controls, though differences in glial
   activation were not statistically significant (ns).

CBD modulates the IDO/cGAS pathway in mice with AD

   IF staining revealed that inhalation of CBD significantly decreased
   cGAS expression in IDO-expressing cells in the entorhinal cortex of
   5XFAD mice with AD, compared with untreated controls ([97]Fig. 2A).
   This reduction in cGAS expression was quantified by measuring the
   colocalization of cGAS and IDO, with pixel intensity analysis showing a
   marked decrease in cGAS/IDO coexpression in CBD-treated AD mice
   relative to untreated controls ([98]Fig. 2B; p < 0.0001). These
   findings suggest that CBD may offer a promising therapeutic approach
   for AD by targeting neuroinflammatory mechanisms, specifically the
   IDO/cGAS pathway. This result has two key implications: First, it
   supports the autoinflammatory theory of AD (AD^2), providing evidence
   that a more immune-targeted treatment strategy could emerge for AD,
   moving beyond the traditional focus on Aβ. Second, our work introduces
   a novel mechanism through the modulation of the IDO/cGAS pathway, which
   warrants further investigation into CBD's potential to alter
   neuroinflammation and immune responses in AD. These results underline
   the importance of CBD as a candidate for treating AD via immune-based
   pathways, expanding our understanding of its therapeutic potential in
   neurodegenerative diseases. We acknowledge that downstream cGAS pathway
   markers such as phosphorylated STING and TBK1 were not evaluated, and
   this remains a limitation to be addressed in future work.

Figure 2.

   [99]Figure 2.
   [100]Open in a new tab

   Inhalation of CBD reduces cGAS/IDO coexpression in 5xFAD mice with AD.
   A, IF staining showed that CBD inhalation significantly reduced cGAS
   expression in IDO-expressing cells within the entorhinal cortex of
   5xFAD mice with AD, compared with untreated controls. The images are
   representative of three independent experimental cohorts. B,
   Quantitative analysis of colocalization between cGAS and IDO, based on
   pixel intensity measurements, revealed a significant decrease in
   cGAS/IDO coexpression in CBD-treated mice relative to untreated
   controls (***p < 0.0001). The graph displays all individual data points
   (n = 10 mice per group), derived from one representative experiment out
   of three biologically independent cohorts, each showing consistent
   results.

Inhaled CBD regulates proinflammatory immune profile in mice with AD

   Flow cytometry analysis was performed to assess immune cell profiles in
   the brains of 5XFAD mice with or without CBD treatment ([101]Fig. 3).
   Panel A shows the total brain cells from untreated (top panel) and
   CBD-treated (bottom panel) 5XFAD mice, with live gating based on
   FSC/SSC parameters. Panel B reveals that CBD treatment significantly
   reduced the frequency of infiltrating macrophages
   (CD45^+/hiCD11b^+TMEM119^−), a population distinguishable from resident
   microglia by their lack of TMEM119 expression, indicating a decrease in
   neuroinflammatory responses. Further analysis (Panel C) showed that CBD
   treatment decreased proinflammatory cytokine production while enhancing
   the anti-inflammatory cytokine IL-10. Panel D quantifies these changes,
   with significant differences (**p < 0.05; ***p < 0.001) observed in the
   cytokine profile. These results suggest that CBD modulates immune
   responses in AD by reducing macrophage infiltration and promoting an
   anti-inflammatory cytokine profile, supporting its potential as a
   therapeutic agent for neuroinflammation in AD.

Figure 3.

   [102]Figure 3.
   [103]Open in a new tab

   Inhaled CBD modulates the proinflammatory immune profile in mice with
   AD. A, Flow cytometry dot plots show total brain cells from untreated
   (top panel) and CBD-treated (bottom panel) 5xFAD mice, with live gating
   based on forward scatter (FSC) and side scatter (SSC) parameters. Data
   are representative of three independent experimental cohorts. B, Flow
   cytometry distinguishes resident microglia (CD45^+/loCD11b^+TMEM119^+)
   from infiltrating macrophages (CD45^+/hiCD11b^+TMEM119^−). CBD
   treatment reduced the frequency of infiltrating macrophages, suggesting
   attenuation of inflammatory responses. C, Intracellular cytokine
   staining revealed a significant reduction in proinflammatory cytokine
   production (IFN-γ, TNF-α, IL-1β) in CBD-treated mice, accompanied by
   enhanced production of the anti-inflammatory cytokine IL-10, relative
   to untreated controls. D, Quantitative analysis of cytokine profiles is
   presented as histograms, displaying all individual data points (n = 10
   mice per group). The dataset shown is representative of three
   biologically independent experiments, each yielding consistent results.
   Significant differences were noted between groups (**p < 0.05;
   ***p < 0.001).

CBD treatment improves cognitive function in mice with AD

   To evaluate whether molecular and immunological changes induced by CBD
   translated into functional benefits, we assessed cognition using the OF
   and NOR tests. CBD-treated 5xFAD mice exhibited significantly improved
   behavioral performance compared with untreated controls ([104]Fig. 4).
   In the OF test, CBD-treated mice spent more time in the central zone
   (290 vs 130 s in untreated mice; p ≤ 0.001), reflecting reduced
   anxiety-like behavior and improved exploratory activity. In the NOR
   test, CBD-treated mice displayed enhanced recognition memory, with a DI
   of 0.5 compared with −0.4 in untreated controls (p ≤ 0.001). Together,
   these findings indicate that inhaled CBD not only modulates
   neuroinflammatory pathways but also improves cognitive outcomes in the
   5xFAD model of AD.

Figure 4.

   [105]Figure 4.
   [106]Open in a new tab

   Inhaled CBD improves cognitive performance in mice with AD. A, Heat
   maps from the OF test show increased central zone exploration in
   CBD-treated 5xFAD mice compared with untreated controls. B,
   Quantification of OF performance demonstrates significantly greater
   time spent in the central zone for CBD-treated mice (p ≤ 0.001). C,
   Heat maps from the NOR test indicate enhanced exploratory preference in
   CBD-treated 5xFAD mice. D, DI analysis confirms improved recognition
   memory with CBD treatment (DI, ∼0.5 vs −0.4; p ≤ 0.001). Data are
   mean ± SEM.

Bioinformatics analysis of IDO/cGAS pathway in AD: CBD as a potential
therapeutic target

   To explore the potential therapeutic role of CBD in AD, we conducted a
   comprehensive bioinformatics analysis focused on the IDO/cGAS pathway
   and its molecular intersection with CBD-targeted proteins ([107]Fig.
   5). Panel A displays a STRING-derived PPI network that positions IDO1
   and cGAS (MB21D1) as central hubs within a broader immune signaling
   architecture implicated in AD-related neuroinflammation. This network
   revealed multiple high-confidence interactions between immune-metabolic
   regulators and CBD-associated proteins. Notably, AKT1, TRPV1, and GPR55
   emerged as key molecular nodes based on their connectivity within the
   network, their STRING confidence scores, and their established roles in
   regulating neuroimmune function. AKT1, a serine/threonine kinase
   involved in the PI3K/AKT signaling cascade, plays a pivotal role in
   immune tolerance, glial survival, and inflammatory cytokine regulation,
   functions that are dysregulated in AD and closely linked to
   cGAS-STING–driven interferon responses ([108]Jia et al., 2021). TRPV1,
   a calcium-permeable ion channel broadly expressed in astrocytes and
   microglia, modulates inflammatory responses, oxidative stress, and
   neuroglial cross talk, thereby influencing the chronic immune
   activation observed in neurodegenerative contexts ([109]Awad-Igbaria et
   al., 2025). GPR55, an orphan G-protein–coupled receptor increasingly
   recognized for its immunomodulatory properties, regulates microglial
   migration, cytokine secretion, and lymphocyte activation and may act as
   an indirect cannabinoid sensor in the CNS ([110]Sumida et al., 2017).
   Panel B of [111]Figure 5 illustrates the enriched co-regulatory
   relationships between these CBD-interacting proteins and the IDO/cGAS
   axis, reinforcing the hypothesis that CBD exerts its effects by
   modulating convergent neuroimmune pathways. Panel C highlights pathway
   enrichment data showing that the top-ranked CBD targets are involved in
   biological processes such as nitric oxide synthase regulation, cytokine
   signaling, calcium ion homeostasis, and immune effector functions, each
   of which plays a critical role in neuroinflammation and neuronal
   integrity in AD. These findings suggest that the therapeutic effects of
   CBD may arise from its ability to influence a coordinated
   immune-metabolic network involving both canonical inflammatory
   mediators (e.g., IDO and cGAS) and regulatory nodes such as AKT1,
   TRPV1, and GPR55. The inclusion of these targets was therefore driven
   not only by STRING-based interaction strength but also by their
   functional integration within pathophysiological processes central to
   AD progression.

Figure 5.

   [112]Figure 5.
   [113]Open in a new tab

   Bioinformatics analysis of the IDO/cGAS pathway in AD: CBD as a
   potential therapeutic target. A, PPI network analysis identifies IDO
   and cGAS as central nodes in neuroinflammatory signaling, along with
   CBD-interacting targets such as AKT1, TRPV1, and GPR55. These targets
   were prioritized based on both their functional relevance to immune
   regulation and their predicted high-confidence interactions with CBD
   from STRING database analysis. B, PPI enrichment analysis demonstrates
   significant coregulation between IDO/cGAS and the selected CBD-targeted
   nodes, suggesting coordinated involvement in neuroimmune and metabolic
   stress pathways implicated in AD. C, Gene ontology-based pathway
   enrichment analysis reveals that CBD-targeted genes participate in
   critical biological processes including nitric oxide synthase
   regulation, immune system modulation, and calcium ion homeostasis, key
   factors that influence glial activation, cytokine signaling, and
   neuroprotection in the context of AD-related neuroinflammation.

Discussion

   Our study provides robust support for the evolving concept of AD as an
   autoinflammatory disorder (AD^2), offering new insights into the
   pathophysiology of AD beyond the Aβ hypothesis ([114]Rayaprolu et al.,
   2021; [115]Weaver, 2023; [116]Arshavsky, 2024). While amyloid plaques
   and tau tangles have historically been considered central to AD
   pathology, emerging evidence suggests that these factors alone cannot
   fully account for the complexity of AD progression ([117]Naseri et al.,
   2019; [118]Zhang et al., 2023; [119]Behl, 2024). Our findings are
   consistent with the immune hypothesis of AD^2, which proposes that
   immune system dysregulation, rather than amyloid deposition in
   isolation, plays a central role in the initiation and progression of
   neurodegenerative processes in AD ([120]Naseri et al., 2019;
   [121]Rayaprolu et al., 2021; [122]Weaver, 2023; [123]Zhang et al.,
   2023; [124]Arshavsky, 2024; [125]Behl, 2024). Specifically, in this
   model, the immune system aberrantly targets the autologous brain
   tissue, exacerbating neuroinflammation, a key feature of AD pathology
   ([126]Latta et al., 2015; [127]Calsolaro and Edison, 2016; [128]Weaver,
   2023). By demonstrating that CBD inhalation modulates immune-related
   pathways in the brain, our results provide compelling evidence
   supporting immune dysfunction as a major contributor to AD progression,
   further validating the AD^2 model.

   The choice of the 5XFAD transgenic mouse model for our studies was
   particularly advantageous, as it closely mimics the hallmark features
   of AD, including Aβ deposition, neuroinflammation, and cognitive
   decline ([129]Oakley et al., 2006; [130]Schneider et al., 2014). This
   model is widely recognized for its ability to reproduce key aspects of
   AD pathology, making it an ideal system for evaluating potential
   therapeutic strategies like CBD. The 5XFAD model provided a robust
   platform to investigate how CBD interacts with immune-related pathways
   in the context of AD, allowing us to generate meaningful insights into
   its effects on neuroinflammation and glial function.

   A critical mechanism implicated in immune dysfunction within the CNS is
   the IDO/cGAS pathway ([131]Lemos et al., 2020; [132]Govindarajulu et
   al., 2023; [133]Gulen et al., 2023). Both IDO and cGAS are integral to
   maintaining immune homeostasis in the brain ([134]Kwidzinski and
   Bechmann, 2007; [135]Baban et al., 2011; [136]Salminen, 2022;
   [137]Govindarajulu et al., 2023). IDO regulates tryptophan metabolism,
   and its overactivation is associated with increased neuroinflammation
   in various neurological conditions, including AD ([138]Liu et al.,
   2018; [139]Sodhi et al., 2021). cGAS, through its role in sensing
   cytosolic DNA, initiates the STING pathway, which, when dysregulated,
   can amplify neuroinflammation ([140]Zhang et al., 2020; [141]Zhang and
   Zhang, 2025). Our study presents novel evidence that CBD effectively
   modulates key components of immune regulation, highlighting its
   potential as a therapeutic strategy to address immune dysfunction at
   the core of AD pathophysiology. While our findings demonstrate that
   total cGAS expression is modulated by CBD treatment, we acknowledge
   that evaluating downstream signaling components such as phosphorylated
   STING (p-STING) and TBK1 (p-TBK1) would offer a more direct readout of
   cGAS pathway activation. However, given the novel focus of this study
   on the intersection of cGAS and IDO signaling as an upstream
   immune-metabolic axis, total cGAS expression served as a relevant
   marker for identifying broader immune dysregulation in AD. Future
   studies incorporating p-STING and p-TBK1 analysis will be essential to
   validate the functional activation status of this pathway and further
   elucidate how CBD impacts specific effector cascades downstream of
   cGAS. By targeting both IDO and cGAS, CBD offers a promising strategy
   to restore immune balance in the CNS and mitigate neurodegenerative
   processes. These findings underscore the emerging importance of the
   cGAS/IDO axis as a central regulatory node in AD-related neuroimmune
   dysfunction. Rather than serving merely as downstream markers of
   inflammation, both cGAS and IDO orchestrate fundamental
   immune-metabolic processes, including interferon signaling, kynurenine
   pathway activation, and glial reprogramming, which drive chronic
   neuroinflammation and neuronal injury. By modulating both DNA-sensing
   innate immune signaling and tryptophan catabolism, CBD exerts a dual
   regulatory effect that may dampen maladaptive immune activation while
   preserving neuroprotective immune tolerance. This layered mechanism
   offers a distinct therapeutic advantage over existing amyloid-centric
   approaches, positioning CBD as a candidate capable of targeting
   upstream inflammatory circuits implicated in both the onset and
   propagation of AD's pathology.

   Our findings are in line with previous studies that have shown
   beneficial effects of CBD in AD models, both in mice and humans
   ([142]Baban et al., 2021; [143]Khodadadi et al., 2021; [144]Xiong and
   Lim, 2021; [145]Singh et al., 2023; [146]Trojan et al., 2023). These
   studies have demonstrated that CBD's anti-inflammatory and
   neuroprotective properties can ameliorate key aspects of AD pathology,
   such as amyloid plaque deposition and cognitive impairment. Our work
   builds on this foundation, specifically highlighting the modulation of
   immune pathways like IDO/cGAS as a novel mechanism by which CBD could
   exert its therapeutic effects in AD. CBD's modulation of the IDO/cGAS
   pathway is accompanied by significant effects on glial cell activity.
   Our results demonstrate that CBD treatment reduces the expression of
   IDO in key glial populations, including microglia and astrocytes, while
   also decreasing the coexpression of IDO and cGAS. Given the central
   role of glial cells in modulating immune responses within the CNS
   ([147]Wang et al., 2022), these findings suggest that CBD directly
   influences glial-mediated immune regulation. In conjunction with this,
   we also observed a CBD-induced increase in microglial activation
   alongside a reduction in astrocytic activation. This dual modulation is
   significant, as elevated microglial activation and lower astrocytic
   activity have been associated with enhanced neuroprotection and
   improved outcomes in AD ([148]Liu et al., 2019). Chronic
   neuroinflammation, driven by the overactivation of immune pathways like
   IDO and cGAS, plays a pivotal role in AD progression ([149]Liu et al.,
   2018; [150]Zhang et al., 2020; [151]Sodhi et al., 2021; [152]Zhang and
   Zhang, 2025). Therefore, CBD's ability to regulate both glial activity
   and immune pathways may not only mitigate neuroinflammation but also
   foster a neuroprotective environment within the brain, offering a
   potential therapeutic benefit for AD.

   Furthermore, the choice of inhalation as the route of CBD
   administration reflects both pharmacological and translational
   considerations. Inhaled delivery circumvents hepatic first-pass
   metabolism and provides a more rapid and reliable systemic exposure
   profile than oral or intraperitoneal routes ([153]Stella et al., 2021;
   [154]Varadi et al., 2023). This delivery method enhances CNS
   bioavailability and has shown improved efficacy in other neurological
   disease models. For example, a recent study in a preclinical model of
   epilepsy demonstrated that CBD administered via inhalation was superior
   to both oral and intraperitoneal routes in reducing seizure severity
   and neuroinflammatory responses ([155]Bhandari et al., 2025). While our
   study focuses on AD, this evidence supports the broader therapeutic
   utility of inhalation for delivering cannabinoids in CNS-targeted
   interventions.

   Our bioinformatic analysis provides deeper mechanistic insight into the
   potential pathways through which CBD may exert therapeutic effects in
   AD, particularly by modulating neuroimmune signaling hubs. The data
   suggest that CBD influences not only upstream inflammatory regulators
   like IDO and cGAS but also engages key molecular effectors such as
   AKT1, TRPV1, and GPR55, each of which plays a distinct but converging
   role in neuroinflammation and neuronal homeostasis. Notably, activation
   of the AKT1 signaling cascade by CBD may promote neuronal survival,
   enhance metabolic resilience, and suppress inflammatory output via
   PI3K-dependent mechanisms ([156]Sun et al., 2015). This is especially
   relevant in the AD context, where impaired AKT1 signaling has been
   associated with increased vulnerability to neurodegeneration. In
   parallel, CBD appears to target TRPV1, a ligand-gated ion channel
   expressed on glial cells that regulates calcium influx and contributes
   to both the initiation and amplification of glial-mediated
   neuroinflammatory responses ([157]Viana et al., 2022). By modulating
   TRPV1 activity, CBD may influence astrocyte–microglial interactions and
   dampen excitotoxic cascades linked to chronic neuroinflammation.
   Furthermore, the orphan receptor GPR55, increasingly recognized for its
   cannabinoid sensitivity and immune-modulatory functions, emerges as a
   compelling CBD-interacting target. GPR55 plays a pivotal role in
   leukocyte recruitment, microglial activation, and cytokine regulation
   within the CNS milieu, and its modulation could have broad implications
   for restoring immune homeostasis in AD ([158]Viana et al., 2022).
   Target selection was informed by both their functional relevance to
   neuroimmune signaling cascades and their predicted high-affinity
   interaction with CBD as determined through STRING network analysis.
   AKT1, TRPV1, and GPR55 collectively represent mechanistically relevant
   and pharmacologically accessible nodes within the CBD-modulated immune
   network, providing a rational foundation for future studies aimed at
   dissecting their individual and combinatorial roles in disease
   modification.

   The ability of CBD to modulate both immune dysfunction and
   neuroinflammation offers a more comprehensive approach to AD treatment
   compared with traditional amyloid-based therapies, which have shown
   limited success in clinical trials ([159]Cummings et al., 2023). CBD's
   effects on the IDO/cGAS pathway, coupled with its ability to influence
   glial activity and neuronal survival, position it as a promising
   candidate for AD therapy. Moreover, CBD's impact on cytokine profiles
   further underscores its therapeutic potential. In our study, CBD
   treatment was associated with a decrease in proinflammatory cytokines,
   such as IFN-γ, IL-1β, and TNF-α, while increasing the production of the
   anti-inflammatory cytokine IL-10. This modulation of the inflammatory
   milieu is particularly important in AD, where chronic neuroinflammation
   plays a central role in disease progression ([160]Lecca et al., 2022;
   [161]C. Wang et al., 2023; [162]M. Wang et al., 2023). By restoring
   immune homeostasis and attenuating neuroinflammation, CBD may help slow
   or halt disease progression, providing a valuable addition to current
   therapeutic strategies. While IFN-γ is classically associated with
   lymphocytes, prior studies have shown that microglia and macrophages
   can express IFN-γ in the inflamed or infected CNS ([163]Suzuki et al.,
   2005; [164]Kawanokuchi et al., 2006). Our findings suggest that chronic
   neuroinflammatory conditions in the 5XFAD model may similarly promote
   IFN-γ expression in these myeloid populations, potentially through
   cytokine-driven activation or cross talk with infiltrating immune
   cells. Notably, the observed reduction in IFN-γ levels following CBD
   treatment may reflect its capacity to reprogram innate immune responses
   in the brain, offering further support for its therapeutic potential in
   modulating aberrant neuroinflammation in AD.

   The current study builds upon previously published work demonstrating
   that CBD treatment improves cognitive performance, reduces Aβ plaque
   burden, and enhances acetylcholine signaling in the 5XFAD mouse model
   of AD ([165]Oakley et al., 2006; [166]Schneider et al., 2014;
   [167]Khodadadi et al., 2021, [168]2024). While the present work focuses
   primarily on the immunoregulatory effects of CBD, particularly its
   modulation of the IDO/cGAS axis, these mechanistic insights are
   conceptually grounded in our earlier findings of CBD's
   disease-modifying potential. Together, this body of work supports the
   hypothesis that neuroimmune modulation may underlie the cognitive and
   neuropathological improvements previously observed, thereby linking the
   emerging autoinflammatory framework of AD to therapeutic response.

   Importantly, the observed behavioral improvements provide functional
   validation of our mechanistic findings. By demonstrating that inhaled
   CBD enhances recognition memory and exploratory behavior in 5xFAD mice,
   these results bridge molecular modulation of the IDO/cGAS axis with
   measurable cognitive benefits. This alignment between immune regulation
   and behavioral outcomes strengthens the translational relevance of our
   work, highlighting inhaled CBD as a promising strategy to
   simultaneously target neuroinflammation and cognitive impairment in AD.

   In conclusion, our findings contribute to the growing body of evidence
   supporting the immune theory of AD^2, demonstrating that CBD modulates
   key immune pathways, particularly the IDO/cGAS axis, which plays a
   critical role in immune dysregulation in AD. Our results highlight the
   potential of CBD as a novel therapeutic agent for AD, offering a
   multifaceted approach to disease management. By modulating immune
   responses, regulating glial activity, and promoting neuronal survival,
   CBD addresses several key aspects of AD pathology. These findings
   warrant further clinical investigation into the use of CBD, either as a
   monotherapy or in combination with other therapeutic strategies, to
   evaluate its potential for modifying the course of AD. Future work will
   extend this research to additional transgenic and sporadic AD models to
   evaluate the generalizability of these findings across diverse
   pathological contexts. Moreover, longitudinal studies will be essential
   to assess the durability and safety of chronic CBD treatment, as well
   as its long-term impact on cognitive decline, neuroinflammation, and
   neuropathological progression.

   While certain methodological and biological caveats remain, as outlined
   in the Limitations section below, these do not diminish the overall
   strength of our findings, which provide novel insight into CBD's impact
   on neuroinflammation and neurodegeneration.

Limitations

   This study has several limitations. Only a single CBD dose was tested
   in 5XFAD mice; future studies should evaluate a broader range.
   Translation to humans remains uncertain due to species differences in
   metabolism and pharmacokinetics, requiring clinical trials. While our
   focus was on microglia and macrophages, additional immune and glial
   populations, including astrocytes, should be examined. Finally,
   reliance on TMEM119 as a microglial marker is a constraint, as its
   expression may be reduced in disease-associated states. Despite these
   caveats, the findings advance understanding of CBD's therapeutic
   potential in neurodegeneration.

Synthesis

   Reviewing Editor: Harald Sontheimer, University of Virginia

   Decisions are customarily a result of the Reviewing Editor and the peer
   reviewers coming together and discussing their recommendations until a
   consensus is reached. When revisions are invited, a fact-based
   synthesis statement explaining their decision and outlining what is
   needed to prepare a revision will be listed below. The following
   reviewer(s) agreed to reveal their identity: John Lukens, Shiva
   shahrara.

   Both reviewers found your work nteresting and innovative and the focus
   on the AD2 theory of Alzheimer's, which highlights the role of immune
   dysfunction and chronic neuroinflammation in disease progression, is
   relevant and supports the rationale for targeting neuroinflammatory
   pathways in AD.

   However, issues and technical shortcomings were noted that warrant
   revsions.

   1. The manuscript should more clearly emphasize the innovative aspect
   of targeting the cGAS/IDO pathway in Alzheimer's. Briefly outline why
   this pathway is a novel target in AD and how it offers potential
   advantages over current therapeutic strategies.

   2. The use of inhalation as the method for CBD administration needs
   further clarification. Please justify why this method was selected over
   others (e.g., oral or intraperitoneal administration), especially in
   the context of pharmacokinetics and therapeutic efficacy.

   3. Expand on the bioinformatics analysis, particularly the selection of
   immune targets such as AKT1, TRPV1, and GPR55. Explain why these
   targets were chosen and their relevance to the neuroinflammatory
   processes in AD. A more detailed discussion of the interactions
   identified through the STRING database will also be useful.

   4. The conclusion would benefit from a mention of the next steps in
   your research, such as investigating CBD's effects in other AD models
   or evaluating long-term effects. This will provide a roadmap for future
   studies and demonstrate the broader potential of CBD in Alzheimer's
   therapy.

   5. The IDO and cGAS pathways are independent pathways and should be
   presented as such in the abstract to limit misinformation.

   6. Unclear if autoimmune disease is correct as this implies that the
   pathology is driven by autoreactive T and B cells, which they have not
   shown. Autoinflammatory disease would be a more correct interpretation
   of their data.

   7. It is unclear as to what they mean by "This aligns with the AD
   theory of AD" in the introduction.

   8. It is strange to group IFN-gamma with cGAS in the introduction as
   type I IFNs, and not IFN-gamma, are downstream of cGAS activation.

   9. Tmem119 is actually downregulated by activated microglia and is not
   a good marker to track microglial responses in AD-related disease as
   most activated microglia around amyloid beta pathology do not express
   Tmem119 in full-blown disease.

   10. Changes in total cGAS expression are not as informative as
   evaluating downstream cGAS signaling such as p-STING and p-TBK1. It is
   difficult to interpret anything meaningful from their cGAS imaging
   data.

   11. It is surprising that they see so much expression of IFN-gamma by
   macrophages/microglia in Figure 3 as it is widely thought that
   macrophages/microglia do not make IFN-gamma. Additional evidence in
   support of this is needed to bolster this potentially controversial
   claim by them.

   12. To increase the rigor and reproducibility of their findings, they
   should present individual data points in their graphs and specifically
   indicate the experimental sample size in each of their experiments.

   13. Without analysis of cognitive behavior, amyloid beta load, neuritic
   dystrophy, or neuronal loss it is difficult to ascertain whether their
   data interpretation is relative to disease pathogenesis.

Author Response

   Rebuttal Letter for Manuscript Revision Manuscript Title: Rethinking
   Alzheimer's: Harnessing Cannabidiol to Modulate IDO and cGAS Pathways
   for Neuroinflammation Control Journal: eNeuro, Manuscript ID# eNeuro
   eN-NWR-0114-25 Corresponding Author: Babak Baban, Ph.D.

   Dear Editors and Reviewers, We would like to thank you for the
   thoughtful and constructive feedback provided on our manuscript. We
   appreciate the opportunity to revise and strengthen our submission.
   Below, we address each comment point-by-point and indicate how we have
   responded in the revised manuscript. Where applicable, we have
   indicated the locations of changes. The reviewers' comments are
   summarized first (in Italic), followed by our replies. All
   modifications made to the revised manuscript have been highlighted in
   YELLOW for ease of reference.

   Reviewer Comment 1: The manuscript should more clearly emphasize the
   innovative aspect of targeting the cGAS/IDO pathway in Alzheimer's.
   Briefly outline why this pathway is a novel target in AD and how it
   offers potential advantages over current therapeutic strategies.

   Response: We are in full agreement with the reviewer and appreciate the
   thoughtful suggestion. In response, we have revised the Abstract,
   Significance Statement, Introduction, and Discussion sections to more
   clearly emphasize the innovative aspect of targeting the cGAS/IDO axis
   in AD.

   Reviewer Comment 2: The use of inhalation as the method for CBD
   administration needs further clarification. Please justify why this
   method was selected over others (e.g., oral or intraperitoneal
   administration), especially in the context of pharmacokinetics and
   therapeutic efficacy.

   Response: We thank the reviewer for this important observation. In
   response, we have revised the manuscript and provided expanded
   justification for the use of the inhalation route in both the Methods
   and Discussion sections.

   Reviewer Comment 3: Expand on the bioinformatics analysis, particularly
   the selection of immune targets such as AKT1, TRPV1, and GPR55. Explain
   why these targets were chosen and their relevance to the
   neuroinflammatory processes in AD. A more detailed discussion of the
   interactions identified through the STRING database will also be
   useful.

   Response: We thank the reviewer for this valuable comment. In response,
   we have expanded both the Results and Discussion sections to provide a
   more detailed explanation of the rationale behind the selection of
   AKT1, TRPV1, and GPR55 as CBD-interacting immune targets. We have also
   elaborated on their relevance to neuroinflammatory processes in AD and
   provided a deeper interpretation of their interactions within the
   IDO/cGAS network based on STRING database analysis.

   Reviewer Comment 4: The conclusion would benefit from a mention of the
   next steps in your research, such as investigating CBD's effects in
   other AD models or evaluating long-term effects.

   Response: We thank the reviewer for this insightful suggestion. In
   response, we have revised the Conclusion section to include future
   research directions, including the evaluation of CBD's long-term
   effects and its therapeutic potential in additional AD models. These
   additions aim to strengthen the translational relevance of our findings
   and outline the next logical steps in this line of investigation.

   Reviewer Comment 5: The IDO and cGAS pathways are independent pathways
   and should be presented as such in the abstract to limit
   misinformation.

   Response: We thank the reviewer for this important clarification. In
   response, we have revised the Abstract to clearly state that the IDO
   and cGAS pathways are mechanistically distinct. We now explicitly note
   that these are independent but complementary immune pathways, both
   modulated by CBD in our study. The revised text aims to maintain
   scientific accuracy while preserving the overall flow and intent of the
   abstract.

   Reviewer Comment 6: Unclear if autoimmune disease is correct as this
   implies that the pathology is driven by autoreactive T and B cells,
   which they have not shown. Autoinflammatory disease would be a more
   correct interpretation of their data.

   Response: We thank the reviewer for this thoughtful clarification and
   have revised the manuscript to use autoinflammatory in place of
   autoimmune where appropriate.

   Reviewer Comment 7: It is unclear as to what they mean by "This aligns
   with the AD theory of AD" in the introduction.

   Response: We thank the reviewer for pointing this out. This was a
   phrasing error, and we have revised the sentence in the Introduction to
   clarify the intended meaning.

   Reviewer Comment 8: It is strange to group IFN-gamma with cGAS in the
   introduction as type I IFNs, and not IFN-gamma, are downstream of cGAS
   activation.

   Response: We thank the reviewer for this important clarification and
   apologize for the unintended implication. Our intent was not to
   categorize IFN-γ and cGAS as part of the same signaling axis, but
   rather to highlight their independent yet converging roles in
   neuroinflammation and their relevance to our study. IFN-γ was mentioned
   specifically for its role in inducing IDO expression, while cGAS was
   discussed as a separate innate immune sensor relevant to the activation
   of type I interferon responses. We have revised the whole segment in
   the Introduction to clarify this distinction and improve the accuracy
   of the immunological framing.

   Reviewer Comment 9: Tmem119 is actually downregulated by activated
   microglia and is not a good marker to track microglial responses in
   AD-related disease as most activated microglia around amyloid beta
   pathology do not express Tmem119 in full-blown disease.

   Response: We thank the reviewer for this important observation. We
   acknowledge that TMEM119 expression can be downregulated in highly
   activated microglia, particularly in proximity to amyloid plaques
   during late-stage AD. In our study, TMEM119 was used primarily to
   identify resident microglia populations for IDO co-localization rather
   than as a definitive marker of activation state. We have now clarified
   this point in the manuscript and have added a note in the limitations
   section.

   Reviewer Comment 10: Changes in total cGAS expression are not as
   informative as evaluating downstream cGAS signaling such as p-STING and
   p-TBK1. It is difficult to interpret anything meaningful from their
   cGAS imaging data.

   Response: We thank the reviewer for this thoughtful and constructive
   comment. We agree that downstream signaling components such as
   phosphorylated STING (p-STING) and TBK1 (p-TBK1) would provide a more
   direct and comprehensive assessment of cGAS pathway activation. In this
   study, our primary objective was to examine total cGAS expression in
   conjunction with IDO, highlighting what we believe to be a novel
   intersection of innate immune sensing and metabolic regulation in the
   context of AD. Our findings suggest that total cGAS expression is
   modulated by CBD treatment and may reflect upstream immune
   dysregulation. Nevertheless, we fully acknowledge the importance of
   evaluating downstream effectors of the cGAS-STING axis in future work
   to better define functional outcomes. We have added a statement in the
   Discussion to clarify this point and emphasize the need for further
   mechanistic investigation.

   Reviewer Comment 11: It is surprising that they see so much expression
   of IFN-gamma by macrophages/microglia in Figure 3 as it is widely
   thought that macrophages/microglia do not make IFN- gamma. Additional
   evidence in support of this is needed to bolster this potentially
   controversial claim by them.

   Response: We thank the reviewer for this excellent and insightful
   comment. We agree that it is not typical for microglia and macrophages
   to be major sources of IFN-γ under physiological conditions. However,
   under certain pathological and inflammatory states, including infection
   and neurodegenerative disease, there is evidence that these myeloid
   populations can express and produce IFN-γ within the CNS. Previous
   studies have demonstrated IFN-γ production by microglia and macrophages
   in the brain in response to inflammatory stimuli. For instance,
   Kawanokuchi et al. (2006) showed that activated microglia can express
   IFN-γ in vitro and in vivo in models of multiple sclerosis. Similarly,
   Suzuki et al. (2005) reported that both microglia and macrophages
   contribute to innate IFN- γ production following Toxoplasma gondii
   infection in the brain. In our AD model, it is possible that chronic
   neuroinflammation or amyloid-driven immune priming induces a heightened
   activation state in microglia and infiltrating macrophages, permitting
   expression of cytokines typically associated with lymphoid populations.
   Potential mechanisms may include local exposure to pro-inflammatory
   cytokines such as IL-12 and IL-18, known inducers of IFN-γ in myeloid
   cells under inflammatory conditions, or cross-talk with infiltrating
   lymphocytes within the neuroinflammatory milieu. While we acknowledge
   that additional functional validation would help strengthen this
   observation, we believe our findings align with emerging evidence that
   myeloid cells in the CNS may exhibit unexpected cytokine profiles in
   chronic neuroinflammatory settings. We have clarified this point in the
   Discussion and included supporting references to ensure appropriate