Table of Contents

Role of the STING-IRF3 Pathway in Ambient GABA Homeostasis and Cognitive Function

  1. Jin Bong Park1
  1. 1Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 08823, Korea
  2. 2Department of Medical Science, College of Medicine, Chungnam National University, Daejeon 35015, Korea
  3. 3Department of Molecular Biology, Dankook University, Cheonan 31116, Korea
  4. 4Departments of Microbiology, College of Medicine, Chungnam National University, Daejeon 35015, Korea
  5. 5Pharmacology, College of Medicine, Chungnam National University, Daejeon 35015, Korea
  6. 6Infectious Biology, College of Medicine, Chungnam National University, Daejeon 35015, Korea
  7. 7Department of Physiology, Eulji University School of Medicine, Daejeon 35233, Korea
  8. 8Department of Physiology, College of Medicine, Chungnam National University, Daejeon 35015, Korea

  1. Author contributions: C.N., R.S., F.F.G., E.-K.J., G-H.C., C.-S.K., and J.B.P. designed research; C.N., R.S., F.F.G., T.L.P., Y.S.K., K.-C.S., G.M.H., and G.-H.C. performed research; C.N., R.S., F.F.G., T.L.P., Y.S.K., B.-E.Y., K.-C.S., G.M.H., G.-H.C., S.S.M., and J.B.P. analyzed data; J.B.P. wrote the paper.

 
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Abstract

Targeting altered expression and/or activity of GABA (γ-aminobutyric acid) transporters (GATs) provide therapeutic benefit for age-related impairments, including cognitive dysfunction. However, the mechanisms underlying the transcriptional regulation of GATs are unknown. In the present study, we demonstrated that the stimulator of interferon genes (STING) upregulates GAT1 and GAT3 expression in the brain, which resulted in cognitive dysfunction. Genetic and pharmacological intervention of STING suppressed the expression of both GAT1 and GAT3, increased the ambient GABA concentration, and therefore, enhanced tonic GABAA inhibition of principal hippocampal neurons, resulting in spatial learning and working memory deficits in mice in a type I interferon-independent manner. Stimulation of the STING-GAT pathway efficiently restored cognitive dysfunction in STING-deficient mice models. Our study uncovered for the first time that the STING signaling pathway regulates GAT expression in a cell autonomous manner and therefore could be a novel target for GABAergic cognitive deficits.

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Significance Statement

GABA concentration in extracellular space is maintained by GABA release and clearance of GABA back to brain cells for degradation. GABA clearance from the synaptic cleft predominantly depends on the level and activity of GATs, and insufficient GABA clearance resulted in an aberrant tonic GABAA inhibition in the brain. In this study, we have identified an unusually high GABA content in the brain of STING-deficient mice, resulting in cognitive impairment. Our results show that STING regulates GAT expression through the STING-TBK1-IRF3 pathway and thus regulates GABAergic tone. This is the first study to indicate that the STING-TBK1-IRF3 signaling pathway maintains GABA homeostasis in the brain, which may offer a novel therapeutic target for modulating GABAergic tone in cases of cognitive dysfunction.

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Introduction

Stimulator of interferon genes (STING), also known as MITA and MPYS, is encoded by TMEM173. It is an endoplasmic reticulum (ER) adaptor that facilitates innate immune signaling (Ishikawa and Barber, 2008). When cyclic GMP-AMP synthase senses cytosolic aberrant DNA species or cyclic dinucleotides, it synthesize cyclic GMP-AMP (cGAMP). cGAMP triggers the translocation of STING from the ER membrane to the cytoplasm and recruits TANK-binding kinase 1 (TBK1), which phosphorylates transcription factors, including interferon regulatory factor 3 (IRF3), to induce several host defense genes that produce type I interferon (IFN-I) and cytokines (Barber, 2015; Dixon et al., 2021). IFN-I plays an important role in the synaptic plasticity and cognitive decline of healthy and neurodegenerative brains (Baruch et al., 2014; Hosseini et al., 2020). Undoubtedly, STING mutations cause cognition deficits in human subpopulations (Hamann et al., 2019, 2020). In addition to immune cell microglia, STING is highly expressed in astrocytes and neurons (Jeffries and Marriott, 2017; Abdullah et al., 2018; Donnelly et al., 2021). However, besides the prominent role of STING-IFN-I signaling, whether and, if so, how the STING-TBK1-IRF3 pathway affects the brain function in neurons and/or astrocytes is not known.

In the brain, γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter, generates two distinct modalities of inhibition in the brain via activation of GABA type A receptors (GABAARs): a fast, phasic, and classical inhibitory postsynaptic current (IPSC), and a tonic form of inhibition (Itonic; Semyanov et al., 2004; Farrant and Nusser, 2005). Itonic, which is primarily mediated by extrasynaptic GABAARs and is directly influenced by extracellular GABA, suppresses neighboring neuronal activity (S. Jo et al., 2014; Yoon and Lee, 2014; Shim et al., 2019; Heo et al., 2020; Pandit et al., 2020). The maintenance of GABA concentration in extracellular space is appropriately preserved by the balanced GABA synthesis and release and its clearance back to the brain cell for degradation. Altered Itonic due to elevated extracellular GABA concentration has been recognized in various brain diseases including cognitive dysfunction (Koh et al., 2023). For example, increased GABA synthesis and release from astrocytes cause aberrantly enhanced Itonic in Alzheimer's disease (AD; S. Jo et al., 2014; Z. Wu et al., 2014). GABA transporters (GATs) reuptake the extracellular GABA into the surrounding cells, thus limiting Itonic to maintain cognitive function in the normal brain. Thus, altered GAT expression is involved in cognitive decline (Banuelos et al., 2014) as well as in anxiety (G. X. Liu et al., 2007) and gait impairment (Chiu et al., 2005). Four subtypes of GATs, named GAT1–3 and betaine GAT, in rats and humans (corresponding in mice to GAT1, 3, and 4 and GAT2, respectively) have been identified (Borden, 1996). GAT1 and GAT3 bind to extracellular GABA and transport GABA to presynaptic neurons or astrocytes. However, the mechanisms underlying the transcriptional regulation of GAT are not known.

In the present study, we found a previously unappreciated role of the STING-TBK1-IRF3 signaling pathway in the transcriptional regulation of GAT expression, showing that STING-GAT is a IFN-I-independent therapeutic target in GABAergic cognitive deficits.

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Materials and Methods

Animal experiments

Animals were housed under diurnal lighting conditions and allowed food and tap water ad libitum. These experiments were approved by the Chungnam National University Animal Care and Use Committee (Registration Number: 2025-1-21). All procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH).

Male wild-type C57BL6/J (STING+/+) mice were purchased from DBL. STING knock-out C57BL6/J (STING−/−) mice (Ishikawa and Barber, 2008), received from Dr. Glen N Barber Laboratory (University of Miami, Miller School of Medicine), were inbred and used for experiments. Male STING heterozygous (STING+/−) derived from STING−/− mice crossing with C57BL6/J (STING+/+) were used for experiments. The age of mice used in this study was 6 weeks unless otherwise mentioned.

In vivo microdialysis

The surgical procedures were performed under 2% avertin anesthesia. Two days before the experiment, mice were surgically prepared for microdialysis of the hippocampus using stereotaxic implantation of a guide cannula (CMA7; Harvard Apparatus), as previously described (Woo et al., 2018). Additionally, anchor screws were placed inside the skull and fixed using zinc polycarboxylate dental cement. After the mice had recovered from anesthesia, a CMA7 microdialysis probe (membrane diameter, 0.24 mm; length, 1 mm; stainless steel shaft diameter, 0.38 mm) was implanted through the guide cannula. The probe was connected to a CMA100 microinjection pump (CMA Microdialysis) with polyethylene tubing (PE 50) and FEP tubing (INSTECH). Then, the probe was perfused with artificial cerebrospinal fluid (aCSF; in mM): 149 NaCl, 2.8 KCl, 1.2 MgCl2, 1.2 CaCl2, and 5.4 glucose, pH 7.4 into the probe inlet at a flow rate of 1.5 μl/min. The perfusates from the tubing outlet were collected in plastic vials in ice. Dialysates were collected at 20 min intervals for 4 h and used for measuring the glutamate and GABA levels from the second sample onward.

The microdialysate (injection volume, 30 μl) was assayed for GABA and glutamate using high-performance liquid chromatography (HPLC) coupled with a fluorescence detector (ESA model 5014). The mobile phase was 75 mM monobasic sodium phosphate, 0.1 mM ethylenediaminetetraacetic acid, 1.4 mM octane sulfonic acid, and 10% acetonitrile, adjusted to a pH of 3.2 using HPLC grade phosphoric acid. The monoamine metabolites were separated using a Waters Nova-Pak C-18 column (4 mm, 150 × 3.9 mm). The flow rate of the system was 0.8 ml/min. The GABA and glutamate levels in the dialysates are expressed as the average of three samples collected from the same mice.

Preparation and treatment of drugs

2′,3′-Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) sodium salt was first dissolved in distilled water (50 mM) and further diluted with normal saline. H-151 was diluted in PBS containing 5% dimethylsulfoxide (DMSO). cGAMP (2 µg/kg, QD for 14 d) or H151 (4 µg/kg for 12 d) or respective vehicles were intraperitoneally injected. The last injection was administered at least 12 h before behavioral test. L-655,708 was dissolved in DMSO (1 mg/ml) and frozen in storage aliquots. The working solution was diluted in normal saline. L-655,708 (1 mg/kg) or vehicles were intraperitoneally injected 30 min before the experiment. All three drugs were purchased from Tocris Bioscience. For the experimental groups treated with IFNAR antibody (IFNAR ab, clone MAR1-5A3, BioLegend), the IFNAR ab was administered to the mice at 50 µg per mouse (i.p., QD) for 10 d.

Electrophysiological recordings and data analysis

Coronal hippocampal slices (300 µm) were prepared to obtain electrophysiological recordings as previously described (Park et al., 2006; J. Y. Jo et al., 2011). Slices were perfused with aCSF (in mM): 126 NaCl, 2.5 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 glucose, 0.4 ascorbic acid, 1 CaCl2, and 2 pyruvic acid, pH 7.3–7.4, saturated with 95%O2–5%CO2 at a flow rate of ∼3 ml/min. Recordings were obtained at 30–32°C using a MultiClamp 700B or an Axopatch 200B amplifier (Molecular Devices). Currents were filtered at 1 kHz and digitized at 10 kHz (Digidata 1440A, pClamp 10 software).

For voltage-clamp experiments, patch pipettes were filled with a high Cl−-containing solution (in mM): 140 KCl, 10 HEPES, 5 Mg2+ATP, 0.9 MgCl2, and 10 EGTA. Spontaneous inhibitory postsynaptic currents (sIPSCs; recorded at −70 mV) were recorded in the presence of an ionotropic glutamate receptor antagonist, kynurenic acid (1 mM). sIPSCs were detected and analyzed using Mini Analysis (Synaptosoft). The holding current (Iholding) was measured in 50 ms epochs of traces lacking PSCs, separated by ∼800 ms, in periods of control aCSF and the presence of drugs and additional GABAA antagonists (n = 40 epochs in each case). The series resistance was monitored at the beginning and end of the experiments and data were discarded if changes >20% were observed.

Input–output (I–O) functions of DGGCs recorded in the current-clamp mode using patch pipettes filled with an internal solution containing the following (in mM):130 K-gluconate, 10 KCl, 10 HEPES, 5 Mg2+ATP, 0.9 MgCl2, and 10 EGTA were plotted as previously described (Neupane et al., 2021). Briefly, the number of evoked spikes versus injected current in response to depolarizing step pulses (of 10 pA from −50 to 60 pA, 1.2 s duration) was plotted and fitted by a Boltzmann function: y = 1 / {1 + exp[(Ih − I50) / k]}, in which y is the number of spikes at a given injected current (Ih), I50 is the current at which half of the maximal spikes are evoked, and k is the slope factor. The resting membrane potential was adjusted for −70 mV in all tested neurons. Drugs were added to the perfusing aCSF solution at known concentrations. The final concentration of DMSO was <0.05% when used to dissolve drugs. All drugs except nipecotic acid (NPA; Tocris) were purchased from Sigma-Aldrich.

Brain tissue collection

Hippocampal and cortical tissues were collected as previously described (Sultan, 2013). Briefly, mice were anesthetized using avertin and decapitated. Their brains were rapidly excised from the skull and transferred to the ice-cold 1× PBS. The brains were hemisected using blades and forceps. Both hemispheres were dissected to excise the hippocampus and cerebral cortex. Each half of the tissues was collected in separate Eppendorf tubes for Western blotting and RT-PCR. The tubes were stored at −80°C until further use. For each Western blot and RT-PCR experiment, at least three mice were used, and specific numbers are described in the respective figure legends.

Western blot

SDS-PAGE and Western blot analysis were performed to determine numerous protein expressions in samples from the brain tissue. The same concentration of proteins was loaded into the SDS-PAGE gel and separated by electrophoresis. Then proteins were transferred to the polyvinylidene difluoride membrane (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline including 0.1% Tween 20 (TBST) for 1 h at room temperature. After being washed twice in TBST for 5 min, membranes were incubated with indicated primary antibodies (1:1,000 diluted in 5% BSA supplemented with TBST) overnight at 4°C. Following three times washing with TBST, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody (1:2,000 diluted in 5% skim milk supplement with TBST) for 2 h at room temperature. After three times washing, the membranes were soaked with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore), and chemiluminescence was detected using a Fusion Solo System (Vilber Lourmat). Band intensity was quantified using ImageJ software (NIH).

RT-PCR

The total RNA from tissue samples was isolated using the TRIzol reagent (Invitrogen). The cDNA was generated using a M-MLV reverse transcriptase kit (Invitrogen Life Technologies) according to the manufacturer's instructions. PCR reactions were performed using a MyCycler (Bio-Rad) for 30 cycles. The primers were designed using the Primer 3 software. The mRNA sequences of the selected genes were obtained from the NCBI website. The primer sequences are summarized in Table 1. The amplified products were electrophoresed in 1.5% agarose gel and visualized using ethidium bromide. The mRNA was quantified using an imaging densitometer (Bio-Rad Laboratories).

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Table 1.

Primer sequences used for RT-PCR

Behavioral tests

For the behavioral tests, mice were brought to the behavioral test room and acclimatized for at least 30 min prior to testing, without the test apparatus being visible to mice. The Y-maze and open field tests were performed in the dimly lit sound-attenuating chamber.

Y-maze test

The Y-maze experiments were performed as previously described (Prieur and Jadavji, 2019) to assess the working and spatial memory. Briefly, the Y-maze apparatus consisted of three arms of acrylic gray plastic that were connected in the middle to form a “Y” shape. Each arm was 50 cm long, 12 cm wide, and 20 cm deep. The arms were interconnected at 120°. Briefly, mice were placed in the center of the maze and allowed to explore freely the three arms of the maze for 8 min. A video of the mice's movements was recorded. Data from mice that made at least 20 entries into the arms were considered valid. Additionally, mice that did not show exploratory activity for more than 2 min were rejected. The maze was cleaned using 60% ethanol-soaked paper after each mouse was tested and left in place for 10 min to allow evaporation of ethanol. Each arm was identified by a letter (A, B, or C). The sequence and number of arm entries were counted manually. The spontaneous alternations consisted of sequential entries into the three arms (e.g., the sequence ABCBCBCA was counted as two with the first sequential ABC and the last sequential BCA). Then, the percentage of spontaneous alternations was calculated by dividing the number of alternations by the number of possible alterations (number of alternations/total possible alternations). The total possible alternations were the number of total arm entries − 2.

Barnes maze test

The Barnes maze experiments were performed as previously described (Pitts, 2018) to assess the spatial learning and memory. Briefly, the Barnes maze consists of a circular table with a diameter of 90 cm and a height of 100 cm. Twenty 5-cm-wide holes are located at the perimeter of the table, at equal distances. Out of 20, one hole (the target hole) has an escape chamber (a black chamber attached underneath to the hole), which could be used by the animal to escape. A high illumination light was provided just over the center of the table to encourage the animal's motivation to search for the target hole. The experimental timeline was divided into habituation (1 d, one trial per animal) and training phase (4 d, three trials each day per animal). At the beginning of each trial (habituation as well as training phase), a loud buzzer sound (90 dB) was played using a loudspeaker to induce escape behavior. The buzzer sound was turned off immediately after the animals entered the escape chamber. Additionally, three visual cues were presented on the walls surrounding the Barnes table. During the habituation phase, which lasted for 1 d, the animal was placed in a black-colored cylinder (start chamber) at the center of the maze. After 10 s, the chamber was lifted up, and the mouse was pretrained to locate the target hole and enter the escape chamber by guiding it and to remain there for 3 min. In this phase, mice were allowed a single trial only. On the next day, the training trials were started. At the beginning of each trial, the mouse was placed in the same start chamber, and 10 s after the onset of a buzzer, the chamber was lifted, and the mouse was allowed to explore the maze freely. In the spatial acquisition phase, mice were given 3 min per trial to identify the target hole and enter the escape chamber. Mice that did not find the escape chamber were trained to enter the escape chamber by guiding it and to remain there for 1 min. Mice were trained using three trials per day for 4 d with an intertrial interval of 20 min. After each trial, a quarter of the maze table was rotated, and the entire maze, including the escape chamber, was cleaned using 60% ethanol-soaked paper and left to stand for 2 min for evaporation of ethanol. After completing the training for 4 d, we evaluated the primary latency (the latency to locate the target hole), escape latency (the latency to enter the escape chamber), primary errors (the number of incorrect holes checked prior to locating the target hole), and total errors (number of incorrect holes checked prior to entering the target hole).

Passive avoidance test

To test fear conditioning and memory retention, mice were subjected to passive avoidance test (Kumar et al., 2009). The passive avoidance chamber consisted of bright and dark compartments by a partition with an automated window with stainless steel rods on the floor connected to an electric shock generator. Before starting, the actual test mice were allowed to move freely in the chamber for 10 min for 2 consecutive days. On the first day of the test, mice were placed in a lighted compartment, after 30 s the window was raised automatically to allow the mouse to freely enter the dark chamber, and the latency to enter the dark compartment was recorded as accusation latency. When the mouse entered a dark chamber, the window immediately closed and an electric shock (0.5 mA, 2 s) was delivered and kept for 20 s before returning to the home cage. After 24 h each mousse was again placed in the light compartment. After 30 s, the window was raised automatically to allow the mouse to freely enter the dark chamber. The latency to enter the dark compartment was recorded as retention latency with a cutoff time of 300 s.

Open field test

The open field test was performed as previously described (Grailhe et al., 1999; Tatem et al., 2014; Chung et al., 2017; Jang et al., 2019) to assess spontaneous locomotor activity and anxiety-like behavior in mice. The open field box consisted of a square acrylic white box (40 × 40 cm). Mice were placed in the corner of the open field box and allowed to explore freely the arena for 1 h. Each test was videotaped from the top of the arena. The boxes were cleaned using 60% ethanol-soaked paper after each mouse was tested and left in place for 10 min to allow evaporation of ethanol. The total locomotor activity in the whole field (40 × 40 cm) and time spent in the center (20 × 20 cm) were analyzed using EthoVision® XT (Noldus).

Rotarod test

The motor learning skills were evaluated using an accelerating speed rotarod apparatus (Scitech Korea). The rotarod consisted of a cylindrical platform (5.7 cm wide, 3 cm diameter) suspended 16 cm above the bottom of the apparatus. On Day 1, mice were placed in the rod and kept for 1 min at a constant speed of 4 rpm to allow habituation to the system. From the next day, the mice were trained for 3 consecutive days using an accelerating speed of 4–40 rpm for 5 min. Each animal was allowed 10 trials per day with a time interval of 10 min. The latency to fall was recorded for each trial and averaged. After each trial, the rod was cleaned using 60% ethanol-soaked paper and left to stand for 2 min to allow evaporation of ethanol.

Experimental design and statistical analysis

Statistical analysis was performed using OriginPro 8.1 (OriginLab, RRID: SCR_002815). Numerical data are presented as mean ± standard error of the mean (SEM). Normality of data was determined using the Shapiro–Wilk test. The statistical significance of normally distributed data was determined between the groups with parametric tests: two-sample t test or ANOVA followed by a post hoc test (e.g., Bonferroni's post hoc test). In case data were not normally distributed, we used a nonparametric test: the Mann–Whitney test. The differences were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001). Animals or group sizes (n) were described in figure legends. Electrophysiological recordings were conducted in three or more animals in each group; and two to three slices were collected per animal. mRNA and protein expression levels were measured in three or more animals and were compared with STING+/+ or with vehicle-treated group. Behavioral experiments were conducted in six or more animals and were compared with STING+/+ or with vehicle-treated group.

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Results

Enhanced tonic GABAA inhibition impairs memory in STING−/− mice

In addition to its role in host defense against viral infections, IFN-I signaling is also important for synaptic plasticity and cognitive function (Hosseini et al., 2020), such as in AD. As an upstream regulator of IFN-I, STING may play a role in cognitive function. Here, we investigated whether alterations of the STING pathway affect cognitive function in an IFN-I-independent manner. The genetic deletion of STING impaired working and spatial memory, leading to a significant decrease in the spontaneous alternations in STING−/− mice compared with STING+/+ mice in the Y-maze test (STING+/+: 61.19 ± 0.95% and STING−/−: 48.21 ± 1.96%, p < 0.001, two-sample t test, n = 10 mice in each group, 6-week-old male mice; Fig. 1A). Working memory deficits were not different in 6-month-old male and 6-week-old male and female STING−/− mice (Fig. 1A). We used 6-week-old male mice in subsequent experiments to avoid the potential effect of physiological change on GABA-inhibitory mechanism in female mice (Jancke, 2018; Gilfarb and Leuner, 2022). To investigate whether IFN-I is involved in memory function, STING+/+ and STING−/− mice were treated with IFNAR blocking antibody (IFNAR ab) for up to 10 d. IFNAR ab minimally affected the spontaneous alternations in both STING+/+ (vehicle: 64.67 ± 1.63%; vs IFNAR ab: 64.72 ± 2.50%, p = 0.57) and STING−/− mice (vehicle: 49.56 ± 2.24%; vs IFNAR ab: 52.75 ± 1.97%, p = 0.10, Bonferroni’s post hoc test following two-way ANOVA; Fig. 1B). Noted that, in accordance with a previous report (Donnelly et al., 2021), IFNAR ab significantly increased basal pain sensitivity in STING+/+ mice but not in STING−/− mice (data not shown). However, IFNAR blocking antibody failed to affect the memory function in both STING+/+ and STING−/− mice, indicating that working and spatial memory deficits in STING−/− mice are IFN-I independent.

Figure 1.
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Figure 1.

Working and learning memory deficits in STING−/− mice. A, The percentage of spontaneous alternations in the Y-maze test in STING+/+ and STING−/− mice at 6 weeks and 6 months (n = 8–10 mice in each group). STING−/− mice exhibited sex and age-independent working memory loss (***p < 0.001, STING+/+ vs STING−/− mice, two-sample t test, n = 8–10 mice in each group). B, The percentage of spontaneous alternations after vehicle or IFNAR antibody (50 µg/mouse, i.p., QD for 10 d) treatment is summarized in the bar graph (n = 6 mice in each group). C–F, During the acquisition training for the Barnes maze test, primary latency, total latency, primary errors, and total errors progressively decreased over 4 d of training in STING+/+ and STING−/− mice. However, STING−/− mice showed delayed latency and an increased number of errors compared with STING+/+ mice (*p < 0.05, **p < 0.01, Bonferroni's post hoc test following two-way RM ANOVA, n = 10 mice in each group). G, Summarized bar graph from the passive avoidance test showing that fear-aggravated learning memory was significantly decreased in STING−/− mice (***p < 0.001, two-sample t test compared with STING+/+ mice, n = 10 mice in each group). H, I, Summarized result from open field test result showing that the total distance traveled (H) and time in center (I) did not differ between the STING+/+ and STING−/− mice (n = 10 mice in each group). J, The latency to fall in the rotarod test (10 trials per day for 3 consecutive days) is presented in the point graph (n = 10 mice in each group). All the summarized data are presented as mean ± SEM.

In addition, the Barnes maze test further showed significant spatial learning and memory dysfunction in STING−/− mice as indicated by differences in performance between STING+/+ and STING−/− mice in primary latency [F(1,72) = 11.30; p < 0.001], escape latency [F(1,72) = 9.07; p < 0.01], primary errors [F(1,72) = 11.30; p < 0.001], and total errors [F(1,72) = 9.20; p < 0.01, two-way RM ANOVA; Fig. 1C–F]. We also found that STING−/− mice had decreased fear conditioning shown by decreased latency to enter in the dark (STING+/+: 4.29 ± 0.26 min; vs STING−/−: 1.70 ± 0.29 min, p < 0.001, two-sample t test, n = 10 mice in each group) in passive avoidance test (Fig. 1G). Meanwhile, locomotor activity and anxiety in the open field test (Fig. 1H,I) and motor skill learning in the rotarod test (Fig. 1J) were unchanged. These findings suggest that cognitive dysfunction in STING−/− mice occurs independently of the type I IFN level and influences on locomotor, emotional, and motor activities.

Basal IFN-I signaling was downregulated in the CNS of STING−/− mice (Donnelly et al., 2021). Because the blockade of basal IFN-I signaling pathway affects cognitive function and increases expression of the astrocytic glutamate–aspartate transporter (Hosseini et al., 2020), the altered levels of ambient neurotransmitters may cause cognitive dysfunction in STING−/− mice. Thus, we used microdialysis to collect interstitial fluid samples from the hippocampal dentate gyrus of freely moving mice (Fig. 2A) and measured the levels of excitatory and inhibitory neurotransmitters. Glutamate levels were not significantly different in STING+/+ and STING+/− mice (STING+/+: 37.29 ±9.17 pM; vs STING−/−: 60.24 ± 17.90 pM, p = 0.22, two-sample t test, n = 10–14 mice in each group), while the GABA level was significantly higher in STING−/− mice than in STING+/+ mice (STING+/+: 1.01 ± 0.18 pM; vs STING−/−: 2.97 ± 0.82 pM, p < 0.05, Mann–Whitney test, n = 12–17 mice in each group; Fig. 2A). Our results suggest that cognitive dysfunction may be caused by enhanced GABAergic tone compared with the excitatory glutamatergic tone in STING−/− mice. In line with this, L-655,708, a partial inverse agonist of extrasynaptic GABAARs (Quirk et al., 1996), restored the spatial memory deficits in STING−/− mice (Fig. 2B,C). Spontaneous alternations were not affected in vehicle (saline)-injected mice in both groups and were comparable to that in baseline (baseline, STING+/+: 59.43 ± 1.15%; vs STING−/−: 48.33 ± 1.33%, p < 0.001, two-sample t test, n = 20–21 mice in each group). By contrast, L-655,708 treatment recovered the decreased spontaneous alternations in STING−/− mice (L-655,708 injection, STING+/+: 61.06 ± 1.58%; vs STING−/−: 57.61 ± 2.37%, p = 0.22, two-sample t test, n = 10–11 mice in each group), whereas it did not affect the performance of STING+/+ mice in the Y-maze test. Overall, our findings showed that increased GABAergic tone caused memory deficits in STING−/− mice.

Figure 2.
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Figure 2.

Increased GABA level and impaired cognitive function in STING−/− mice. A, Schematic illustration of microdialysis and summary plot for glutamate (bottom left panel; n = 10–14 mice in each group) and GABA (bottom right panel; *p < 0.05, STING+/+ vs STING−/− mice, Mann–Whitney test, n = 12–17 mice in each group) levels in the hippocampal microdialysate. B, Schematic illustration of the experimental timeline of the Y-maze test and injection of the GABAA receptor antagonist L-655,708 (1 mg/kg, i.p.). C, Summary plot for spontaneous alternations in STING+/+ and STING−/− mice before (baseline, ***p < 0.001, STING+/+ vs STING−/− mice, two-sample t test, n = 20–21 mice in each group) and after the injection of vehicle (saline, ***p < 0.001, or L-655,708; p = 0.270, STING+/+ vs STING−/− mice, two-sample t test, n = 10–11 mice in each group in both cases). D, Representative traces of sIPSCs from hippocampal DGGCSs of STING+/+ and STING−/− mice. E, Summary plots for sIPSC frequency and amplitude (n = 20–22 neurons from 7 to 8 mice in each group). F, Representative traces of averaged sIPSCs. G, Summary plot of decay time in STING+/+ and STING−/− mice (n = 20–22 neurons from 8 to 9 mice in each group). H, Representative traces of tonic current from hippocampal DGGCs of STING+/+ and STING−/− mice. I, Summary plot of tonic current in each group (***p < 0.001, STING+/+ vs STING−/− mice, two-sample t test, n = 8–9 neurons from 4 mice in each group). J, Representative traces of action potentials evoked by the depolarizing step current before and after the application of BIC (20 µM) in DGGCs of STING+/+ and STING−/− mice. The number of spikes was significantly increased in STING−/− mice after BIC application. Vm was maintained at −70 mV under all conditions. K, Mean number of spikes produced by the depolarizing step current before and after the application of BIC in STING+/+ and STING−/− mice. Blockade of tonic GABAA inhibition significantly shifted the I–O relationship curve to the left compared with controls (n = 10 neurons from 3 mice in each group).

Next, we compared the phasic and tonic GABAA currents of DGGCs in STING+/+ and STING−/− mice. The sIPSCs of DGGCs were blocked by bicuculline (BIC) in both strains. The main characteristics of sIPSCs, including their frequency, amplitude, and decay time, were not different between STING+/+ and STING−/− mice (Fig. 2D–G), suggesting that genetic deletion of STING has minimal effects on the basal synaptic GABAA transmission in DGGCs. These results exclude the possibility that GABA spillover from the synaptic IPSCs affects the ambient GABA level or tonic GABAA inhibition of DGGCs in STING−/− mice. However, the Itonic of DGGCs, measured as the change in the baseline current after application of BIC, a GABAA receptor antagonist, was significantly larger in STING−/− mice than in STING+/+ mice (Itonic, STING+/+: 8.14 ± 0.92 pA; vs STING−/−: 21.47 ± 2.69 pA, p < 0.001, two-sample t test, n = 7–9 cells from three to four mice in each group; Fig. 2H,I).

Next, we investigated the functional significance of increased GABAergic tone on synaptic efficacy in hippocampal DGGCs. The number of action potentials of DGGCs was determined as a function of the injected current at a holding potential of −70 mV in STING+/+ and STING−/− mice (Fig. 2J,K). The evoked firing rate was similar in the two groups as shown by the similar half-maximal current (I50; STING+/+, 43.90 ± 1.25 pA; vs STING−/−, 42.50 ± 2.15 pA, p = 0.22, Bonferroni’s post hoc test following two-way ANOVA, n = 7–8 neurons from three mice in each group). Bath application of BIC significantly sifted the I–O function curve to the left, which was larger in STING−/− mice than in STING+/+ mice (STING+/+: −5.38 ± 1.51 pA; vs STING−/−: −9.71 ± 1.23 pA, p < 0.05, two-sample t test; Fig. 2J,K), suggesting that enhanced GABAA inhibition suppresses the excitability of DGGCs more in STING−/− than in STING+/+ mice (Brickley et al., 1996).

GABAA receptor compositions were minimally affected in STING−/− mice

To investigate whether changes in extrasynaptic GABAARs (eGABAARs) increase Itonic in STING−/− mice, we recorded the Itonic after administration of 30 µM GABA and compared it between STING+/+ and STING−/− mice (Fig. 3A,B). After administration of 30 µM GABA, the high-affinity eGABAARs, but not synaptic GABAARs, are near-saturated (Mortensen et al., 2011). We found that an increased ambient GABA level after exogenous administration of 30 µM GABA eliminated the difference in Itonic between the groups (total Itonic; STING+/+: 58.27 ± 13.74; vs STING−/−: 63.98 ± 16.75, p = 0.76, two-sample t test, n = 8 neurons from three mice in each group), whereas IGABA was not significantly different between STING+/+ and STING−/− mice. These findings suggest that the increased ambient GABA level in STING−/− mice enhanced the Itonic level.

Figure 3.
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Figure 3.

GABAA receptors were not altered in STING−/− mice. A, Representative traces of tonic current in STING+/+ and STING−/− mice after exogenous GABA administration. B, Summary plot of basal and total tonic current (n = 8 neurons from 3 mice in each group). C, Representative current traces showing the effects on Iholding before and after the application of 1 µM THIP in the presence of 5 µM GABA in DGGCs of STING+/+ and STING−/− mice. D, Mean current amplitude of THIP-induced current and basal tonic currents are summarized in bar graphs (n = 8 neurons from 3 mice in each group). *p < 0.05; two-sample t test compared with STING+/+ mice.

Although we found an increased extracellular GABA level in STING−/− mice, Itonic may also be increased by changes in the composition of high-affinity eGABAARs in STING−/− mice. We found that the administration of THIP, a GABAAR δ-subunit agonist, generated similar Itonic in STING+/+ and STING−/− mice (ITHIP, STING+/+: −14.61 ± 1.49; vs STING−/−: −13.79 ± 1.76, p = 0.72, two-sample t test, n = 8–9 neurons from three to four mice; Fig. 3C,D). These results suggest that STING mutations have minimal effects on the composition of eGABAARs that generate the Itonic in DGGCs.

Overall, our results showed that an increased ambient GABA activated a greater number of eGABAARs, thus increasing Itonic in the hippocampus, leading to working and spatial memory deficits in STING−/− mice.

GAT expression was reduced in STING−/− mice

The balance between presynaptic GABA release and GABA reuptake by GATs determines the ambient GABA level in the normal hippocampus (Pandit et al., 2020). Considering that GABA spillover from the synaptic IPSCs does not increase the Itonic (Fig. 2D–G), altered GAT activity may affect the extracellular GABA concentration and subsequently influence the Itonic in STING−/− mice. To investigate this hypothesis, we compared the Itonic after the administration of GAT inhibitors between STING+/+ and STING−/− mice (Fig. 4).

Figure 4.
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Figure 4.

Itonic in STING−/− mice is augmented due to GATs. A, Schematic illustration of the target of GAT blockers. B, Representative traces of tonic current in STING+/+ and STING−/− mice after administration of the nonselective GAT inhibitor, NPA (100 µM). C, D, Summary plot of NPA-induced, INPA, (C) and total tonic current (D; n = 8–9 neurons from 3 mice in each group). E, Representative traces of tonic current in STING+/+ and STING−/− mice after sequential application of the GAT1 inhibitor, NO-711; 5 µM, and GAT3 inhibitor, SNAP-5114; 300 µM. F–H, Summary plot of GAT inhibitor-induced (INO-711 and ISNAP) and total tonic current (n = 8–9 neurons from 3 mice in each group). I, Representative image (left) of Western blot showing protein expression of STING-p-TBK1-TBK1-p-IRF3-IRF3-GAT1-GAT3 in the hippocampus of STING+/+ and STING−/− mice. Normalized protein expressions (right) are summarized in bar graph. J, Representative images of RT-PCR (top) showing the mRNA expressions of STING, GAT1, and GAT3 in the hippocampus. Normalized mRNA expressions (bottom) are summarized in bar graph. K, Representative image (left) of Western blot showing protein expression of STING-p-TBK1-TBK1-p-IRF3-IRF3-GAT1-GAT3 in the cerebral cortex from same group as in I (n = 3–4 mice in each group). Normalized protein expressions (right) are summarized in bar graph. L, Representative images of RT-PCR (top) showing the mRNA expressions of STING, GAT1, and GAT3 in the cerebral cortex. Normalized mRNA expressions (bottom) are summarized in bar graph. *p < 0.05, **p < 0.01, ***p < 0.001; two-sample t test, compared with STING+/+ mice.

The administration of NPA, a nonselective GAT inhibitor, created an inward current, INPA, in both groups (Fig. 4B), suggesting that GAT reversal is not the cause of enhanced Itonic. Thus, GAT did not function in the reverse mode, and the GAT inhibition prevented GABA reuptake into cells, leading to an extracellular GABA accumulation, although the reverse mode transport of GABA via the GATs is responsible for the tonic GABA release (Y. Wu et al., 2007). Importantly, NPA caused a significantly smaller Itonic in STING−/− mice compared with STING+/+ mice (INPA, STING+/+: −42.10 ± 4.82 pA; vs STING−/−: −17.67 ± 2.83 pA, p < 0.001, two-sample t test, n = 8–9 neurons from three to four mice; Fig. 4B–D), indicating that the augmented Itonic in STING−/− mice was due to the decreased GAT activity.

Similar results were observed with sequential administration of NO-711, a selective GAT1 inhibitor (Suzdak et al., 1992), and SNAP-5114, a selective GAT3 inhibitor (Borden et al., 1994; Fig. 4E–H). In the hippocampus, the regulation of basal Itonic by ambient GABA largely depends on the activity of the neuronal isotype of GAT, GAT1, with an additional role of the glial isotype of GAT, GAT3 (Kersante et al., 2013). The administration of NO-711 and SNAP-5114 created a significantly smaller Itonic in STING−/− mice than in STING+/+ mice (INO-711, STING+/+: −93.97 ± 17.81; vs STING−/−: −22.93 ± 5.73, p < 0.01 and ISNAP, STING+/+: −74.56 ± 20.91; vs STING−/−: −24.13 ± 6.45, p < 0.05, two-sample t test, n = 8–9 neurons from three to four mice in both cases). Taken together, these findings indicate that the augmented Itonic of DGGCs is a result of the attenuated GABA reuptake via GAT1 and GAT3 in STING−/− mice.

In subsequent experiments, we investigated whether the decreased GAT activity is a result of the decreased GAT1 and GAT3 protein levels. We found that GAT1 and GAT3 protein and mRNA expression were significantly decreased in the hippocampus of STING−/− mice compared with the STING+/+ mice (protein expression, GAT1: p < 0.05, GAT3: p < 0.01 and mRNA expression, GAT1: p < 0.001, GAT3: p < 0.001 compared with STING+/+ mice, two-sample t test, n = 3–4 mice in each group; Fig. 4I,J). Consistently, protein and mRNA expressions of GAT1 and GAT3 were significantly decreased in the cerebral cortex, of STING−/− mice, compared with the STING+/+ mice (protein expression, GAT1: p < 0.05, GAT3: p < 0.01 and mRNA expression, GAT1: p < 0.001, GAT3: p < 0.001 compared with STING+/+ mice, two-sample t test, n = 3–4 mice in each group; Fig. 4K,L).

During immune response, STING activates TBK1, which phosphorylates IRF3 to induce the transcription of genes encoding various peptides (Tanaka and Chen, 2012; S. Liu et al., 2015; Zhang et al., 2019). It is plausible to test whether the STING-TBK1-IRF3 pathway plays a role in regulating GAT expression in the brain. We found that the levels of phosphorylated TBK1 and IRF3 were significantly decreased in STING−/− along with GAT1 and GAT3 (protein expression, p-TBK1; p < 0.01 and p-IRF3; p < 0.05 compared with STING+/+ mice, two-sample t test, n = 3–4 mice in each group for both the hippocampus and cortex; Fig. 4I,K).

Overall, our results suggest that the attenuated GAT expression enhanced the Itonic in STING−/− mice and that the basal GAT expression is under the transcriptional regulation of STING activity and expression in the normal brain via the STING-TBK1-IRF3 pathway.

Activation of STING-GAT pathway restored cognitive impairment in STING-deficient mice

Our results suggest that impairment of the STING-TBK1-IRF3 pathway increased the Itonic level and cognitive dysfunction. In subsequent experiments, we used STING+/− mice to investigate whether the STING agonist restored cognitive dysfunction in a TBK1-IRF3-GAT-dependent manner.

First, we compared the basal protein expression and effects of cGAMP on the STING-GAT pathway between STING+/+ and STING+/− mice. The protein expressions of p-TBK1, p-IRF3, GAT1, and GAT3 were significantly decreased in STING+/− mice compared with STING+/+ mice (p < 0.05, Bonferroni's post hoc test following two-way ANOVA in all cases). The STING agonist cGAMP increased phosphorylation of TBK1 and IRF3 proteins, as well as the total protein expressions of GAT1 and GAT3, in STING+/+ and STING+/− mice (p < 0.01, Bonferroni's post hoc test following two-way ANOVA in all cases, n = 5 mice in each group for both the hippocampus and cortex; Fig. 5A–D), suggesting that STING activation enhanced the GAT1 and GAT3 expressions in the brain. As in protein expression, mRNA expressions of GAT1 and GAT3 were significantly less in STING+/− mice compared with STING+/+ mice (p < 0.05, Bonferroni's post hoc test following two-way ANOVA in both cases). cGAMP enhanced the GAT1 and GAT3 mRNA level of STING+/+ and STING+/− mice (mRNA expression, saline vs cGAMP, p < 0.05, Bonferroni's post hoc test following two-way ANOVA in all cases, n = 5 mice in each group for both the hippocampus and cortex; Fig. 5E,F).

Figure 5.
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Figure 5.

The STING agonist cGAMP restored the GABAergic tone in STING+/− mice. The STING+/+ and STING−/− mice were treated with saline or cGAMP (2 µg/mouse, i.p., QD for 14 d). A, B, Hippocampal (A) and cerebral cortex (B) lysates from the STING+/+ and STING+/− mice were immunoblotted with the indicated antibodies. C, D, Summary graph of the protein expression (n = 5 mice in each group). E, F, Representative image of RT-PCR showing mRNA expressions of GAT1 and GAT3 after saline or cGAMP treatment in STING+/+ and STING+/− mice (E). Normalized mRNA expressions of GAT1 and GAT3 are summarized in bar graphs (F; n = 5 mice per group). G, H, cGAMP restored the enhanced Itonic in STING+/− mice. Representative traces (G) and summary graph (H) for the tonic current in each condition (n = 8 neurons from 3 mice in each group). I, Summary graphs for spontaneous alternations in the Y-maze test (right panel; n = 10 mice in each group). *p < 0.05, **p < 0.01, ***p < 0.001; Bonferroni's post hoc test following two-way ANOVA.

In line with the altered GAT expression, Itonic of DGGCs in STING+/− mice was significantly increased compared with STING+/+ (STING+/+-saline: 6.98 ± 1.82 pA; vs STING+/−-saline: 13.86 ± 1.82 pA, p < 0.01, Bonferroni's post hoc test following two-way ANOVA; Fig. 5G, H). As expected, cGAMP reversed the augmented Itonic of DGGCs in STING+/− mice (saline: 13.86 ± 1.82 pA; vs cGAMP: 4.40 ± 0.94 pA, p < 0.001, Bonferroni's post hoc test following two-way ANOVA). Interestingly, cGAMP did not affect the Itonic of DGGCs in STING+/+ mice (saline: 6.98 ± 1.82 pA; cGAMP: 4.59 ± 0.62 pA, p = 0.94, Bonferroni's post hoc test following two-way ANOVA n = 8 neurons from three mice in each group; Fig. 5G, H). Additionally, the main characteristics of sIPSC did not differ between the groups (Table 2). The administration of cGAMP restored the working and spatial memory deficits, leading to increased spontaneous alternations in the Y-maze test, in STING+/− mice but not in STING+/+ mice (Fig. 5I).

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Table 2.

Properties of sIPSC in DGGCs from saline- and cGAMP-treated mice

The effects of cGAMP on spatial learning and memory were further investigated in the Barnes maze test (Fig. 6A–D). Learning memory deficiency in STING+/− mice was observed by an increased primary latency [F(15,112) = 9.94; p < 0.001], escape latency [F(15,112) = 7.12; p < 0.01], primary errors [F(15,112) = 15.74; p < 0.001], and total errors [F(15,112) = 9.03; p < 0.001 compared with STING+/+ mice, two-way RM ANOVA] in the Barnes maze test. cGAMP restored learning memory deficiency in STING+/− mice shown by decreased primary latency (p < 0.01), escape latency (p < 0.01), primary errors (p < 0.001), and total errors (p < 0.001, Bonferroni's post hoc test following two-way RM ANOVA compared with the saline-injected group). In contrast, cGAMP failed to affect the parameters in STING+/+ mice. As a result, all parameters were not different in cGAMP-injected STING+/+ and STING+/− groups (p > 0.9 in all four parameters; Fig. 6A–D), suggesting that cGAMP improved spatial learning and memory deficiency in STING+/− mice to normal STING+/+ mice level.

Figure 6.
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Figure 6.

The STING agonist cGAMP restored cognitive dysfunction in STING+/− mice. A–D, The Barnes maze test showed that the primary latency (A), total latency (B), primary errors (C), and total errors (D) progressively decreased over the 4 d of training in both saline- and cGAMP-treated mice. STING+/− mice showed delayed latency and an increased number of errors compared with STING+/+ mice (*p < 0.05,**p < 0.01; Bonferroni's post hoc test following two-way RM ANOVA compared with the saline-treated STING+/+ mice), meanwhile cGAMP treatment significantly improved the latencies and decreased the number of errors in STING+/− mice (#p < 0.05, ##p < 0.01, Bonferroni's post hoc test following two-way RM ANOVA compared with the saline-treated STING+/− mice). E, Summarized bar graph from the passive avoidance test showing that cGAMP significantly improved fear-aggravated learning memory deficits in STING+/− mice (*p < 0.05, **p < 0.01, Bonferroni's post hoc test following one-way ANOVA as indicated in graph, n = 8 mice in each group). F, G, Open field test showed that the total distance traveled (F) and time in center (G) were not different between the saline- and cGAMP-treated mice.

In the passive avoidance test (Fig. 6E), cGAMP significantly increased latency to enter in the dark room in STING+/− mice [F(3, 28) = 4.78; p < 0.05, Bonferroni's post hoc test following two-way ANOVA] but not in STING+/+ mice, suggesting that cGMAP also restored the reduced fear conditioning in STING+/− mice. Meanwhile, the locomotor activity and anxiety in the open field test were not affected by cGAMP in both groups (Fig. 6F,G).

Pharmacological inhibition of STING-GAT pathway impaired cognitive function

Our results suggest that restoration of the STING-GAT pathway normalized the enhanced Itonic and, in turn, impaired learning and memory in STING-deficient mice. Similar results were observed for mice with pharmacologically induced STING deficiency (Fig. 7). A selective STING inhibitor, H-151 (Haag et al., 2018), induced cognitive impairment in control STING+/+ mice [F(5,42) = 9.45; p < 0.01; Fig. 7A]. A significant working and spatial memory deficit in the Y-maze test was observed at 12 d (12D-PI: spontaneous alteration percentage, vehicle: 61.64 ± 1.64; vs H151: 51.86 ± 1.61, p < 0.001, Bonferroni's post hoc), but not at 7 d (7D-PI: vehicle: 60.78 ± 2.63; vs H151; 55.66 ± 0.95, p = 0.10, Bonferroni's post hoc test, n = 7–10 mice in each group), after H-151 injection (Fig. 7A).

Figure 7.
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Figure 7.

STING antagonist H-151 mimics the altered GABAergic tone and cognitive dysfunction in STING+/+ mice. A, Summary graphs for spontaneous alternations after H-151 treatment (4 µg/kg, i.p., QD, n = 8–10 mice in each group). Spontaneous alternations in the Y-maze test were recorded at Postinjection Day 7 (7D-PI) and 12 (12D-PI). B, C, H-151 attenuated the Itonic in STING+/+ mice. Representative traces (B) and summary graph (C) for tonic current in each condition (n = 6 neurons from 3 mice for 7D-PI and n = 8–10 neurons from 4 mice for 12D-PI in each group). D, Representative image (left) of Western blot showing protein expression of STING-p-TBK1-TBK1-p-IRF3-IRF3-GAT1-GAT3 in the hippocampus of saline- and H-151-injected groups. Normalized protein expressions (right) are summarized in a bar graph. E, Representative image (left) of Western blot showing protein expression of STING-p-TBK1-TBK1-p-IRF3-IRF3-GAT1-GAT3 in the cerebral cortex from the same group as in H (n = 3–4 mice in each group). Normalized protein expressions (right) are summarized in a bar graph. F, G, Representative images of RT-PCR showing the mRNA expressions (left) and summary graph (right) for the mRNA level of STING, GAT1, and GAT3 in the hippocampus (F) and cerebral cortex (G). mRNAs were measured using semiquantitative PCR analysis from the same group as in A and B. H, Summary graphs for spontaneous alternations after an additional GABAAR antagonist L-655,708 treatment to H-151 injected mice (n = 10 mice in each group). *p < 0.05, **p < 0.01, ***p < 0.001; two-sample t test compared with vehicle injected STING+/+ mice.

In line with the time-dependent changes in working and spatial memory, H-151 significantly increased the Itonic of DGGCs in the 12D-PI group (vehicle: 8.35 ± 1.02; vs H151: 14.01 ± 2.49, p < 0.01, two-sample t test, n = 8–10 neurons from three to four mice in each group), with minimal changes in the 7D-PI group (p = 0.34, two-sample t test, n = 6 neurons from three mice in each group; Fig. 7B,C). Meanwhile, H-151 minimally affects the sIPSC characteristics of DGGCs (Table 3). In addition, H-151 decreased the GAT1 and GAT3 protein expression levels in the hippocampus and cortex. Consistently, H-151 suppressed the phosphorylation levels of TBK1 and IRF3 at 12D-PI in the hippocampus and cerebral cortex, resulting in decreased GAT1 and GAT3 expression (protein expression, p-TBK1: p < 0.001, p-IRF3: p < 0.01, GAT1: p < 0.01 and GAT3: p < 0.05 compared with STING+/+ mice, two-sample t test, n = 3–4 mice in each group for both the hippocampus and cortex; Fig. 7D,E). H-151 also suppressed GAT1 and GAT3 mRNA levels in the brain (mRNA expression, GAT1: p < 0.01, GAT3: p < 0.01 compared with STING+/+ mice, two-sample t test, n = 3–4 mice in each group for both the hippocampus and cortex; Fig. 7F,G). Finally, L-655,708 reversed the working and spatial memory impairment and resulted in increased spontaneous alternations in H-151-treated STING+/+ mice (spontaneous alteration percentage, vehicle; 51.54 ± 2.41%; vs L-655,708; 63.39 ± 2.35%, p < 0.001, two-sample t test, n = 7–10 animals in each group; Fig. 7H).

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Table 3.

Properties of sIPSC in DGGCs from the vehicle- and H-151-treated mice

Using the Barnes maze test, H-151-induced learning memory deficiency was confirmed in the 12D-PI group (Fig. 8A–D). As observed in genetically modified STING-deficient mice, STING inhibition by H-151 significantly decreased the primary latency [F(1,64) = 11.27; p < 0.001], escape latency [F(1,64) = 7.78; p < 0.01], primary errors [F(1,64) = 11.61; p < 0.001], and total errors [F(1,64) = 8.58; p < 0.01 compared with saline group, two-way RM ANOVA] at 12D-PI. H-151 also decreased fear conditioning as shown by the decreased latency to enter in dark (vehicle: 4.35 ± 0.26 min; vs H-151: 2.65 ± 0.44 min, p < 0.001, two-sample t test, n = 8 mice in each group) in passive avoidance test (Fig. 8E). However, STING inhibition by H-151 did not affect the locomotor activity and anxiety in the open field test (Fig. 8F,G).

Figure 8.
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Figure 8.

Pharmacological inhibition of STING impaired spatial learning and memory in STING+/+ mice. A–D, The Barnes maze test showed that the primary latency (A), total latency (B), primary errors (C), and total errors (D) progressively decreased over the 4 d of training in both vehicle- and H-151-treated mice. However, H-151 significantly delayed the latencies and increased the number of errors compared with the vehicle-treated mice. E, Summarized bar graph from the passive avoidance test showing that fear-aggravated learning memory was significantly decreased H-151-treated mice. F, G, Open field test showed that the total distance traveled (F) and time in center (G) were not different between the vehicle- and H-151-treated mice. Summarized data are shown as means ± SEM. *p < 0.05, **p < 0.01, Bonferroni's post hoc test following two-way RM ANOVA compared with the vehicle-treated group (A–D) and two-sample t test (E); n = 8–9 mice in each group.

Taken together, our results showed that STING inhibition by H-151 induced learning and memory dysfunction, which is independent of locomotor activities or anxiety-like behavior as in STING-deficient mice.

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Discussion

In the present study, we found a role of the noncanonical STING-IRF3 signaling pathway in cognitive function. The STING-TBK1-IFR3 signaling pathway acts as a gatekeeper of the innate immune system and transcriptionally regulates the expression of neuronal and glial GATs, thus maintaining the homeostasis of tonic GABAA inhibition in the hippocampus and, in turn, the normal learning and memory functions. Pharmacological and genetic modifications of STING impaired spatial learning and memory in a TBK1-IRF3-GAT activity-dependent manner. Although the altered tonic GABAA inhibition in various neurological diseases is under the tight control of GAT activity and/or expression, the molecular mechanisms of regulation of GAT expression are unknown. To the best of our knowledge, this is the first study to identify the STING-TBK1-IRF3-GAT signaling cascade as a novel pathway that connects the innate immune pathway to maintain GABAergic tone in the brain, which is particularly important for cognitive function.

STING-IRF3 pathway regulating GAT expression and cognitive function in the brain

The STING-TBK1-IRF3 pathway triggers IFN-I production, which plays a crucial role in innate immune responses, and, thus, has emerged as a key player in the CNS functions. Despite the relatively low baseline of IFN-I level in the CNS, its blockade affects CNS functions, such as cognitive function (Hosseini et al., 2020) and pain sensitivity (Donnelly et al., 2021). Because the activation of IFN-I signaling involves the recognition of IFN-α and IFN-β by their cognate receptors, it is interesting that the synapse number and synaptic plasticity related to spatial learning memory are impaired in the hippocampus of healthy IFNAR-deficient mice (Hosseini et al., 2020). In the present study, we did not detect altered synaptic GABA transmission in the STING-deficient mice (Fig. 2), although further studies are needed to determine whether there may be subtle differences in the synapse number or synaptic plasticity in STING-deficient mice with their cognitive dysfunction. However, blocking of IFN-I receptors using a type I IFNR antibody did not alter working and spatial memory functions in STING+/+ and STING−/− mice in the present study (Fig. 1B). Importantly, STING deficiency induced aberrant tonic GABAA inhibition mediated by extrasynaptic GABAARs in the hippocampus (Fig. 2), and the attenuation of aberrant tonic GABAA inhibition improved the spatial memory impairment in STING-deficient models (Fig. 5). Overall, these results showed that, in addition to the IFN-I signaling, STING-IRF3 signaling cascade facilitates tonic GABAA inhibition in the normal brain physiology, thus shedding light on a new path for developing novel therapeutics for learning and memory dysfunction.

STING-IRF3 pathway regulating GAT expression in nonimmune cells

As the primary producers of IFNs-I, microglia produce an antiviral IFN-I response. In an agreement, the physiological expression of STING is enriched in microglia compared with astrocytes and neurons (Reinert et al., 2016). However, in addition to the primary immune cell microglia and the immune effector cell astrocytes, “nonprofessional” immune cell neurons are involved in the STING-IRF3 system. Although microglia confer STING-dependent antiviral signals to neurons and astrocytes during viral infections (Nazmi et al., 2012; Reinert et al., 2016), neuronal STING regulates the immune cell-mediated inflammatory signaling following traumatic brain injury (Abdullah et al., 2018; Sen et al., 2020). Furthermore, neuronal STING regulates the sensory input from peripheral nociceptors via IFNs-I signaling (Donnelly et al., 2021) in dorsal root ganglion neurons without any infection or inflammatory stimuli. In the present study, our results showed that STING is involved in the transcriptional regulation of both neuronal (GAT1) and astrocytic GAT subtype (GAT3) in the normal brain. These results, for the first time, demonstrate that the innate immune STING-IRF3 signaling pathway in nonimmune cells is involved in the brain function, in which altered GAT expression causes aberrant GABAA tonic inhibition in the brain diseases including cognitive dysfunction.

Of the four GAT subtypes (Kristensen et al., 2011), GAT1 plays the most significant role in normal brain physiology. Thus, GAT1−/− mice exhibited impaired hippocampus-dependent learning and memory (Gong et al., 2009) with increased tonic GABAA inhibition in the brain. Although it is expressed in oligodendrocytes (Fattorini et al., 2017), CNS immune cell microglia (Fattorini et al., 2020), and peripheral macrophages (Wang et al., 2008), GAT1 is predominantly a neuronal GAT, which is particularly abundant in the brain regions rich in GABAergic neurons, such as the cerebellum, hippocampus, and neocortex (Guastella et al., 1990; Chiu et al., 2002). In the present study, we found that the STING-IRF3 pathway transcriptionally regulates GAT expression in the brain, the tonic GABAA inhibition in the hippocampal neurons, and in turn, memory. Combined with the result that the working and spatial memory dysfunction was independent on IFN-I receptors (Fig. 1B), these results showed that learning and memory are tightly controlled by the neuronal STING-IRF3 pathway, which transcriptionally regulates GAT expression and activity to maintain the GABAergic homeostasis in the normal brain.

Functional significance of the STING-GAT pathway in brain diseases

Given that TBK1 phosphorylation is essential in the STING-IRF3 pathway, it is interesting that the conditional neuronal deletion of TBK1 leads to cognitive and locomotor deficits in mice (Duan et al., 2019). Furthermore, episodic memory loss and disorientation in time and/or space are common early symptoms in carriers of a pathogenic TBK1 mutation (Van Mossevelde et al., 2016), resulting in a diagnosis of AD in some carriers (Pottier et al., 2015; Van Mossevelde et al., 2016). These results suggest that altered TBK1 activity contributes to cognitive impairment and locomotor deficits. Similarly, TBK1 gene mutations cause frontotemporal dementia (FTD; Le Ber et al., 2015; Freischmidt et al., 2017; van der Zee et al., 2017). Haploinsufficiency of TBK1 or loss of interaction of the C-terminal TBK1 coiled-coil domain with its adaptor proteins can cause FTD and familial amyotrophic lateral sclerosis (ALS; Freischmidt et al., 2015). Combined with our finding that downregulation of the STING-TBK1-IRF3 pathway causes cognitive deficits, these results suggest that a possible role for TBK1 activation as a linker in the STING-IRF3-GAT pathway could be a potential target for cognitive impairment in neurodegenerative diseases. However, TBK1 activation is a gatekeeper of neuroinflammation and is related to the IFN pathway in various neurodegenerative diseases, including AD and ALS/FTD. The activation of the STING signaling pathway in the brain may be detrimental or neuroprotective, depending on the inflammatory state.

It has been reported that the IFN-I initiated by the STING signaling pathway inhibits synaptic efficacy, which may be a novel pathophysiological mechanism underlying the memory impairments in the aging brain. In addition to their role in the normally aged brain, IFNs cause neuroinflammation and synapse loss in neurodegenerative diseases, such as AD (Roy et al., 2020). Recent studies have consistently shown that abnormal tonic GABAA inhibition in hippocampal neurons can disturb learning and memory function in AD (S. Jo et al., 2014; Z. Wu et al., 2014). The effects of aberrant tonic GABAA inhibition in cognition function are not limited to AD brain. The detrimental effect of aberrant tonic GABAA inhibition in hippocampal neurons inducing cognitive dysfunction has been reported in rheumatoid arthritis mouse model (Won et al., 2022) and mouse model of social deprivation (Cheng et al., 2023) as well. The maintenance of ordinary GABA content in extracellular space relies on effective GABA clearance by GATs. Thus, altered GAT3 expression and/or activity contributing to the aberrantly enhanced tonic GABAA inhibition induced memory impairments in neurodegenerative diseases such as Parkinson's disease and AD (Z. Wu et al., 2014; Chazalon et al., 2018). Furthermore, GATs release GABA from the neurons or reactive astrocytes in the pathophysiological conditions, including AD, while they reuptake GABA into the cells in the normal forward mode. Thus, GATs are involved in a far more complex regulation of tonic GABAA inhibition and/or cognitive function than expected from only an increase in their expression in the AD brain. For example, it is interesting to note that the STING agonist cGAMP improved cognitive function, with increased Aβ clearance and microglia polarization in APP/PS1 mice (Xu et al., 2019). In the present study, our results showed that, upon activation, the STING-IRF3 signaling pathway upregulates GATs to suppress tonic GABAA inhibition and improve cognitive dysfunction. Overall, these results are in agreement with that innate immunity should be boosted to diminish the neurodegenerative disease progress in AD. Further studies are required to determine whether the STING-GAT signaling pathway could be a novel pathophysiological mechanism underlying learning and memory impairments in neurodegenerative diseases, including AD.

Neurotransmitters provide the molecular basis for integrated, bidirectionally coordinated neuroimmune responses to homeostatic disturbance induced by stress, inflammation, or infection (Miller and Raison, 2016). In the present study, we found that the STING-TBK1-IRF3 pathway plays a central role in neuronal and astrocytic GAT expression, which is involved in the GABA homeostasis of the brain for cognitive function. To the best of our knowledge, this is the first study to demonstrate that GABA is one of the “biochemical languages” integrating the immune system into the CNS even in the absence of infection and inflammation, for normal brain function in the context of E/I balance.

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Footnotes

    • Received September 24, 2023.
    • Revision received August 12, 2024.
    • Accepted August 22, 2024.

  • We thank Dr. Glen N. Barber for providing the STING−/− mice. This work was supported by the National Research Foundation of Korea to J.B.P. (2023R1A2C100524812) and Ascending SNU Future fellowship through Seoul National University to R.S. (550-20240062).

  • ↵*C.N., R.S., and F.F.G. contributed equally to this work.

  • The authors declare no competing financial interests.

  • C.N.’s present address: Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095-1751; F.F.G.’s present address: Stem Cell Research and Cellular Therapy Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524000, China; T.L.P.’s present address: Department of Histopathology, Hai Phong University of Medicine & Pharmacy, Hai Phong 042-12, Vietnam

  • Correspondence should be addressed to Jin Bong Park at jbpark{at}snu.ac.kr.
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  1. Published online before print 3 September 2024, doi: 10.1523/JNEUROSCI.1810-23.2024 The Journal of Neuroscience, 44(41): e1810232024
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ORCID

  1. Profile for Neupane, C.https://orcid.org/0000-0003-2719-9331
  2. Profile for Sharma, R.https://orcid.org/0000-0002-8563-6109
  3. Profile for Yoon, B. E.https://orcid.org/0000-0002-1506-2437
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