Infralimbic Cortex Microglial TREM-1 Mediates Neuroinflammation and Exacerbates Depressive-Like Behaviors in Parkinson’s Disease Model Mice

Abstract

Depression is a common nonmotor feature of Parkinson’s disease (PD) that severely compromises the quality of life of patients, yet its pathogenesis remains elusive. Triggering receptor expressed on myeloid cells 1 (TREM-1) is an immunoglobulin family receptor present on myeloid cells that amplifies neuroinflammatory cascades. However, the contribution of TREM-1 to the depressive-like behaviors associated with PD remains unclear. In a subacute model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) administered at a dose of 30 mg/kg/day for five consecutive days, we evaluated depressive-like behaviors and the expression of microglial TREM-1 in the infralimbic cortex (IL) on Days 3, 7, 14, and 21 following the final MPTP injection. Microglial TREM-1 expression in the IL peaked on Day 14, which coincided with the peak severity of depressive-like behaviors. Both genetic knockout and pharmacological blockade of TREM-1 attenuated proinflammatory cytokines production and reversed depressive-like behaviors. Together, these findings suggested that TREM-1 is a pivotal mediator of microglia-driven neuroinflammation and depression in PD model mice, underscoring its potential as a therapeutic target for nonmotor symptoms.

Introduction

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder worldwide [1], primarily characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and pathological aggregation of α-synuclein [2–4]. While motor deficits are predominant in clinical diagnoses, nonmotor symptoms, including cognitive impairment, autonomic dysfunction, and depression, substantially impair quality of life and often precede the onset of motor symptoms [5–7]. Depression affects approximately 40% of PD patients and is associated with accelerated disease progression and poor therapeutic outcomes. However, the underlying mechanisms driving PD-related depression remain largely elusive [8, 9].

Neuroinflammation has emerged as a central player in the pathophysiology of PD, with microglia, the resident immune cells of the central nervous system, serving as key effectors [10]. Upon sensing pathological stimuli, activated microglia release proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which exacerbate neuronal damage and synaptic dysfunction [11]. Although microglial activation in the SNpc contributes to dopaminergic neurodegeneration, increasing evidence suggests that region-specific microglial responses, particularly in emotion-related cortical regions, may underlie nonmotor symptoms such as depression [12, 13].

The medial prefrontal cortex (mPFC) plays a critical role in emotional regulation and stress responses [14]. Within the mPFC, the infralimbic cortex (IL) has garnered particular attention for its sensitivity to antidepressant interventions and its involvement in mood regulation [15]. Recent studies have shown increased microglial reactivity in the mPFC in models of depression [12]. However, the molecular drivers of IL microglial activation in PD-associated depression remain undefined, and it is unclear whether modulating this process could alleviate affective symptoms.

In the central nervous system (CNS), microglia exhibit remarkable heterogeneity and adopt a spectrum of activation states in response to diverse cues. Within this broader framework, microglial activation was historically simplified into the “M1-like” and “M2-like” phenotypes: M1-like cells produce IL-1β, IL-6, TNF-α and iNOS to amplify inflammation and neuronal injury, whereas M2-like cells secrete IL-10, TGF-β and arginase-1 to promote repair and resolution of inflammation [16, 17]. Although this binary paradigm is still noted in the literature, accumulating evidence indicates that it oversimplifies the highly dynamic and condition-specific nature of microglial states and functions, In this context, triggering receptor expressed on myeloid cells 1 (TREM-1) is predominantly associated with M1-like proinflammatory activation, while its paralog triggering receptor expressed on myeloid cells 2 (TREM-2) is linked to M2-like anti-inflammatory processes and phagocytosis [18, 19].

Mechanistically, TREM-1 amplifies inflammatory responses via the SYK/CARD9/NF-κB pathway and the NLRP3 inflammasome [20], and its upregulation has been implicated in stroke and Alzheimer’s disease (AD) [21, 22]. In PD models, peripheral monocyte-derived TREM-1 exacerbates dopaminergic injury in the SNpc [23]. However, whether microglial TREM-1 in the IL drives depressive-like behaviors in PD remains largely unexplored.

In the present study, we utilized a subacute 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP) model of PD to investigate whether microglial TREM-1 in the IL mediates neuroinflammation and contributes to depressive-like behaviors. Through a combination of longitudinal behavioral assessments, targeted microglial depletion, genetic ablation of TREM-1, and pharmacological inhibition, we provide evidence that TREM-1 is a key regulator of microglia-driven inflammation in the IL and a critical contributor to affective dysfunction in PD model mice. These findings establish a mechanistic link between innate immune activation and nonmotor symptoms in PD and identify TREM-1 as a promising therapeutic target.

Materials and Methods

Animals

Male wild-type (WT) C57BL/6J mice (8 weeks old, weighing 25–30 g) and TREM-1 knockout (KO) mice (B6/JGpt-TREM-1^em1Cd60266026/Gpt) on the C57BL/6 background were procured from Gempharmatech Corporation (Nanjing, China). The animals were maintained at a temperature of 22 ± 1 °C and a humidity level of 50 ± 10% under a standard 12-hour light/dark cycle, and they were housed in groups of 6 to 8 mice per cage with free access to food and water. All the experiments and analyses were conducted in a double-blinded manner. Morphological and immunofluorescence analyses were performed with sample sizes ranging from 3 to 6 mice, whereas the behavioral experiments involved a total of 8 mice in each case. All procedures complied with the guidelines set forth by the National Institutes of Health for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee of Xuzhou Medical University.

Drugs and Treatments

For the subacute MPTP model, MPTP (MedChemExpress, Shanghai, China) was dissolved in 0.9% saline and administered via daily intraperitoneal injections at a dosage of 30 mg/kg/day for five consecutive days. The control group received an equivalent volume of saline only [24]. The TREM-1 inhibitory peptide (LP17: LQVTDSGLYRCVIYHPP) and scrambled control peptide (TDSRCVIGLYHPPLQVY) were chemically synthesized as previously reported (GenScript, Nanjing, China). On Day 10 following the final MPTP injection, the mice were treated with 20 mg/kg peptide in 10 µl of saline by intranasal administration. For the microglial depletion experiments, the mice were subjected to stereotactic injection of 1 µl of clodronate liposome (CLP) to deplete microglia or PBS liposomes.

Antibodies and Chemicals

Behavioral Tests

Rotarod Test

Prior to the experiment, the mice underwent rotarod acclimatization consisting of three daily training sessions (30 min/session). During the formal assessment, the mice were placed in individual compartments of an accelerating rotarod apparatus. The protocol began at a baseline speed of 4 rpm and increased linearly to 40 rpm over a duration of 300 s. The latency to fall from the rod was recorded, with a maximum trial duration of 300 s.

Pole Test

The pole test was used to evaluate the severity of bradykinesia in the mice. Two days prior to model establishment, the mice were trained to descend accurately from the top to the bottom of a self-constructed vertical rod measuring 60 cm in length and 1 cm in diameter. During the testing phase, the time taken for each mouse to reach the bottom of the pole was recorded. Each animal underwent three consecutive trials, with a 5-minute interval between trials. The average time from the three trials was subsequently calculated for statistical analysis.

Open Field Test

The open field test (OFT) was performed to assess locomotor activity as previously described [25]. Briefly, the mice were placed individually in a 50 cm × 50 cm × 50 cm open-top box and allowed to move freely for 10 min under bright illumination. Between each test, all surfaces were thoroughly cleaned with 75% ethanol to eliminate any residues. The behavior of the mice was recorded using the ANY-maze video tracking system, and the total distance traveled was measured.

Sucrose Preference Test

The sucrose preference test (SPT) is widely utilized to assess anhedonia in mice. The mice were housed individually during the experiment. On the first day, the mice were provided with two identical bottles containing tap water. On the second day, one of the water bottles was replaced with a bottle filled with a 1% sucrose solution. The positions of the two bottles were swapped on the third day to control for side preference. Following the training period, the mice were deprived of food and water for 24 h. The mice were subsequently presented with two bottles—one containing the 1% sucrose solution and the other containing tap water. The positions of the bottles containing drinking water or the sucrose solution were assigned randomly before the experiment. After 12 h, the positions of the bottles were swapped again. The volume of liquid consumed from each bottle was measured after 24 h. Sucrose preference was calculated as the change in weight of the sucrose solution divided by the total change in weight of the two bottles. The sucrose preference ratio was determined using the following equation: sucrose preference ratio (%) = sucrose intake/(sucrose intake + water intake) × 100% [26].

Forced Swimming Test

The forced swim test (FST) was conducted in a plastic cylinder. Prior to the experiment, the water temperature in the cylinder was maintained between 23 °C and 25 °C, and the water depth was calibrated according to the body weight of the animals, ensuring that their tails remained at a safe distance from the bottom of the cylinder. The mice were considered immobile when they ceased struggling and passively floated, with their heads above the water [27]. The forced swimming session lasted for 6 min, with immobility recorded during the final 4 min.

Tail Suspension Test

Mouse tails were immobilized using adhesive tape and suspended 50 cm above the platform. Behavioral recording commenced following a 120-second acclimatization period. Immobility was defined as passive hanging without struggling. The duration of immobility during the last 4 min of the 6-minute period was recorded. For ethical compliance, two blinded observers performed real-time monitoring and immediately terminated the experiment upon signs of distress.

Stereotactic Injections

The mice were anesthetized with 1% pentobarbital sodium (60 ml/kg) and subsequently secured in a stereotactic apparatus (68046, RWD Life Science). CLP (5 mg/ml, 1 µl/site, Sigma, USA) and PBS liposomes were injected bilaterally into the IL using coordinates relative to the bregma as follows: (AP: +2.0 mm; ML: ±0.30 mm; DV: −3.30 mm). Following the injection, the mice were allowed to rest for 10 min to ensure complete diffusion of the solution. Postoperatively, the mice were maintained under thermal support at 37 °C for 1 h, followed by surgical wound closure with 6‒0 nylon sutures and topical antibiotic ointment.

Western Blot Analysis

Following deep anesthesia with 3% isoflurane, the mice were decapitated using sharp surgical scissors, and the substantia nigra and infralimbic cortexes were dissected on ice-cold platforms. The tissue samples were flash-frozen in liquid nitrogen within 30 s of extraction before being stored at −80 °C. The brain tissue was thoroughly homogenized and lysed in RIPA tissue lysis buffer (500 µl/mg tissue) supplemented with the phenylmethylsulfonyl fluoride (1 mM) phosphatase inhibitor. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatants (total protein) were collected. The protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were loaded into each lane, separated by SDS‒PAGE, and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature (RT) for 2 h. The membranes were subsequently incubated overnight at 4 °C with various primary antibodies against tyrosine hydroxylase (TH), TREM-1, Iba1, IL-1β, IL-6, and TNF-α. After three washes with TBST (5 min each), the membranes were incubated with HRP-conjugated secondary antibodies at RT for 1 h. The protein bands were visualized using the BeyoECL Moon kit and analyzed with ImageJ software (NIH, USA). All primary and secondary antibodies used herein are listed in Table 1.

Table 1.

Experimental antibodies used in this article

Name of antibody	Host	Manufacturer, Catalog Number	Application	Working dilutions
TREM-1	Rat	Abcam, ab217161	WB, IF	1:400
Iba1	Rabbit	Cell signaling, 17198 S	WB, IF	1:400
Anti-rabbit IgG, Alexa 594	Donkey	Invitrogen, A-21207	IF	1:500
Anti-mouse IgG, Alexa 488	Donkey	Invitrogen, A-21202	IF	1:500
Anti-Rat IgG, Alexa 594	Goat	Invitrogen, A-11007	IF	1:200
IL-1β	Rabbit	Proteintech, 26048-1-AP	WB	1:2000
IL-6	Rabbit	Proteintech, 26049-1-AP	WB	1:2000
TNF-α	Rabbit	Bioss, bs-2081R	WB	1:2000
GAPDH	Rabbit	HUABIO, ET1601-4	WB	1:5000
Tubulin	Mouse	Affinity, DF7967	WB	1:5000
Beta Actin	Rabbit	Affinity, AF7018	WB	1:5000
Anti-rabbit IgG, HRP	Goat	Proteintech, SA00001-2	WB	1:2000
Anti-mouse IgG, HRP	Goat	Proteintech, SA00001-1	WB	1:2000
APC anti-mouse CD45	Mouse	BioLegend, 103112	Flow Cytometry	1:200
FITC anti-mouse/human CD11b	Mouse	BioLegend, 101206	Flow Cytometry	1:500
PE anti-mouse TREM-1	Mouse	Invitrogen, MA5-28221	Flow Cytometry	1:400

Immunofluorescence

Following behavioral assessments, the mice were transcardially perfused with 0.9% cold saline (100 ml/100 g), followed by 4% paraformaldehyde (PFA) (100 ml/100 g). The brains were subsequently fixed in the same PFA solution at 4 °C for 24 h and then stored in a 30% sucrose solution for 48 h. A Leica microtome (CM1800, Leica, Germany) was used to cut 30 μm thick coronal cryosections for immunofluorescence staining. The selected sections were washed three times with PBS (5 min each) before being blocked in PBS containing 0.4% Triton X-100 and 10% normal donkey serum at RT for 2 h. The sections were subsequently incubated overnight at 4 °C with primary antibodies against TH, Iba1, and TREM-1. After washing, the sections were incubated with the appropriate secondary antibodies at RT for 2 h. The cell nuclei were stained with DAPI, and images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan). The proportion of Iba1⁺ microglia expressing TREM-1 was quantified within predefined ROIs and averaged per animal. The corresponding y-axis label in statistical graphs was expressed as “Iba1⁺/TREM-1⁺ microglia (%)”. All primary and secondary antibodies used in this study are listed in Table 1.

Flow Cytometry and Analysis

After harvesting the IL tissue, single-cell suspensions were prepared through enzymatic digestion in a 37 ℃ water bath using DNase I (VIC115, Vicmed) and collagenase II (VIC080, Vicmed) for 30 min. The cell suspensions were filtered through nylon mesh and then stained with FITC-conjugated anti-mouse/human CD11b, APC-conjugated anti-mouse CD45, PerCP/Cyanine5.5-conjugated anti-mouse Ly-6G, and APC-Cy7-conjugated anti-mouse TREM-1 antibodies. The staining process was conducted for 30 min at 4 °C in the dark. All the antibodies used in this study are listed in Table 1. Sample collection was performed using a FACS Canto II (BD Biosciences, USA), and data analysis was conducted with FlowJo X software.

Microglia Morphological Analysis

Sholl analysis is a well-established quantitative neuroanatomical technique that facilitates a rigorous evaluation of microglial branching complexity through concentric sphere sampling [28]. Using an Olympus BX53 equipped with a 60× oil immersion lens, z-stack images of individual microglia were obtained with a step size of 0.86 μm. The analyzed images were skeletonized using the skeleton analysis feature of ImageJ, allowing measurement of the number of microglial terminals and the length of their maximum branches. Morphometric validation was performed through blind analysis of at least 30 cells per group, excluding those with truncated processes.

Statistical Analysis

Data were analyzed using GraphPad Prism 10.1.2. Unpaired two-tailed t-test, one-way ANOVA with Tukey’s multiple comparisons test, or two-way ANOVA (group × time) followed by Tukey’s multiple comparisons test were applied as appropriate. Correlation analysis was conducted using Pearson’s correlation coefficient, and results are presented as r values with corresponding p values. Data distribution was visually inspected using quantile–quantile (Q-Q) plots in Prism, which applies parametric tests under the assumption of normality and homogeneity of variance. Significance levels were denoted as *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

Establishment of PD Model Mice with Depressive-Like Behaviors

A subacute MPTP model in mice was established through daily intraperitoneal injections of MPTP (30 mg/kg) for five consecutive days. Following the final MPTP injection, a series of behavioral assessments was conducted, including OFT, rotarod test, and pole test, to evaluate motor function. Concurrently, depressive-like behaviors were assessed using the FST, TST and SPT (Fig. 1a). Because dopaminergic neurons are predominantly localized to the SNpc, neurodegeneration in this region was quantified by assessing positivity of TH—the rate-limiting enzyme in dopamine synthesis and a canonical dopaminergic neuron marker. Compared with saline injection, MPTP injection markedly diminished the TH⁺ neuronal density (Fig. 1b, c), and Western blot analysis confirmed a decrease in TH protein expression in the SNpc (Fig. 1d). Motor function evaluations conducted on Day 1 following the final injection revealed that MPTP-treated mice traveled significantly shorter distances in the OFT than the saline-treated mice (Fig. 1e, f). This finding was further evidenced by a reduced latency to fall in the rotarod test and increased descent time in the pole test (Fig. 1g, h). Tests for depressive-like behaviors (FST, TST and SPT) were performed on Days 3, 7, 14, and 21 following the final injection. The immobility time in the FST and TST progressively increased from Day 3 to Day 21, peaking on Day 14 (Fig. 1i, j). Additionally, sucrose preference continuously decreased from Day 3 to Day 21, reaching its lowest point on Day 14 (Fig. 1k). In summary, these results demonstrated progressively worsening depressive-like behaviors in PD model mice, with peak severity on Day 14.

Fig. 1.

Subacute MPTP injection induces dopaminergic neurodegeneration and progressive depressive-like behaviors. (a) Experimental timeline of MPTP injection and behavioral tests in mice (n = 8). (b, c) Representative immunofluorescence images and quantitative analysis of TH⁺ neurons in SNpc (Red: TH; Blue: DAPI). Scale bars: 200 μm for the top row and 50 μm for the bottom row (n = 3). (d) Western blot and quantitative analysis of TH expression in the SNpc (n = 3). (e) Representative movement paths from 10-min open field test sessions. (f) Total distance traveled in the OFT (n = 8). (g) Latency to fall in rotarod test (n = 8). (h) Latency to descend in the pole (n = 8). (i) Temporal progression of immobility time in the FST (n = 8). (j) Temporal progression of immobility time in the TST (n = 8). (k) Dynamic changes in sucrose preference ratio (n = 8). Data are presented as mean ± SEM. Statistical analyses: (b-h): unpaired Student’s t-test; (i-k): two-way ANOVA (group × time) with Tukey’s multiple-comparisons test. (*P < 0.05, **P < 0.01, and ***P < 0.001)

Microglial Activation and Elevated Proinflammatory Cytokines in the IL of PD Model Mice with Depressive-Like Behaviors

Clinical evidence indicates that elevated proinflammatory cytokines in the cerebrospinal fluid of PD patients are correlated with the severity of depressive symptoms [29, 30], suggesting a potential role of neuroinflammation in depressive-like behaviors. To investigate this link, we assessed neuroinflammation in the IL of PD model mice by quantifying microglial activation and proinflammatory cytokines. Compared with saline-treated mice, MPTP-treated mice displayed microglial activation characterized by somal hypertrophy and reduced branching complexity, as evidenced by Iba1 immunofluorescence (Fig. 2a) and Sholl analysis (Fig. 2b). Furthermore, Western blot analysis revealed pronounced upregulation of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in MPTP-treated mice (Fig. 2c, d). Notably, correlation analyses confirmed that proinflammatory cytokine levels were positively correlated with immobility time in both the FST (Fig. 2e) and TST (Fig. 2f) but inversely associated with sucrose preference in the SPT (Fig. 2g). These correlations underscored the pathological connection between neuroinflammation and depression severity in PD model mice.

Fig. 2.

Neuroinflammation in the IL correlates with depressive-like behaviors in PD model mice. (a) Representative immunofluorescence images of Iba1⁺ microglia in the IL (Green: Iba1; Blue: DAPI). Scale bars: 50 μm. (b) Sholl analysis of microglial morphology in Saline and MPTP-treated mice (n = 3, 10 cells per mouse). (c, d) Western blot and quantitative analysis of IL-1β, IL-6, and TNF-α protein expression in the IL (n = 3). (e, f) Pearson correlation analysis between cytokines expression and immobility time in both FST and TST. (g) Pearson correlation analysis between cytokine expression and sucrose preference ratio in SPT. Data are presented as mean ± SEM. Statistical analyses: (b, d): unpaired Student’s t-test; (e-g): Pearson correlation test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Upregulation of Microglial TREM-1 in the IL Coincides with the Peak Severity of Depressive-Like Behaviors in PD Model Mice

To evaluate the impact of TREM-1 on the severity of depressive-like behaviors, the temporal expression of TREM-1 was investigated in PD model mice. Western blot analysis revealed progressive TREM-1 upregulation from Day 7 to Day 21 following the final MPTP injection, peaking on Day 14 (Fig. 3a, b). This peak coincided with the peak severity of depressive-like behaviors, suggesting a pathological interplay between TREM-1 expression and depressive-like behaviors. Thus, all subsequent experiments were conducted on Day 14. Flow cytometry analysis of CD11b⁺CD45ˡᵒʷ microglia revealed significantly elevated mean fluorescence intensity (MFI) of TREM-1 in MPTP-treated mice (Fig. 3c, d). In addition, double-label immunofluorescence in the IL demonstrated TREM-1 expression in Iba1⁺ microglia and showed a higher percentage of double-labeled Iba1⁺/TREM-1⁺ microglia in MPTP-treated mice (Fig. 3e, f). These findings suggested that microglial TREM-1 upregulation in the IL is associated with the pathogenesis of depression in PD.

Fig. 3.

TREM-1 expression dynamics in the IL microglia in PD model mice with depressive-like behaviors. (a, b) Western blot and quantitative analysis of TREM-1 expression in the IL at different time points (n = 3). (c) Representative histograms of microglial TREM-1 expression (Red: MPTP group; Black: Saline group). (d) MFI of TREM-1 in the IL microglia at peak severity (n = 6). (e) Representative double-label immunofluorescence of TREM-1 and Iba1 in the IL (Red: TREM-1; Green: Iba1; Blue: DAPI). Scale bar: 50 μm. (f) Quantification of the percentage of double-labeled Iba1⁺/TREM-1⁺ microglia in the IL (n = 3). Data are presented as mean ± SEM. Statistical analyses: (b): two-way ANOVA (group × time) with Tukey’s multiple-comparisons test. (d, f): unpaired Student’s t-test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Microglial Depletion in the IL Attenuates Neuroinflammation and Rescues Depressive-Like Behaviors in PD Model Mice

To investigate the contribution of microglia to neuroinflammation and depressive-like behaviors, IL microglia were depleted in MPTP-treated PD mice using CLP (macrophage-depleting agents). Bilateral stereotactic CLP liposome (5 mg/ml, 1 µl/side) or PBS liposome were administered into the IL (AP: +2.0 mm; ML: ±0.30 mm; DV: −3.30 mm) on Day 10 [31] (Fig. 4a). A significant reduction in Iba1 protein expression was observed in the MPTP + CLP group (Fig. 4b) and immunofluorescence quantification revealed near-complete ablation of Iba1⁺ microglia in the IL of MPTP + CLP group compared to the MPTP + PBS group (Fig. 4c, d). This targeted depletion of microglia concurrently attenuated TREM-1 expression, as validated through both Western blot and flow cytometric analyses (Fig. 4e-g). Western blot analysis confirmed pronounced reductions in the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) in the MPTP + CLP group compared with those in the MPTP + PBS group (Fig. 4h, i). Four days after the injection of CLP, the immobility times were markedly shorter in both the FST and TST (Fig. 4j, k), whereas the sucrose preference ratio was significantly greater in the SPT (Fig. 4l). These results demonstrated that CLP-mediated microglial ablation markedly attenuates neuroinflammation, effectively rescuing depressive-like behaviors in PD model mice.

Fig. 4.

Microglial depletion in the IL attenuates neuroinflammation and rescues depressive-like behaviors in PD model mice. (a) Experimental timeline for bilateral stereotactic injection of CLP or PBS-liposome into the IL on day 10. (b) Western blot and quantitative analysis of Iba1 expression in the IL (n = 3). (c) Representative double-label immunofluorescence of TREM-1 and Iba1 in the IL (Red: TREM-1; Green: Iba1; Blue: DAPI). Scale bar: 50 μm. (d) Quantification of the percentage of double-labeled Iba1⁺/TREM-1⁺ microglia in the IL (n = 3). (e) Western blot and quantitative analysis of TREM-1 expression in the IL (n = 3). (f) Representative histograms of microglial TREM-1 expression (Red: MPTP + PBS group; Green: MPTP + CLP group). (g) MFI of TREM-1 in the IL microglia (n = 6). (h, i) Western blot analysis and quantitative analysis of IL-1β, IL-6, and TNF-α expression (n = 3). (j) Immobility time in the FST (n = 8). (k) Immobility time in the TST (n = 8). (l) Sucrose preference ratio in the SPT (n = 8). Data are presented as mean ± SEM. Statistical analyses: unpaired Student’s t-test (*P < 0.05, **P < 0.01, and ***P < 0.001)

TREM-1 Knockout Alleviates Neuroinflammation and Depressive-Like Behaviors in PD Model Mice

Previous studies have established a critical role for TREM-1 in amplifying neuroinflammation in neurodegenerative diseases. To investigate the possible involvement of TREM-1 in depressive-like behaviors in PD model mice, WT and TREM-1 KO mice were compared. Western blot analysis showed that TREM-1 protein was markedly reduced in TREM-1 KO mice (Fig. 5a). Moreover, TREM-1 deficiency did not significantly alter the expression of Iba1 or proinflammatory cytokines (IL-1β, IL-6, and TNF-α) under basal conditions (Fig. 5b, c). To further investigate the pathological function of TREM-1 in PD, Western blot analysis was performed, which revealed significant upregulation of TREM-1 expression in MPTP-treated WT mice (Fig. 5d). In addition, the protein levels of IL-1β, IL-6, and TNF-α were markedly greater in MPTP-treated WT mice than in MPTP-treated TREM-1 KO mice (Fig. 5e, f). Consistently, MPTP-treated WT mice exhibited a significantly higher proportion of Iba1⁺/TREM-1⁺ microglia in the IL relative to TREM-1 KO mice (Fig. 5g, h). Compared with MPTP-treated WT control mice, MPTP-treated KO mice presented reduced immobility times in the FST and TST (Fig. 5i, j), as well as an increased sucrose preference ratio in the SPT (Fig. 5k). The findings in PD model mice revealed that TREM-1 deficiency significantly attenuates MPTP-induced neuroinflammation and alleviates depressive-like behaviors.

Fig. 5.

TREM-1 knockout alleviates neuroinflammation and depressive-like behaviors in PD model mice. (a) Western blot and quantitative analysis of TREM-1 expression in the IL of WT and TREM-1 KO mice (n = 3). (b, c) Western blot and quantitative analysis of IL-1β, IL-6, TNF-α, and Iba1 expression in the IL of WT and TREM-1 KO mice (n = 3). (d) Western blot and quantitative analysis of TREM-1 expression on day 14 following the final MPTP injection (n = 3). (e, f) Western blot and quantitative analysis of IL-1β, IL-6, and TNF-α expression on day 14 following the final MPTP injection (n = 3). (g) Representative double-label immunofluorescence of TREM-1 and Iba1 in the IL (Red: TREM-1; Green: Iba1; Blue: DAPI). Scale bar: 50 μm. (h) Quantification of the percentage of double-labeled Iba1⁺/TREM-1⁺ microglia in the IL (n = 3). (i) Immobility time in the FST (n = 8). (j) Immobility time in the TST (n = 8). (k) Sucrose preference ratio in the SPT (n = 8). Data are presented as mean ± SEM. Statistical analyses: unpaired Student’s t-test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Pharmacological Inhibition of TREM-1 Attenuates Neuroinflammation and Depressive-Like Behaviors by Suppressing Microglial TREM-1/SYK/CARD9/NF-κB Signaling in PD Model Mice

Based on the genetic evidence from TREM-1 KO mice, we further assessed the therapeutic potential of targeting TREM-1. LP17, a TREM-1 inhibitor [32], was intranasally administered at a dose of 20 mg/kg (10 µl) on Day 10 following the final MPTP injection (Fig. 6a). Double-label immunofluorescence showed a significant reduction in the proportion of Iba1⁺ microglia expressing TREM-1 in the IL in the MPTP + LP17 group compared with the MPTP + control peptide group (Fig. 6b, c). Western blot analysis confirmed that LP17 treatment significantly reduced the protein levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and drove TREM-1 protein toward baseline relative to the control peptide group (Fig. 6f, g). To investigate the underlying mechanism, we examined the TREM-1/SYK/CARD9/NF-κB signaling pathway. LP17 treatment in MPTP mice resulted in decreased SYK activation (lower p-SYK/total SYK), downregulated CARD9, and attenuated NF-κB signaling, as evidenced by reduced phosphorylated NF-κB (p-NF-κB) relative to total NF-κB (Fig. 6h-l). These findings suggest that pharmacological inhibition of TREM-1 disrupts the SYK/CARD9/NF-κB cascade, which amplifies inflammatory gene expression in microglia. In behavioral assays, LP17 significantly reduced immobility time in both the FST and TST (Fig. 6m, n) and increased sucrose preference in the SPT (Fig. 6o). Overall, these data indicate that the reduction in neuroinflammation through TREM-1/SYK/CARD9/NF-κB signaling inhibition is associated with alleviation of depressive-like behaviors in PD model mice.

Fig. 6.

Pharmacological inhibition of TREM-1 attenuates neuroinflammation and depressive-like behaviors in PD model mice. (a) Experimental timeline for intranasal administration of LP17 (20 mg/kg in 10 µl) or control peptide on Day 10. (b) Representative double-label immunofluorescence images of Iba1⁺/TREM-1⁺ microglia in the IL (Red: TREM-1; Green: Iba1; Blue: DAPI). Scale bar: 50 μm. (c) Quantification of the percentage of double-labeled Iba1⁺/TREM-1⁺ microglia in the IL (n = 3). (d, e) Western blot and quantitative analysis of IL-1β, IL-6, and TNF-α expression in the IL. (f) Western blot analysis of TREM-1, SYK, and p-SYK expression in the IL. (g) Quantification of TREM-1 expression (n = 3). (h) Quantification of SYK expression (n = 3). (i) Quantification of p-SYK expression (n = 3). (j) Western blot analysis of CARD9, NF-κB, and p-NF-κB expression in the IL. (k) Quantification of CARD9 expression (n = 3). (l) Quantification of NF-κB activation, represented by the p-NF-κB/total NF-κB ratio (n = 3). (m) Immobility time in FST (n = 8). (n) Immobility time in TST (n = 8). (o) Sucrose preference ratio in SPT (n = 8). Data are presented as mean ± SEM. Statistical analyses: one-way ANOVA with Tukey’s multiple comparisons test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Discussion

Our study establishes microglial TREM-1 in the IL as a pivotal mediator of neuroinflammation and depressive-like behaviors in a subacute MPTP model of PD. Using a combination of genetic, pharmacological, and microglial depletion methods, we demonstrate that microglial TREM-1 amplifies proinflammatory cytokines (IL-1β, IL-6, TNF-α) in the IL, exacerbating depressive-like behaviors. These results not only reveal a novel mechanism underlying depression in PD but also position TREM-1 as a potential therapeutic target for addressing nonmotor symptoms in PD.

The prefrontal cortex, particularly the IL region, plays a critical role in emotion regulation, decision-making, and mood disorders [33, 34]. Furthermore, the IL along with the prelimbic area work together to modulate stress responses, emotional regulation, and cognitive functions [35]. Research has shown that the IL is particularly vulnerable to alterations in depression, with structural and functional changes observed in both animal models and human patients [36, 37]. For instance, reductions in the IL volume and activity are frequently reported in individuals with major depressive disorder (MDD) and have been linked to impaired emotional regulation [38]. Additionally, studies using optogenetic or pharmacological manipulations have demonstrated that disrupting IL activity in animal models induces depressive-like behaviors [39], while stimulation of the IL often produces antidepressant-like effects [40]. Although the role of neuroinflammation in the IL in mood regulation is well-established, its specific involvement in PD-related depression has been less thoroughly explored. In our study, we observed that neuroinflammation in the IL was closely associated with the onset and severity of depressive-like behaviors in MPTP-treated mice, with peak severity occurring on Day 14. By contrast, a partial improvement was evident by Day 21. This later recovery likely reflects a time-dependent attenuation of IL microglial activation and proinflammatory cytokines expression after MPTP, with compensatory adaptations in surviving dopaminergic neurons further helping to preserve synaptic output despite neuronal loss [41]. This temporal correlation suggests that neuroinflammation in the IL plays a critical role in the development of depression in PD, highlighting its importance in linking the neurodegenerative processes of PD with its psychiatric manifestations.

Prior studies have shown that microglial activation contributes to depressive-like behaviors in both stress and neurodegenerative models [42–45]. In PD, microglial activation are known to exacerbate dopaminergic neuronal loss in the SNpc and contribute to nonmotor symptoms such as depression [23, 46]. In our study, we extend these observations by demonstrating that microglial activation in the IL is tightly associated with the onset of depressive-like behaviors in MPTP-induced PD model mice, thereby linking region-specific neuroinflammation to affective dysfunction in PD.

Beyond general microglial activation, specific receptors on microglial cells are crucial in driving neuroinflammation and depression. For instance, receptors such as CD11b/CD18, P2X7, and Toll-like receptors (TLRs) have been implicated in microglial activation and inflammatory responses. Moreover, TREM-1 is well established as a key mediator of neuroinflammation in neurological diseases, such as ischemic stroke and AD [22, 47]. Nevertheless, whether TREM-1 within the IL contributes to depressive-like behaviors in PD has remained unclear. In this context, TREM-1 is distinguished in the IL as an upstream amplifier of inflammatory signaling. Through the adaptor DAP12/TYROBP, TREM-1 activates SYK, which then engages the CARD9–BCL10–MALT1 complex and culminates in activation of NF-κB (p65) [19, 48, 49]. Notably, recent work in AD also demonstrated that TREM-1 orchestrates crosstalk with SYK to amplify neuroinflammation [22], underscoring the translational relevance of this pathway. This cascade promotes transcriptional upregulation of IL-1β, IL-6, and TNF-α and consolidates an M1-like microglial polarization state. To further substantiate this framework, additional Western blot analyses demonstrated increased p-SYK/SYK, elevated CARD9, and increased p-p65/p65 following the final MPTP injection, with each change attenuated by the TREM-1 inhibitory peptide LP17 (Fig. 6f–l), supporting TREM-1–dependent engagement of this signaling axis in the IL.

Independent work by Shen et al. demonstrated that microglial TREM-1–SYK signaling activates CARD9/NF-κB and that both LP17 and the SYK inhibitor R406 mitigate MPTP-induced neuroinflammation [50]. In that study, activation of the TREM-1/SYK pathway increased IL-1β and IL-6 release, exacerbated dopaminergic neuronal loss, and reinforced the same pathway in peripheral neutrophils, suggesting a positive feedback loop between the brain and periphery. Taken together, the temporal upregulation of microglial TREM-1 in the IL, along with the attenuation of proinflammatory cytokines and depressive-like behaviors in TREM-1 KO mice or after LP17 treatment, provides consistent evidence. These findings support the interpretation that TREM-1 likely functions as an upstream driver of M1-like polarization rather than merely reflecting downstream changes.

In our study, we found that TREM-1 upregulation in the IL microglia coincided with the peak severity of depressive-like behaviors in MPTP-treated mice. Genetic deletion of TREM-1 significantly attenuated neuroinflammation and ameliorated depressive-like behaviors, confirming that TREM-1 plays a pivotal role in mediating the neuroinflammatory and affective dysfunction in PD. Moreover, pharmacological inhibition of TREM-1 using the peptide LP17 led to a reduction in both inflammatory markers and depressive-like behaviors, and stereotaxic injection of CLP into the IL to deplete microglia likewise diminished neuroinflammation and ameliorated behavioral deficits. Collectively, these convergent manipulations implicate microglial TREM-1 as a causal driver in the IL and support its candidacy as a therapeutic target for PD-related depression.

Several limitations of this study should be acknowledged. First, we employed heterozygous rather than homozygous TREM-1 knockout mice, which may underestimate the full impact of TREM-1 deficiency. Second, colocalization was assessed by double labeling of Iba1⁺/TREM-1⁺ microglia instead of pixel-based quantitative colocalization analysis, which may reduce spatial precision. Third, some assays such as Western blot and immunofluorescence were performed with relatively small sample sizes (n = 3), which may limit statistical power. In addition, potential off-target effects of LP17 cannot be excluded, and blood–brain barrier penetration was not measured [51]. CLP depletion was nonselective, limiting clinical translatability despite its utility for establishing cellular necessity [31]. Finally, the SYK/CARD9/NF-κB pathway was validated in the IL, but NLRP3 inflammasome activation was not assessed, and cytokines profiling was limited to IL-1β, IL-6, and TNF-α. These constraints do not alter the main conclusions but delineate priorities for future work.

In line with clinical observations in PD patients, where elevated cerebrospinal fluid cytokines are often correlated with depressive symptoms [52], our study identifies IL microglial TREM-1 as a critical mediator of neuroinflammation and depressive-like behaviors in PD. These results advance our understanding of PD mechanisms and support approaches targeting TREM-1 to modulate IL microglia-driven neuroinflammation and treat PD-related depression, with potential benefits for other nonmotor features. Furthermore, our findings broaden current paradigms of PD pathogenesis and open avenues for combinatorial strategies that address both motor and nonmotor symptom domains through modulation of inflammation.

Author Contributions

All the authors contributed significantly to this work. Yuan-qing Chu, Rong Hua and Yong-mei Zhang conceived and designed the experiments. Yuan-qing Chu, Wei Song, Zhi-jing Song, Ying-qi Huang, Ling-jing Gu and Jia-xuan Lian performed the experiments and analyzed the data. Yuan-qing Chu wrote the manuscript, Rong Hua and Yong-mei Zhang revised and edited the manuscript. All authors approved the final manuscript and agreed to take responsibility for all aspects of the work.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 82471244; No. 82271257), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX25_3240; No. KYCX25_3308), Open Project of Jiangsu Provincial Key Laboratory of Xuzhou Medical University (XZSYSKF2022032), and the Xuzhou Municipal Health Commission Science and Technology Project (XWKYSL20220446).

Data Availability

The data that support the findings of this study are available on request from the corresponding author, Y. M., upon reasonable request.

Declarations

Ethics Approval and Consent to Participate

The animal protocols were approved by the Institutional Animal Care and Use Committee of Xuzhou Medical University (approval No. 202010A017). Clinical trial number: not applicable.

Consent for Publication

The manuscript has been approved by all authors for publication.

Competing Interests

The authors declare no competing interests.