Mutant LRRK2 increases immune reaction and brain cell damage in long-term experimental gut inflammation

All animal experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by the Bioethical Committee of the KU Leuven (Belgium) (ECD projects 051/2020 and 156/2022). For this study 8–10-week-old male C57BL/6 J (NTG, JAX 000664, RRID: IMSR_JAX:000664) and B6.Cg-Lrrk2tm1.1Hlme/J (GS LRRK2, JAX:030961, RRID:IMSR_JAX:030961) were used. Animals were housed in individually ventilated cages with free access to food and water, under a controlled light–dark cycle (12-h light–12-h dark) and temperature (21 ± 1 °C).

DSS (molecular weight, 40 kDa, TdB Labs) was added to the drinking water at a concentration range of 0.5–2% (w/v) for 28 days. The DSS concentration was gradually increased by 0.5% w/v every 5 days and replaced with regular animal facility water for 2 days in between).

C57BL/6-Ly5.1 (B6.SJL-PtprcaPepcb/BoyCrCrl, Charles river) and B6.Cg-Lrrk2tm1.1Hlme/J were used as donors. Donor mice were euthanised by cervical dislocation without anaesthesia and the humerus, femur and tibia were used for the bone marrow (BM) isolation. Upon aseptic conditions, BM cells were isolated, filtered through a 100 μm cell strainer, washed in ice-cold PBS, and resuspended in serum free medium (RPMI, Fisher scientific). Ten-week-old recipient C57BL/6 J and B6.Cg-Lrrk2tm1.1Hlme/J mice, lethally irradiated at 9.5 Gy for 10 min, were transplanted with approximately 10 × 10⁶ cells/mouse by retro-orbital injection. Recipient mice were treated with 1 mM sulfamethoxazole and 0.18 mM trimethoprim in the drinking water for 2 weeks. Twelve weeks post transplantation the DSS administration protocol was initiated.

Mice were fed with food pellets containing either MLi-2 (MedChemExpress) at a dose of 25 mg/kg of body weight per day (Research Diets, Inc.) or placebo starting 2 days prior to DSS administration and maintained for the duration of the experiment. Animals were randomly assigned to the compound or placebo groups.

All surgical procedures were performed using aseptic techniques and mice were treated with ketamine (60 mg/kg, i.p., Ketalar, Pfizer, Puurs) and medetomidine (0.4 mg/kg, Dormitor, Pfizer) anaesthesia. Following anaesthesia, the rodents were placed in a stereotactic head frame (Stoelting, Wood Dale, IL). Injections were performed with a 30-gauge needle and a 10-μl Hamilton syringe (Hamilton, Bonaduz, GR, Switzerland). LRRK2 GS or WT mice were injected with 2 μl rAAV2/7 αSYN vector (CMVenh-synapsin-intron-Hs αSyn WT) at a dose of 3 × 10⁹ genome copies/ml. The right SNpc was targeted using the following stereotactic coordinates: anteroposterior − 3.1 mm, mediolateral − 1.2 mm, and dorsoventral 4.3 mm (counting from the skull) using bregma as reference. The injection rate was 0.25 μl/min, with a 5-min interval following initial needle placement. The needle was left in place for an additional 5 min period after the injection procedure before being slowly retracted.

The motor phenotype of the rAAV2/7 αSYN vector-injected mice was assessed one month post-injection. One week prior to behavioural analysis, mice were handled on a daily basis to minimise stress and anxiety. Before each behavioural test, mice were allowed to acclimate to the testing room for a minimum of 30 min. Two behavioural tests were implemented to evaluate locomotion and forelimb asymmetry. For the rotarod, mice were placed on 5 rotating rods and were left walking face forward under steady rotation speed (4 rpm), for at least one minute (revolutions per minute). Past training time, the speed of the rotation was progressively increased from 4 to 40 rpm over 5 min. Latency to fall or passive rotation was recorded. Three trials were conducted with 1 h resting intervals in between. The mean latency for all three trials was used for analysis. For the cylinder test, mice were placed in a transparent glass cylinder and the front paw contacts to the cylinder wall were recorded as left, right, or both paws, with a total of 25 contacts per animal. The number of contralateral forelimb contacts was expressed as a percentage of total forelimb contacts.

The animals were killed with an overdose of sodium pentobarbital (Dolethal, 200 mg/kg, i.p.), followed by intracardial perfusion with ice-cold saline. Half brains were dissected and enzymatically digested with Collagenase D (Merck Life Science BV) and DNAse I (Roche) at 37 °C, followed by passage through a 70 μm cell strainer. Cells were washed with Hank’s Balanced Salt Solution (HBSS) and centrifuged at 1000 g for 10 min. The pellet was resuspended with 38% Percoll (GE Healthcare Bio-Sciences) and centrifuged for 25 min at 800 g with slow acceleration and no brake. Cells were collected from the bottom of the tube and washed once with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS) and once in FBS stain buffer (BD Biosciences).

Equal parts of distant colon samples were dissected and cleaned. After incubation in HBSS with 10% FBS and 5 mM EDTA at 37 °C, the colon pieces were washed with DMEM and minced finely. Then, each sample was enzymatically digested with Collagenase D and DNAse I at 37 °C followed by passage through a 100 μm cell strainer. Cells were washed in DMEM with 10% FBS and centrifuged at 1000 g for 10 min. The pellet was resuspended in a 40% Percoll in DMEM underlaid by a layer of 80% Percoll in DMEM and centrifuged for 25 min at 800 g with slow acceleration and no brake. The interphase was collected and washed in FBS stain buffer.

For both protocols, the cells were then blocked with mouse anti-rat FcγII receptor (CD32) (D34-485, BD Biosciences) in FBS stain buffer and stained for the following surface markers: CD45 30-F11, PE-Cy7 (Biolegend); CD11b M1/70, PerCPCy5.5- (Biolegend); Ly-6G 1A8, PE (BioLegend); CD4 GK1.5, AlexaFluor 488 (BioLegend); CD8a 53–6.7, Pacific Blue (BioLegend); CD25 PC61, PE-Cy5 (BioLegend); MHC II M5/114.15.2, APC (BioLegend); Ly-6C HK1.4, Brilliant Violet 605 (BioLegend); CD19 6D5, Brilliant Violet 570 (BioLegend); CD11c, eFluor™ 506 (ThermoFisher eBioscience^™); CD161 PK136, Brilliant Violet 650 (BioLegend); CD68 FA-11, Brilliant Violet 711 (BioLegend); CD3 17A2, Brilliant Violet 750 (BioLegend); CD45.2 104, APC (BioLegend); CD45.1 A20, FITC (BioLegend). A fixable viability dye eFluor 780 (eBioscience) was used in each condition. Cells were fixed in 4% PFA, and data were acquired on the following day. Flow cytometry/FACS was performed at the VIB-KU Leuven FACS Core Facility using a BD FACSymphony A5 cytometer with the FACSDiva software. Data were analysed using FlowJo v10.0.7 software. Fifty thousand to one hundred thousand events were collected for each brain sample and one hundred fifty thousand events for the colon samples.

Mice were killed with an overdose of sodium pentobarbital (200 mg/kg, i.p., Dolethal, Vetoquinol) followed by intracardial perfusion with saline and/or 4% PFA in PBS. After perfusion and fixation, colons were embedded in paraffin blocks, cut into 6 μm sections and mounted on glass slides. Slides were stained using standard immunohistochemistry as described below. Slides were de-paraffinised with 2 sequential 5-min washes in xylenes, followed by 5-min washes in a descending series of ethanol concentrations: 100%, 100%, 95%, 70% and finally incubated in deionised water for 5 min prior to antigen retrieval. Slides were stained for haematoxylin and eosin (H & E) staining. The histological score for the colitis was assessed based on different parameters as follows; crypts architecture: from normal morphology to loss of entire crypts morphology (0–3); presence of oedema: oedema (0–3); goblet cell depletion (0–1). The infiltration of immune cells into the lamina submucosa was scored from 0 to 3. The total histological score was calculated as the sum of the individual scores of these parameters.

For immunohistochemical analysis slides were treated with 3% hydrogen peroxide for 10 min and incubated overnight with rat anti-mouse MHC Class II (I-A/I-E) (eBioscience™) primary antibody in 10% rabbit serum (DakoCytomation) and as secondary antibody we used biotinylated anti-rabbit IgG (DakoCytomation), followed by incubation with streptavidin–horseradish peroxidase complex (DakoCytomation). MHC II immunoreactivity was visualised using 3' diaminobenzidine tetrahydrochloride (DAB; Merck Life Science BV). For immunofluorescent staining the slides were incubated overnight in PBS-0.1% triton X-100 with 10% donkey serum (Jackson ImmunoResearch Laboratories) and the following antibodies: goat Myeloperoxidase (MPO, eBioscience^™) and rat F4/80 (Biolegend). After three rinses in PBS-0.1% triton X-100, the sections were incubated in the dark for 2 h in fluorochrome-conjugated secondary antibodies: donkey anti-goat Alexa 488 (Abcam) (MPO) and donkey anti-rat Alexa 555 (Abcam) (F4/80).

For the immunohistochemical stainings of the brain, after the post-fixation overnight in 4% PFA, 25 µm thick coronal brain sections were made with a vibrating microtome (HM 650 V, Microm). Immunohistochemistry was performed on free-floating sections using antibodies against tyrosine hydroxylase (TH, rabbit polyclonal, Merck Millipore). Sections were pretreated with 3% hydrogen peroxide for 10 min and incubated overnight with primary antibody in 10% rabbit serum (DakoCytomation) and as secondary antibody we used biotinylated anti-rabbit IgG (DakoCytomation), followed by incubation with streptavidin–horseradish peroxidase complex (DakoCytomation). TH immunoreactivity was visualised using Vector SG (SK-4700, Vector Laboratories).

For the assessment of the integrity of the SNpc, the number of TH + cells was determined by stereological measurements using the optical fractionator method in a computerised system as described before [3] (Stereo Investigator; MicroBrightField, MBF). Every 8th section throughout the entire SNpc and a total of 5 sections for each animal, was analysed for TH immunoreactivity. The coefficients of error, calculated according to the procedure of Schmitz and Hof as estimates of precision, varied between 0.05 and 0.10. Percentages represent the loss of dopaminergic neurons compared to the contralateral, non-injected side. All analyses were performed by an investigator blinded to the different groups.

Colon pieces were freshly isolated, weighted and snap-frozen. Samples were homogenised in 20 volumes, respectively, of RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton-X100, 0.5% (w/v) Sodium Deoxycholate, 1.0 mM EDTA pH 7.4) containing a protease inhibitor cocktail (Roche cOmplete) and phospho-STOP EASYPACK (Roche) using a tissue homogeniser (TH, Omni Tissue Homogenizer). After 30-min incubation at 4 °C, the samples were spun for 10 min at 3000 g at 4 °C, the supernatant and was further spun for 30 min at 20.000 g at 4 °C, and the resulting supernatants represented the protein fraction. A second colon piece was homogenised in 10 volumes of urea buffer (8 M urea, 100 mM Tris–HCl, 10% glycerol, 1% SDS, 5 mM DTT, 1 mM EDTA, 1 mM EGTA, pH 6.8) containing protease and phosphatase inhibitor using a tissue homogeniser. After 30-min incubation at 4 °C, the samples were spun for 10 min at 6000 g at 4 °C, and the resulting supernatants represented the protein fraction. Protein sample concentration was determined by BCA protein assay (Thermo Scientific, MA, USA) according to the manufacturer’s directions.

To assess LRRK2 and LRRK2 P-S1292 by Western blot analysis, 40 μg protein of colon samples extracted in urea buffer were loaded on NuPAGE 3–8% Tris–acetate gradient gels (Invitrogen, Waltham). Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Nonspecific binding sites were blocked for 15 min in PBS with 0.1% Triton X-100 (PBS-T) and 5% nonfat milk. After overnight incubation at 4 °C with primary antibodies (anti-LRRK2 N241A/34, NeuroMab; anti-LRRK2 (P-S1292), Abcam; anti-vinculin V9131, Sigma), blots were washed 3 times with PBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Dako) for 1 h and washed again 3 times. Bands were visualised using Clarity Western ECL (Bio-Rad) and developed with a GE ImageQuant 800 (GE Healthcare). Densitometric analysis was performed using ImageQuant software (GE Healthcare).

Pro- and anti-inflammatory proteins were analysed in the protein fraction of the colon homogenates using a 96-well V-PLEX Mouse cytokine 19-plex (Proinflammatory Panel-1 and Cytokine Panel-1) (#K15255D; Meso Scale). Each mouse colon extract (homogenised in RIPA buffer) was diluted two fold with diluents and incubated for 2 h at RT. After washing with 0.05% Tween‐20 in 0.01 M PBS, the plates were incubated with detection antibodies for 2 h at RT. Data were generated from the V-PLEX Mouse cytokine 19-plex kits by washing away detection antibody solution and adding reading buffer to generate an electrochemiluminescence signal measured in a Meso Quickplex SQ120 (Meso Scale) and quantified with the Discovery Workbench 4.0 software.

H&E slides were scanned using an Aperio ScanScope CS whole slide scanner at 40X magnification. Fluorescent staining images were obtained with Leica DM6 B upright microscope using Leica Application Suite 3.6 Download. For the counting of F4/80 and MPO positive cells, tiled images of three distant colon sections/animal were counted using QuPath 0.3.2 software. For the imaging analysis, Fiji v2.0.0 software was utilised.

Graph creation and statistical analysis were performed using GraphPad Prism for Windows (GraphPad Software, Inc.), version 9.0. Results are presented as means ± standard error of the mean. Normality of data was tested using the Shapiro–Wilk test. Data not showing normal distribution were transformed before applying the parametric ANOVA. Statistical significance was assessed using t-test or when multiple groups were analysed simultaneously, one-way ANOVA followed by Tukey’s multiple comparison post hoc test, two-way ANOVA followed by Sidak’s post hoc test, or three-way ANOVA followed by Sidak’s post hoc test. Significance was represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Administration of DSS in mice induces disruption of the distal colonic intestinal epithelial layer, resulting in histological damage with morphological and symptomatic resemblance to human UC. The severity and acuteness of the inflammation can be modulated by the dose and administration pattern of DSS. In this study, we first established a chronic and progressive colitis model by administration of increasing concentrations of DSS during a period of 28 days and compared the response between knock-in mice carrying the G2019S LRRK2 mutation (GS LRRK2) and control wild-type mice (WT LRRK2) (Fig. 1a). In agreement with the literature, our preliminary analysis suggested that female mice are partially protected against DSS [2, 22]. Since analysing both sexes separately was deemed necessary for a thorough investigation, we decided to exclusively utilise male mice in all subsequent experiments to ensure the appropriate statistical power.

Our DSS protocol moderately reduced the body weight but did not cause lethality, with no differences between GS LRRK2 and WT LRRK2 mice (Sup Fig. 1a). The severity of the colitis was scored three times per week by assessing the stool consistency and presence of blood in the stool (Sup Fig. 1b, c). Both parameters, as well as the combined score (disease index score), show that our chronic administration scheme induced mild experimental colitis in WT mice (Fig. 1b). Nevertheless, a significantly increased sensitivity was observed in the GS LRRK2 mice, which developed severe colitis symptoms. Additionally, the GS LRRK2 mice presented severe shrinkage of the colon, which is linked to the severity of the inflammation (Sup Fig. 1d). The histological score based on crypt architecture, infiltration of immune cells, presence of oedema and goblet cell depletion also demonstrated more damage to the colonic epithelium after DSS in the GS LRRK2 mice compared to the controls (Fig. 1c, d).

The G2019S LRRK2 mutation has been reported to increase kinase activity by approximately 4 times [65]. No difference in the basal LRRK2 protein levels was found in the colon of untreated GS and WT LRRK2 mice (Fig. 1e, f). In response to DSS, however, a significant increase in LRRK2 was found in the colon of the GS LRRK2 mice. In order to assess the kinase activity of LRRK2, we measured the autophosphorylation on Serine 1292 (Fig. 1g). Phosphorylation on S-1292 was significantly increased in GS LRRK2 mice, but to the same extent in control and DSS conditions, indicating that the kinase activity of LRRK2 remains unaltered during colitis.

To characterise the inflammation in the colon we first used flow cytometry immunophenotyping (Fig. 2a). Different populations of immune cells were quantified according to the gating strategy shown in the Supplementary Fig. 2. An overall increase in leukocytes (CD45⁺ cells) was found in the GS animals after DSS treatment. When looking at different subpopulations, we observed a significantly higher response in the GS LRRK2 animals treated with DSS for the non-classical (Ly6C⁻) monocytes, dendritic cells (CD11c⁺) and the lymphoid population (CD45^hi, CD11b⁻), more specifically the CD8⁺ and CD4⁺ T cells. A similar trend that did not reach statistical significance was found for the myeloid cells (CD45^hi, CD11b⁺), the classical (Ly6C⁺) monocytes and the granulocytes (defined as Ly6G⁺ , Ly6C⁺ , CD11b⁺ cells).

Next, we performed immunostaining in the distal colon for macrophages (F4/80), neutrophils (MPO) and for the major histocompatibility complex class II (MHC II) (Fig. 2b). In agreement with the higher level of inflammation detected by flow cytometry, the three markers were significantly increased in the GS LRRK2 mice treated with DSS (Fig. 2c, d, e). Finally, a panel of cytokines was measured in colon homogenates from these animals (Fig. 3). An overall increase in cytokine levels was present in GS LRRK2 mice after experimental colitis. More specifically, significant differences were found between WT and GS LRRK2 mice for IL-33, CXCL10, CCL2, CCL3, CXCL2, IL-10, IL-12p70, IL-2 and TNF-α, as well as a consistent trend for IFN-γ, IL-1β, IL-5, IL-6 and CXCL1.

We hypothesised that the presence of mutant LRRK2 specifically in the immune cells might be responsible for the exacerbated inflammatory response of the GS mice towards DSS. Therefore, we transplanted the bone marrow of WT mice into lethally irradiated WT (WT^WT) and GS LRRK2 mice (GS^WT), as well as GS bone marrow into recipient GS LRRK2 (GS^GS) mice. To control for proper engraftment, we transplanted in parallel bone marrow from C57BL/6-Ly5.1 mice carrying the CD45.1 alloantigen of CD45 to our GS and WT LRRK2 mice which express CD45.2. 10 weeks post transplantation, engraftment was evaluated through flow cytometry (Fig. Sup. 3a). An average of 98.7% of engraftment was measured in every control animal. Next, we induced experimental colitis in the WT^WT, GS^GS and GS^WT mice 10 weeks after bone marrow transplantation. The disease index score of the GS^WT group was improved compared to the GS^GS group and reaching similar levels as the WT^WT mice (Fig. 4a). The colon length was normalised in the GS^WT mice treated with DSS (Fig. Sup. 3b). Also, the histological score confirmed a reduction in the colonic epithelial damage in GS^WT mice (Fig. 4b; Fig. Sup. 3c).

Flow cytometry analysis of the colon revealed overall comparable immune cell numbers in the WT^WT and GS^WT animals in contrast to the increase seen in the GS^GS mice (Fig. 4c). More specifically, a rescue was found by the WT bone marrow transplantation in the GS^WT mice for total myeloid cells (CD45⁺ CD11b⁺), Ly6C⁺ and Ly6C⁻ monocytes, granulocytes (Ly6G⁺), dendritic cells (CD11c⁺), MHC II + leukocytes, NK cells (CD161⁺), CD4⁺ T cells, CD25⁺ expressing CD4 T cells and B cells (CD19⁺). These results demonstrate that the increased inflammation present in the GS LRRK2 mice after DSS is completely rescued by the bone marrow transplantation, suggesting that the DSS response is modulated by the activity of LRRK2 in the immune cells.

To determine whether or not the increased LRRK2 kinase activity is responsible for the enhanced DSS response in the GS mice, we treated the animals with the LRRK2 inhibitor MLi-2 [19]. MLi-2 was administered using an in-diet treatment protocol as previously described for 30 days starting 2 days before the DSS treatment [49]. The dose of ingested compound was estimated at 25 mg/kg/day (mean per animal). The treatment was well tolerated and no differences in animal weight between the treatment and placebo groups were observed (Fig. Sup 4a). LRRK2 inhibition was confirmed by quantifying LRRK2 dephosphorylation at S935 in colon through western blot (Fig. Sup 4b). Our protocol results in a reduction of 80% in the phosphorylation of S935, which suggests partial inhibition of LRRK2.

Pharmacological inhibition of LRRK2 significantly ameliorated the colitis phenotype based on the disease index score, especially in the last two weeks of the treatment (Fig. 5a). Nevertheless, protection with the treatment was partial as the MLi-2-treated animals still responded more severely than the WT LRRK2 mice. The length of the colon was not significantly altered by the inhibition of LRRK2, suggesting that LRRK2 kinase inhibition at this dose and administration route could not completely counteract the GS mutation effect (Fig. Sup. 4c).

Quantification of the immune cells in the colon by flow cytometry revealed a reduction in the MLi-2 treated mice for the different immune populations tested, with the exception of Ly6C⁻ monocytes, which did not reach statistical significance (Fig. 5b). Overall, the results suggest a reduction of inflammation in the MLi-2-treated GS mice.

Inflammatory bowel disease has been reported to increase the risk for developing PD by 20–90% [48, 54, 60]. Furthermore, anti-inflammatory treatment of IBD patients with anti-TNF-α significantly reduces this risk [50]. Despite being protected by the blood–brain barrier, communication between the brain and the peripheral organs has been described. Inflammation in the gastrointestinal tract can have an effect on the central nervous system (CNS) homeostasis and induce neuroinflammation [26, 57]. To study the neuroinflammatory status after experimental colitis, we performed flow cytometry of the brain (Fig. 6a; Fig. Sup. 5). Following our chronic DSS administration protocol, modest neuroinflammation was observed in the GS LRRK2 but not WT mice. While no significant differences were found in the total number of microglial cells (CD45^med, CD11b⁺), a higher number of microglia expressing MHC II were detected in the GS animals compared to the WT mice. This microglial response was accompanied by the infiltration of peripheral immune cells. Indeed, the GS mice presented a higher number of total myeloid cells (CD45^hi, CD11b⁺) including both Ly6C⁺, Ly6C⁻ and MHC II⁺ monocytes. In addition, the infiltration of lymphocytes (CD45^hi, CD11b⁻) was also increased in the GS mice, with similar trends found for both CD4⁺ and CD8⁺ T cells. We hypothesise that LRRK2 might be a key player in this gut-to-brain communication. Thus, we decided to explore whether DSS-induced inflammation could potentially compromise neuronal health. First, we checked dopaminergic cell viability after chronic DSS treatment and found no differences in TH⁺ cell counts in WT or GS mice (Fig. Sup. 6). Since dopaminergic neurodegeneration might require a combination of deleterious triggers, we decided to combine the DSS-colitis with our in-house PD mouse model based on human α-synuclein overexpression in the substantia nigra using recombinant adeno-associated vectors (rAAV 2/7). This model displays progressive dopaminergic neurodegeneration, behavioural deficits and neuroinflammation [46]. Experimental colitis was induced using the chronic DSS administration protocol starting 7 days after vector injection (Fig. 6b). The disease index score confirmed the significantly stronger phenotype in the GS LRRK2 mice (Fig. Sup.7).

Motor behaviour was examined using different tests. Rotarod test was used to evaluate the muscular coordination and grip strength of the animals 5 weeks after rAAV injection (Fig. 6c). Remarkably, a significant decrease in motor performance was found in the GS injected animals compared to the WT controls in the absence of DSS. DSS increased the motor phenotype in the WT mice, but no additional effect was observed in the GS mice. In parallel, the cylinder test was used to measure the forelimb asymmetry at 3 and 5 weeks post-injection (Fig. 6d). We observed a progressive reduction in the use of the contralateral left paw which only reached significance at the last time point between the WT and GS mice injected with α-synuclein rAAV combined with DSS, suggesting a synergistic effect between the α-synuclein overexpression and the colon inflammation.

Stereological quantification of the dopaminergic neurons in the substantia nigra corroborated the behavioural data (Fig. 6e, f). A trend towards increased neurodegeneration was observed in the GS LRRK2 mice after α-synuclein overexpression. Furthermore, DSS significantly aggravated this TH⁺ cell loss in the GS LRRK2 mice but not in the WT control mice. These results confirm that in GS LRRK2 mice, DSS-experimental colitis can aggravate α-synuclein-mediated dopaminergic neurodegeneration.

Below is the link to the electronic supplementary material.

The authors declare that they have no competing interests.

All animal experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by the Bioethical Committee of the KU Leuven (Belgium) (ECD projects 051/2020 and 156/2022).

Authors consent for the publication. Manuscript was read and approved by all authors.

All data generated or analysed during this study are included in this published article [and its supplementary information files].