Table of Contents
Liver biopsy tissues
PD patients carrying GBA1 variants (GBA1-PD) were recruited from the Department of Neurology of the First Affiliated Hospital of Zhengzhou University, and the gender-matched PD patients without GBA1 variants (nGBA1-PD) were recruited as positive controls. The GBA1 variants were confirmed using polymerase chain reaction (PCR) followed by Sanger sequencing. All the patients were diagnosed by two experienced neurologists. Additionally, the liver biopsy tissues from non-PD patients with abnormal liver function but no neurological disorders were used as controls (nPD-Control). Informed consent was obtained from all participants. Moreover, the acquisition of paraffin biopsy specimens was approved by the ethics committees of the First Affiliated Hospital of Zhengzhou University and the First Affiliated Hospital of University of South China.
Animals
The Gba1L444P/+ mice were constructed with a C57BL/6J background by Shanghai Model Organisms Center, Inc. (Shanghai, China) using CRISPR/Cas9 technology. Based on the gene structure of Gba1, the Gba1-202 transcript was chosen for analysis. This transcript consists of 12 exons, with protein translation starting at exon 3 and ending at exon 12. Through analysis of human GBA1 L444P, it was found that the corresponding mouse mutation is L462P, which is located in exon 11 of the Gba1-202 transcript. The mutation information can be described as follows: the wild-type sequence is GAC-TTG-GAA (D-L-E), while the mutant sequence is GAC-CCG-GAA (D-P-E). In brief, in vitro transcription of Cas9 mRNA was performed using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, TX, USA), followed by purification with the MEGAclearTM Kit (ThermoFisher, USA). The desired Cas9-targeted guide RNA with the sequence 5’-GGCCAGTGAGAGCACTGACTTGG-3’ was transcribed and subsequently purified in vitro. The obtained F0 mice were characterized by PCR and sequencing using primer pairs: forward, 5’-GGGAAGTGACAAGGTGGCAT-3’ and reverse, 5’-GTGACACGGTCTCCAGGAAG-3’. F0-positive mice were bred with C57BL/6 J mice to generate the F1 generation, with genotypes confirmed by PCR and sequencing. C57BL/6J mice (WT) were used as normal controls. All mice were maintained under specific pathogen-free conditions and fed a standard chow diet consisting of 24.2% protein, 12.4% fat, and 63.4% carbohydrates. Animal care and experimental procedures were conducted in accordance with the guidelines of the Care and Use Committees of Zhengzhou University and approved by the Institutional Ethics Committees of the First Affiliated Hospital of Zhengzhou University (2021-KY-1013-002).
GCase enzyme activity assay
The liver tissue was processed according to the ratio of tissue mass (g) to extraction volume (mL) of 1:5 ~ 10 (approximately 0.2 g of tissue was taken and added to 1 mL of extraction solution). The mixture was homogenized on an ice bath and then centrifuged at 15,000 × g, 4 °C for 20 min. The supernatant was collected and kept on ice for subsequent analysis. The GCase enzyme activity was then measured using a β-glucosidase Activity Assay Kit (BC2560, Solarbio, Beijing, China) following the manufacturer’s protocols.
Mice peripheral blood smear
Peripheral blood smears were prepared from mouse blood samples and stained with Giemsa solution (C0131, Beyotime, China). After air drying, the smears were fixed in 70% ethanol for 10 min. The slides were then stained with a 1× modified Giemsa working solution for 45 min. Staining time or solution concentration was adjusted as necessary to avoid over- or under-staining. After staining, the slides were thoroughly rinsed with distilled water, air dried, and examined under an Olympus IX51 microscope equipped with a DP71 digital camera for image capture.
Dairy diet-fed mice model
Besides the feed needed for growth, the diet of the mice was managed through changing the composition of the drinking water. 3-month-old Gba1L444P/+ and WT mice were fed with a commercially available pure milk, which contains 3.8 g protein, 4.6 g fat, and 5.5 g carbohydrates per 100 mL. For the dairy-free diet, moderate dairy diet, and dairy-rich diet mouse models, the mice were supplemented with purified water, 20% volume fraction of the pure milk, and 100% volume fraction of the pure milk, respectively. Age-matched Gba1L444P/+ and WT mice were fed with a standard laboratory normal chow diet served as controls. Each mouse is fed with 10 mL of the milk every day, and the milk is changed every 24 h.
Lactose, whey protein, calcium, casein, and calcium/casein diet-fed mice models
Three-month-old Gba1L444P/+ and WT mice were fed a diet consisting of 20% lactose (L8911, Solarbio, Beijing, China), 20% hydrolyzed whey protein (LP0048B, Solarbio, Beijing, China), 5% of a 1 mol/L sterile calcium chloride solution (G0070, Solarbio, Beijing, China), 20% hydrolyzed casein (LP0041B, Solarbio, Beijing, China), and a combination of 20% hydrolyzed whey protein and 5% of a 1 mol/L sterile calcium chloride solution, respectively. Age-matched Gba1L444P/+ and WT mice receiving a standard laboratory chow diet served as controls. Each mouse was administered 10 mL of the aforementioned solution daily, with the solution replaced every 24 h.
Immunohistochemical staining
Liver, colon, and brain tissues from perfused WT and Gba1L444P/+ mice were fixed in 70% ethanol solution and a mixture of 30% sucrose and 4% paraformaldehyde solution, respectively, after which the tissues were embedded in paraffin blocks and cut into 4 μm-thick sections using the Rotary Microtome (Leica RM2235, Leica, Nussloch, Germany) and then the sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Next, the tissues were placed in a 3% hydrogen peroxide solution and treated with citrate buffer at 100 °C for 5 min. The tissues were then blocked with normal goat serum at room temperature for 30 min. Next, primary antibodies were added, and the tissues were incubated overnight at 4 °C. Following that, corresponding secondary antibodies were added and incubated for 1 h at room temperature. Finally, the signal was visualized using 3,3’-diaminobenzidine, and images were captured using an Olympus IX51 microscope equipped with a DP71 Olympus digital camera.
Protein extraction and WB analysis
Liver and brain samples, including DNV, SNC, STR, and PFC, were collected. The samples were homogenized in 5× v/w RIPA buffer, which consisted of 50 mM Tris–HCl (pH 8.0), 1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, and 0.1% SDS. To preserve the protein integrity, a protease inhibitor cocktail (Roche, Switzerland) was added to the RIPA buffer. Additionally, the cultured cells were lysed in cell lysis buffer (#P0013, Beyotime, China).
For WB analysis, the homogenized samples were boiled for 10 min to denature the proteins, and then separated on 8-12% SDS-PAGE gels. Subsequently, the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The PVDF membranes were then blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature with slow agitation. After blocking, the membranes were incubated with primary antibodies overnight at 4 °C. Following the primary antibody incubation, the membranes were then washed with TBST three times and incubated with HRP-linked secondary antibodies for 2 h at room temperature. Finally, the membranes were visualized using an enhanced chemiluminescence kit (ThermoFisher Scientific, USA).
Tissue immunofluorescence and liver function detection
Liver and colon tissues were embedded in paraffin blocks and cut into 4-μm slices using a Rotary Microtome (Leica RM2235, Leica, Germany). The slices were deparaffinized and rehydrated in a graded ethanol series. Antigen retrieval was performed by subjecting the slices to citrate buffer at 100 °C for 5 min, after which the slices were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 0.5% BSA for 30 min at room temperature. Then, primary antibodies were added and incubated overnight at 4 °C. Following the primary antibody incubation, the slices were washed three times with PBS for 5 min each at room temperature. Subsequently, fluorochrome-conjugated secondary antibodies were applied and incubated for 3 h at room temperature. After washing off the unbound secondary antibodies, the slices were stained with Hoechst 33258 to label the nuclei. Finally, the immunofluorescence images were captured using an LSM-980 confocal laser microscope (Carl Zeiss, Germany) to visualize the target proteins within the liver tissues. Blood samples were centrifuged at 3000 rpm for 15 min at 4 °C to obtain serum, and liver function was assessed using ALT (#MAK052, Sigma-Aldrich) and AST (#MAK055, Sigma-Aldrich) assay kits.
Proteomic TMT labeling and UHPLC-MS/MS analysis
TMT-based quantitative proteomic analysis was conducted to identify and quantify proteins in liver tissues. Liver samples from DF Gba1L444P/+ and CC Gba1L444P/+ mice were individually ground in liquid nitrogen and lysed in SDT buffer (containing 100 mM NaCl) with 1/100 volume of DDT, followed by 5 min of ultrasonication on ice. Samples were incubated at 95 °C for 10 min and then cooled on ice for 2 min. The lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatants were treated with an excess of IAM at room temperature in the dark for 1 h. Next, the samples were mixed with four times their volume of pre-cooled acetone and incubated at −20 °C for 2 h. The samples were centrifuged again at 12,000 × g for 15 min at 4 °C, and the precipitates were washed with 1 mL of cold acetone and dissolved in Dissolution Buffer. For TMT labeling, the samples were reconstituted with 100 μL of 0.1 M TEAB buffer. Subsequently, 41 μL of acetonitrile-dissolved TMT labeling reagent was added, and the mixture was shaken for 2 h at room temperature. The reaction was terminated by adding 8% ammonia. To ensure equal volumes, all labeled samples were combined, followed by desalting and lyophilization. Finally, UHPLC-MS/MS analyses were performed using an EASY-nLC TM 1200 UHPLC system (Thermo Fisher, Germany) coupled with a Q ExactiveTM HF-X (Thermo Fisher, Germany) at Novogene Co., Ltd. (Beijing, China).
Cell culture
Immortalized mouse KCs were purchased from BeiNa Biological Company (BNCC340733, Beijing, China). KCs were cultured in RPMI-1640 complete medium supplemented with 10% (v/v) FBS at 37 °C with 5% CO2.
Lentivirus construction
The lentiviruses were constructed by Shanghai Genechem Co. (Shanghai, China). Briefly, the lentiviral expressing shRNA targeting the sequence of Gba1 gene (the target sequences were: shRNA#1, gcATTGCTGTTCACTGGTATA; shRNA#2, gcACAGGGTTACTACTCACTT; and shRNA#3, ctTATGCTAAGTATGGCCTAA) and NC (TTCTCCGAACGTGTCACGT) were synthesized and cloned into GV493 (hU6-shRNA-CBh-gcGFP-IRES-puromycin) vector with AgeI and EcoRI sites. And the recombinant vectors were detected by DNA sequencing.
Lentivirus infection
KCs were infected with the lentiviruses according to the manufacturer’s instructions. Specifically, when the cell confluence reached 20-30%, KCs were infected with lentiviruses at a multiplicity of infection (MOI) of 10, in the presence of 40 μL/mL Hitrans GP. After 16 h, the old medium was replaced with fresh complete medium, and the culture was continued until 72 h after infection.
Detection of oxidative stress activation
Intracellular ROS level was detected using a kit according to the manufacturer’s instructions (S0033S, Beyotime, China). Briefly, KCs without treatment and treated with calcium/casein were washed three times with PBS, and then the DCFH-DA probe was added with a final concentration of 10 μM to each well, incubated at 37 °C for 30 min. The cells were then washed three times with PBS to remove DCFH-DA that did not enter the cells. The ROS level was estimated according to the fluorescence.
FITC-α-syn PFFs preparation and imaging
To obtain PFFs, mouse FITC-α-syn (Cusabio, Wuhan, China) was dissolved in assembly buffer (20 mM Tris-HCl, 100 mM NaCl, pH 7.4) at a concentration of 1 mg/mL. The solution was transferred into a sealed 2-mL sterile polypropylene tube and incubated on an orbital thermomixer with a heating lid at 37 °C, shaking at 1,000 rpm, for 7 days. After incubation, the solution was sonicated for 45 s using an ultrasonic cell disruptor at 20% amplitude (Scientz-IID). α-syn PFFs were then characterized using a JEOL 1400 transmission electron microscope equipped with Gatan Orius CCD cameras. Briefly, the PFFs were adsorbed onto carbon-coated 200-mesh grids and stained with 1% uranyl acetate, keeping the grids dry throughout. Prior to use, α-syn PFFs were stored at −80 °C.
Cell immunofluorescence
After treatment, cells on coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 for 20 min, and blocked with 5% FBS for 1 h. Cells were then incubated with primary antibodies overnight at 4 °C. Following washes, nuclei were stained with Hoechst 33258, and images were acquired using an LSM-980 confocal laser microscope (Carl Zeiss, Germany). Immunofluorescence intensity was quantified by averaging the fluorescence signals from cells in 6 randomly selected fields at ×400 magnification. Each experiment was independently repeated three times.
KCs isolation and identification
To isolate mouse KCs, liver tissue was perfused with Hank’s buffer, enzymatically digested with 0.5 mg/mL collagenase IV, and minced in a culture dish. The tissue was incubated at 37 °C for 20 min, then filtered to obtain a single-cell suspension. Cells were washed with PBS by centrifugation at 300 × g for 5 min at 4 °C. The pellet was resuspended in PBS and centrifuged at 50 × g for 5 min at 4 °C, followed by another centrifugation step at 300 × g for 5 min at 4 °C. The final pellet was resuspended in buffer. For KCs separation, a gradient was prepared with a 2 mL bottom layer of 70% Percoll, a 2 mL middle layer of 30% Percoll, and a 2 mL top layer of the cell suspension, centrifuged at 600 × g for 20 min at 4 °C. The middle layer containing a white membrane-like fraction was collected and washed twice with PBS by centrifugation at 300 × g for 5 min at 4 °C to remove residual Percoll. The final cell pellet was resuspended in complete DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, then plated onto culture dishes. After 4 h of incubation at 37 °C with 5% CO2, the medium was replaced to remove non-adherent cells, leaving the adherent KCs population.
For KCs identification and purity assessment, the isolated cells were characterized through immunofluorescence staining for CD68 (a lysosomal marker highly expressed in KCs) and multiparameter flow cytometry analysis using a panel of surface markers including CD31 (endothelial cell marker for negative exclusion), CD45 (leukocyte marker), F4/80, and CD11b (myeloid cell marker), with the population of CD31-CD45+F4/80+CD11bint cells being identified as KCs. This combined approach allowed for both morphological visualization and quantitative analysis of KCs purity at single-cell resolution.
NADP+/NADPH detection
The NADP+/NADPH colorimetric detection kit (S0179, Beyotime, China) was used according to the manufacturer’s protocol. Briefly, isolated mouse KCs were resuspended in NADP+/NADPH extraction buffer, followed by centrifugation at 12,000 × g for 10 min at 4 °C. The supernatant was collected for subsequent analysis. The preparation of the NADPH standard solution, the generation of the standard curve, and the G6PDH working solution were performed as instructed. To degrade NADP+, 100 μL of the supernatant was heated at 60 °C in a water bath. The blank control, NADPH standard, and samples were then transferred to a 96-well plate, with 100 μL of G6PDH working solution added to each well. After incubation at 37 °C in the dark for 10 min, 10 μL of the color reagent was added, followed by further incubation at 37 °C for 15 min. Absorbance was measured at 450 nm, and the NADP+/NADPH ratio was calculated according to the kit instructions.
Behavioral tests
The rotarod test was conducted using the rotarod apparatus (Rotarod YLS-4C; YiYan Science and Technology Development Co., Ltd., Shandong, China) to evaluate the mice’s motor coordination and balance ability. Prior to the test, the mice were given a 30-min acclimation period in the testing room. They were then placed on the rotarod device, which had an accelerating speed mode (increasing from 4 to 40 rpm in 5 min). Three trials were conducted each day, with a 15-min break between each trial. The mean latency to fall off the rotarod was calculated for data analysis. The hanging wire test was conducted to assess muscular strength. The mice were taken to the testing room and given 30 min to adjust before the test. Each mouse was placed on a wire mesh and gently shaken to enable it to grasp the wire and invert. The time it took for the mouse to fall from the wire was recorded, with a maximum time limit of 300 s. Three trials were performed for each mouse, and the average time was used for statistical analysis. The elevated plus maze test was carried out to evaluate anxiety-like behavior. The device was raised 40 cm above the ground, comprising two open arms (25 × 5 × 15 cm), two closed arms (25 × 5 × 15 cm), and a central square area (5 × 5 cm). Each mouse was placed alone in the central square area of the elevated plus maze device and was allowed to explore freely for 10 min. The time spent on the open arms was automatically recorded by the VisuTrack software and analyzed. The open field test was utilized to evaluate the spontaneous motor function and anxiety behavior. Each mouse was put individually in the middle of an open field device and was enabled to explore freely for 10 min. The tests were conducted in a dim and quiet room. The overall distance and the time spent in the central region were automatically recorded by the VisuTrack software and analyzed. The new object recognition test was carried out to inspect the memory capacity. On the first day, the mice were placed in a bright testing arena (40 × 40 × 40 cm) and allowed to explore for 10 min in order to become accustomed to the new environment. After 24 h, two identical objects (A1 and A2) were placed in the arena at the relative position, and the mice were allowed to explore the objects for 10 min. On the third day, one of the identical objects (A2) was substituted with a new object (B), and the mice were placed into the arena once again to explore the new object within 10 min. The time spent on the familiar object (A1) and the new object (B) was recorded.
Mouse liver transplantation
Orthotopic mouse liver transplantation was performed using a combined cuff and suture technique without hepatic artery reconstruction under inhalation anesthesia, as described in detail60. For the liver transplantation experiment, Gba1L444P/+ mice fed with a calcium/casein diet for one month were used as donors, and WT mice were used as recipients. WT mice that received liver transplantation were sampled at postoperative week 6. The pα-syn pathology of the liver and brain was evaluated by immunohistochemistry.
Hepatic denervation
An upper midline abdominal incision was made to expose the hepatic vagal and sympathetic nerves in 2-month-old Gba1L444P/+ mice under inhalation anesthesia. The common hepatic branch of the vagal nerve was isolated and transected. For hepatic sympathetic nerve denervation, a sterile cotton-tipped applicator saturated with a 10% phenol solution in ethyl alcohol was applied carefully to the surface of the hepatic artery and portal vein bundle. The abdominal cavity was then rinsed with 0.9% saline solution to remove any residual phenol. In the sham operation group, the common hepatic branch of the vagal nerve was exposed but not cut, and a sterile cotton-tipped applicator soaked in 0.9% saline was applied similarly to the experimental group. Following nerve dissection, Gba1L444P/+ mice were fed a diet high in calcium and casein and were sampled three months postoperatively.
Antibodies
The antibodies that used in this study are summarized in Supplementary Table 1.
Statistical analyses
The mean fluorescence intensities were measured and calculated using Image-Pro Plus 6.0 software (Media Cybernetics, USA). Protein bands and immunohistochemical analyses were performed by calculating the gray value and positive staining area using ImageJ software (version 2.0, National Institutes of Health), respectively. Statistical analyses were conducted and figures were generated using GraphPad 9.0 Prism (GraphPad Software, USA). Prior to analysis, normality tests and homogeneity tests of variance were performed on continuous variables. The Shapiro-Wilk normality test was conducted to assess whether the groups were drawn from a normally distributed population. For normally distributed datasets, statistical comparisons were performed as follows: unpaired two-tailed Student’s t-tests for comparing two groups; one-way ANOVA with Dunnett’s post hoc test for multiple comparisons when analyzing more than three groups with a single variable; and two-way ANOVA with Tukey’s post hoc test for datasets involving more than three groups with two variables. A significance level of P < 0.05 was considered statistically significant. The levels of significance were denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns (not significant).
