Journal: Frontiers in Neuroscience

Article Title: Exploring the relationship between GBA1 host genotype and gut microbiome in the GBA1 L444P/WT mouse model: implications for Parkinson’s disease pathogenesis

doi: 10.3389/fnins.2025.1546203

Figure Lengend Snippet: Gut microbiome alterations in GBA1 L444P/WT and GBA1 WT/WT mice. (A) Differences in species (MSP) richness between groups (WT/WT and L444P/WT) at 3 months and 6 months. (B) Differences in species (MSP) richness within the WT/WT group and the L444P/WT group over time. PCoA performed on the Bray–Curtis dissimilarity index at 3 months (C) and 6 months (D) . (E) Differences in the Bray–Curtis dissimilarity index between the WT/WT and L444P/WT groups at different time points. (F) Weighted UniFrac distance metric within the WT/WT and L444P/WT groups at different time points. (G) Microbial species abundance between WT/WT and L444P/WT animals at 6 months: left, diverging bar plots show the relative abundance of species in WT/WT and L444P/WT according to the effect size measured by the Cliff’s Delta; right, bar charts show the percentage of prevalence of species between groups. Significant differences, determined using Wilcoxon’s signed-rank test and Fisher’s exact test, are indicated by a star above each box plot and bar plot (* = p < 0.05, ** = p < 0.01, and *** = p < 0.001).

Article Snippet: Experimental model: Organism/ strain , GBA1 L444P/WT , B6;129S4-Gba1tm1Rlp/Mmnc (strain name); Mutant Mouse Resource & Research Centers (vendor); 000117-UNC (catalog number); RRID:MMRRC_000117-UNC (RRID) , https://www.mmrrc.org/catalog/sds.php?mmrrc_id=117 , reuse , .

Techniques:

Journal: NPJ Parkinson's Disease

Article Title: Dairy-rich diet triggers hepatic α-synuclein pathology via the liver-brain axis in GBA1 -related Parkinson’s disease

doi: 10.1038/s41531-025-01211-9

Figure Lengend Snippet: a Representative immunohistochemistry images of pα-syn in the liver sections from n GBA1 -PD and two GBA1 L444P-PD patients (left), and quantification of pα-syn positive staining area (right). Black arrows point to the region of positive pα-syn staining. Scale bars, 50 μm. Data are presented as means ± s.d. (error bars), n = 5 microscope fields of view per subject. P -values from a two-tailed Student’s t -test. b Representative immunohistochemistry images of pα-syn in the liver of 1-month DF Gba1 L444P/+ , MD Gba1 L444P/+ , and DR Gba1 L444P/+ mice, respectively (left), and quantification of pα-syn positive staining area (right). Black arrows point to the region of positive pα-syn staining. Scale bars, 50 μm. n = 6 mice/group, 3 males and 3 females. P -values from One-way ANOVA followed by Dunnett’s multiple comparisons test. c Representative immunohistochemistry images of pα-syn in the livers of DF and DR WT mice, as well as DF, lactose, whey protein, calcium, casein, and CC Gba1 L444P/+ mice (up), and quantification of the pα-syn positive staining area (lower left). Black arrows point to the region of positive pα-syn staining. Scale bars, 50 μm. Data are presented as means ± s.e.m (error bars), n = 4 mice per group (2 males and 2 females). P -values from One-way ANOVA followed by Dunnett’s multiple comparisons test. Comparison of hepatic pα-syn positive staining area between DR Gba1 L444P/+ and CC Gba1 L444P/+ mice (lower right). P -values from a two-tailed Student’s t -test. d Representative WB analysis of pα-syn in DF, lactose, whey protein, calcium, casein, and CC Gba1 L444P/+ mice (up), and quantification of the pα-syn expression levels (down). GAPDH was used as a loading control. Data are presented as means ± s.d. (error bars), based on 6 panels from 3 independent experiments. P values from One-way ANOVA followed by Dunnett’s multiple comparisons test. e Representative double-labeled immunofluorescence images of pα-syn (red) co-stained with F4/80 (green), CYP2E1 (green), or Desmin (green) in the liver of DF Gba1 L444P/+ mice (i, ii, and iii) and CC Gba1 L444P/+ mice (iv, v, and vi). The white arrow points to the region of colocalization. Hoechst 33258 (blue) was used for nuclear staining. Scale bars, 20 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The Gba1 L444P/+ mice were constructed with a C57BL/6J background by Shanghai Model Organisms Center, Inc. (Shanghai, China) using CRISPR/Cas9 technology.

Techniques: Immunohistochemistry, Staining, Microscopy, Two Tailed Test, Comparison, Expressing, Control, Labeling, Immunofluorescence

Journal: NPJ Parkinson's Disease

Article Title: Dairy-rich diet triggers hepatic α-synuclein pathology via the liver-brain axis in GBA1 -related Parkinson’s disease

doi: 10.1038/s41531-025-01211-9

Figure Lengend Snippet: a Schematic diagram of the quantitative proteomics analysis process. b , c Heatmap ( b ) and volcano plots ( c ) results of liver proteomic analysis from DF Gba1 L444P/+ and CC Gba1 L444P/+ mice. n = 3 mice/group, 2 males and 1 female. d Representative WB analysis of NNT in KCs treated with PBS or calcium/casein, respectively (left), and quantification of NNT expression levels (right). Blots were probed for β-actin as a loading control. e Representative immunofluorescence images of ROS particles (green) in KCs treated with PBS or calcium/casein, respectively (left), and quantitative analysis of the relative fluorescence intensities (right). Scale bars, 10 μm. Data are presented as means ± s.e.m (error bars), n = 6 independent culture holes. P -values from a two-tailed Student’s t -test. f Diagram of KCs extraction from mouse liver. g Relative NADP + /NADPH levels of KCs extracted from DF WT, CC WT, DF Gba1 L444P/+ , and CC Gba1 L444P/+ mice, respectively. Data are presented as means ± s.d. (error bars), n = 6 independent experiments. P -values from Two-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05, **** P < 0.0001.

Article Snippet: The Gba1 L444P/+ mice were constructed with a C57BL/6J background by Shanghai Model Organisms Center, Inc. (Shanghai, China) using CRISPR/Cas9 technology.

Techniques: Quantitative Proteomics, Expressing, Control, Immunofluorescence, Fluorescence, Two Tailed Test, Extraction

Journal: NPJ Parkinson's Disease

Article Title: Dairy-rich diet triggers hepatic α-synuclein pathology via the liver-brain axis in GBA1 -related Parkinson’s disease

doi: 10.1038/s41531-025-01211-9

Figure Lengend Snippet: a Representative WB analysis of LC3, Beclin-1, and p62 in KCs infected with NC and shRNA#1 lentiviruses (left), and quantification of LC3-II/I ratio, Beclin-1, and p62 expression levels (right). Blots were probed for β-actin as a loading control. Data are presented as means ± s.d. (error bars), n = 6 panels in 3 independent experiments. P -values from a two-tailed Student’s t -test. b Schematic diagram of the cell processing procedure. c Representative double-labeled immunofluorescence images of F4/80 (red) and FITC-α-syn (green) in KCs infected with NC (i) or ShRNA#1 (ii) lentivirus, and co-cultured with FITC-α-syn PFFs (iii and iv), and further treated with calcium and casein (v and vi), respectively. The white arrow points to the region of colocalization. Hoechst 33258 (blue) was used for nuclear staining. Scale bars, 10 μm. d Quantification of pα-syn expression levels in cells. Blots were probed for β-actin as a loading control. Data are presented as means ± s.d. (error bars), n = 6 panels in 3 independent experiments. P -values from Two-way ANOVA followed by Tukey’s multiple comparisons test. e Representative WB analysis of NNT, LC3, Beclin-1, p62, and pα-syn in KCs of DF WT, CC WT, DF Gba1 L444P/+ , and CC Gba1 L444P/+ mice, respectively (left), and quantification of NNT, LC3-II/I ratio, Beclin-1, p62, and pα-syn expression levels (right). Blots were probed for GAPDH as a loading control. Data are presented as means ± s.d. (error bars), n = 6 panels in 3 independent experiments. P values from Two-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The Gba1 L444P/+ mice were constructed with a C57BL/6J background by Shanghai Model Organisms Center, Inc. (Shanghai, China) using CRISPR/Cas9 technology.

Techniques: Infection, shRNA, Expressing, Control, Two Tailed Test, Labeling, Immunofluorescence, Cell Culture, Staining

Journal: NPJ Parkinson's Disease

Article Title: Dairy-rich diet triggers hepatic α-synuclein pathology via the liver-brain axis in GBA1 -related Parkinson’s disease

doi: 10.1038/s41531-025-01211-9

Figure Lengend Snippet: a Representative immunohistochemistry images of pα-syn in the liver of DF WT, CC WT, DF Gba1 L444P/+ , and CC Gba1 L444P/+ mice (left), and quantification of the pα-syn positive staining area (right). Black arrows point to the region of positive pα-syn staining. Scale bars, 50 μm. Data are presented as means ± s.e.m (error bars), n = 6 mice per group (3 males, 3 females). P -values from Two-way ANOVA followed by Tukey’s multiple comparisons test. b Representative immunohistochemistry images of pα-syn in the DNV, SNC, STR, and PFC of DF WT, CC WT, DF Gba1 L444P/+ , and CC Gba1 L444P/+ mice (left), and quantification of the pα-syn positive staining area (right). Black arrows point to the region of positive pα-syn staining. Scale bars, 50 μm. Data are presented as means ± s.e.m (error bars), n = 6 mice per group (3 males, 3 females). P -values from Two-way ANOVA followed by Tukey’s multiple comparisons test. c Representative WB analysis of pα-syn in the DNV, SNC, STR, and PFC of DF WT, CC WT, DF Gba1 L444P/+ , and CC Gba1 L444P/+ mice (left), and quantification of the pα-syn expression levels (right). GAPDH was used as the loading control. Data are presented as means ± s.d. (error bars), with n = 6 samples from 3 independent experiments. P -values from Two-way ANOVA followed by Tukey’s multiple comparisons test. d Behavioral comparisons, including latency to fall in the rotarod test (i), latency to fall in the hanging wire test (ii), time spent in the open arms of the elevated plus maze (iii), total distance traveled (iv), time spent in the center during the open field test (v), and new object recognition time in the novel object recognition test (vi). Behavioral assessments were conducted at baseline, 1 month, and 3 months in DF WT, CC WT, DF Gba1 L444P/+ , and CC Gba1 L444P/+ mice. Data are presented as means ± s.d. (error bars), with n = 6 mice per group (3 males, 3 females). P -values from Two-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The Gba1 L444P/+ mice were constructed with a C57BL/6J background by Shanghai Model Organisms Center, Inc. (Shanghai, China) using CRISPR/Cas9 technology.

Techniques: Immunohistochemistry, Staining, Expressing, Control

Journal: NPJ Parkinson's Disease

Article Title: Dairy-rich diet triggers hepatic α-synuclein pathology via the liver-brain axis in GBA1 -related Parkinson’s disease

doi: 10.1038/s41531-025-01211-9

Figure Lengend Snippet: a Schematic representation of the mouse liver transplantation model (left) alongside representative immunohistochemistry images of pα-syn in the liver, DVN, SNC, STR, and PFC in WT mice post-liver transplantation (right). Scale bars, 50 μm. b Schematic diagram illustrating vagotomy and sympathectomy performed in the liver of Gba1 L444P/+ mice (left), with representative immunohistochemistry images of pα-syn in the liver, DVN, SNC, STR, and PFC in the mice fed with a calcium/casein diet (right). Scale bars, 50 μm. c Schematic depiction of how a rich-dairy diet initiates the propagation of pα-syn pathology along the “liver-brain” axis in CC Gba1 L444P/+ mice (right), compared to the normal physiological condition in DF WT mice (left).

Article Snippet: The Gba1 L444P/+ mice were constructed with a C57BL/6J background by Shanghai Model Organisms Center, Inc. (Shanghai, China) using CRISPR/Cas9 technology.

Techniques: Transplantation Assay, Immunohistochemistry

Journal: Human molecular genetics

Article Title: TFEB regulates lysosomal proteostasis.

doi: 10.1093/hmg/ddt052

Figure Lengend Snippet: Figure 1. Sucrose treatment enhances L444P GC trafficking and activity in GD patient-derived fibroblasts. (A) Time course of relative L444P GC activities in cells treated with a range of concentrations of sucrose for 8 days. GC activities were normalized to the activity of untreated cells (left y-axis) (P , 0.01). The corresponding fraction of wt GC activity is reported (right y-axis). Data are reported as mean+SD (n ¼ 3). (B) Western blot analysis of the total protein content from L444P GC fibroblasts treated with sucrose (150 mM for 4 days) and with EndoH, and detected using a GC antibody. The solid and dashed arrows indicate EndoH-resistant and EndoH-sensitive bands, respectively. Quantification of GC bands was obtained with ImageJ analysis software. Quantification of the lower MW, EndoH-sensitive band corresponding to ER-retained GC is reported in the white portion of the bars; quantification of higher MW, EndoH-resistant bands corresponding to lysosomal GC is reported in the black portions. (C and D) Confocal microscopy analysis of GC and CNX (an ER marker) (C) and GC and LAMP-1 (a lysosomal marker) (D) in L444P GC fibroblasts. Cells were treated with sucrose (150 mM) for 4 days. Colocalization of CNX (blue, column 1) and GC (red, column 2) is indicated in purple (column 3) color. Colocalization of LAMP-1 (blue, column 1) and GC (red, column 2) is indicated in purple (column 3) color. Heatmaps of co-localization images were obtained with ImageJ analysis software (column 4). Hot colors represent positive correlation (co-localization), whereas cold colors represent negative correlation (exclusion). Unt, untreated; Suc, sucrose. The scale bar is 10 mm. (E) Annexin V binding affinity change (%) in L444P GC patient-derived fibroblasts treated with sucrose (150 mM) or MG-132 (0.6 mM) for 16 h normalized to untreated cells (P , 0.01). (F) PI binding population change (%) of cells treated with sucrose (150 mM) or MG-132 (0.6 mM) for 16 h normalized to untreated cells (P , 0.01). The number of total counted cells was 10 000. The data are reported as the mean+SD (n ¼ 3).

Article Snippet: Proteostasis regulators mediate an increase in L444P GC activity by inducing both GBA upregulation and enhancement of the cellular folding capacity (14–16).

Techniques: Activity Assay, Derivative Assay, Western Blot, Software, Confocal Microscopy, Marker, Binding Assay

Journal: Human molecular genetics

Article Title: TFEB regulates lysosomal proteostasis.

doi: 10.1093/hmg/ddt052

Figure Lengend Snippet: Figure 2. Sucrose treatment promotes TFEB nuclear localization in GD patient-derived fibroblasts. (A) Confocal microscopy analysis of TFEB subcel- lular localization in wt and L444P GC fibroblasts transfected with a plasmid encoding mRuby-TFEB and treated with 150 mM sucrose for 24 h. Colocaliza- tion of DAPI (blue, column 1) and mRuby-TFEB (red, column 2) is shown in purple color (column 3). Heatmaps of co-localization images were obtained as described in Figure 1. The scale bar is 5 mm. (B) Percentage of cells with nuclear localization of mRuby-TFEB upon sucrose treatment. Representative fields containing 50–100 cells were analyzed to calculate the percentage of cells with mRuby-TFEB nuclear localization in cells that express mRuby-TFEB (P , 0.01). Unt, untreated. Data are reported as mean+SD (n ¼ 3).

Article Snippet: Proteostasis regulators mediate an increase in L444P GC activity by inducing both GBA upregulation and enhancement of the cellular folding capacity (14–16).

Techniques: Derivative Assay, Confocal Microscopy, Transfection, Plasmid Preparation

Journal: Human molecular genetics

Article Title: TFEB regulates lysosomal proteostasis.

doi: 10.1093/hmg/ddt052

Figure Lengend Snippet: Figure 4. TFEB activation mediates rescue of L444P GC activity in GD patient-derived fibroblasts. (A) Relative L444P GC activities in L444P GC fibroblasts transfected with TFEB and treated with 150 mM sucrose for 4 days. Cell lysates were normalized to the same total protein concentration and GC activities were normalized to the activity of untreated control sample (cells transfected with empty vector pcDNA4TM/TO) (P , 0.01). (B) Relative GC activities of wt fibro- blasts transfected as in (A) and treated with 150 mM sucrose for 3 days. GC activities were calculated as described in (A) (P , 0.01). (C) Relative L444P GC activities of L444P GC fibroblasts transfected with plasmids encoding wt TFEB, TFEB S142A and TFEB-NLS. GC activities were calculated as described in (A) (P , 0.01). (D) Relative GC activities of L444P GC fibroblasts incubated with TFEB siRNA and sucrose (black bars). GC activities were normalized to the activity measured in control cells (control siRNA) (P , 0.05). TFEB mRNA expression level was evaluated by qRT–PCR (white bars) (P , 0.05). Data are reported as mean+SD (n ¼ 3).

Article Snippet: Proteostasis regulators mediate an increase in L444P GC activity by inducing both GBA upregulation and enhancement of the cellular folding capacity (14–16).

Techniques: Activation Assay, Activity Assay, Derivative Assay, Transfection, Protein Concentration, Control, Plasmid Preparation, Incubation, Expressing, Quantitative RT-PCR

Journal: Human molecular genetics

Article Title: TFEB regulates lysosomal proteostasis.

doi: 10.1093/hmg/ddt052

Figure Lengend Snippet: Figure 5. TFEB activation upregulates expression of lysosomal targeting genes. (A) Relative mRNA expression levels of representative genes of the folding and trafficking network in L444P GC fibroblasts treated with 150 mM sucrose for 2, 4 and 7 days. HSPA5 (BiP), CNX, CRT, CHOP, ATF4, LIMP2, M6PR and SORT1 mRNA expression levels were obtained by qRT–PCR, and calculated as described in Fig. 3F (P , 0.05). (B) Relative mRNA expression levels of trafficking genes in L444P GC fibroblasts transfected with TFEB. GBA, LIMP2, M6PR, SORT1 and HGF mRNA expression levels in cells transfected with TFEB were obtained by qRT–PCR, corrected by the expression of the house-keeping genes GAPDH and ACTB, and normalized to those of cells transfected with an empty vector (P , 0.01). (C–F) Relative mRNA expression levels of trafficking genes in L444P GC fibroblasts upon TFEB silencing. Relative mRNA expres- sion levels of GBA (P , 0.01) (C), LIMP2 (P , 0.01) (D), M6PR (P , 0.05) (E) and SORT1 (P , 0.05) (F) in L444P GC fibroblasts treated with TFEB siRNA and sucrose were obtained by qRT-PCR, corrected to the expression of the house-keeping genes GAPDH and ACTB, and normalized to the activity of cells treated with control siRNA. Data are reported as mean+SD (n ¼ 3).

Article Snippet: Proteostasis regulators mediate an increase in L444P GC activity by inducing both GBA upregulation and enhancement of the cellular folding capacity (14–16).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Activity Assay, Control

Journal: Human molecular genetics

Article Title: TFEB regulates lysosomal proteostasis.

doi: 10.1093/hmg/ddt052

Figure Lengend Snippet: Figure 6. LIMP2 mediates TFEB-dependent rescue of L444P GC activity in GD patient-derived fibroblasts. (A) Relative L444P GC activities of cells overex- pressing LIMP2 and treated with 150 mM sucrose for 4 days. GC activities were calculated as described in Fig. 4A (P , 0.01). (B) Relative L444P GC activities of L444P GC fibroblasts treated with LIMP2 siRNA and sucrose. GC activities were calculated as described in Fig. 4D (P , 0.01). (C) Relative L444P GC activities of cells overexpressing LIMP2 and treated with EerI (6 mM) or MG-132 (0.6 mM). GC activities were normalized to the activity of untreated cells trans- fected with an empty vector (P , 0.05). Data are reported as mean+SD (n ¼ 3).

Article Snippet: Proteostasis regulators mediate an increase in L444P GC activity by inducing both GBA upregulation and enhancement of the cellular folding capacity (14–16).

Techniques: Activity Assay, Derivative Assay, Plasmid Preparation