pc13n_n-hibit-gba1 donor plasmids l444p  (Addgene inc)

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: 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: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: The primers used for mutagenesis are shown.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Mutagenesis, Plasmid Preparation

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: The donor construct and the two sgRNAs used to create mutations in the Gba1b gene.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Construct

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Primers used for amplifying the mutation containing Gba1b gene.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Mutagenesis, Plasmid Preparation

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Design of Drosophila Gba1b L444P and Gba1b D370S genes. ( A ). Multiple sequence alignment of GBA1 fragments from different organisms containing the two amino acids that were mutated in the present study. The N370 is highlighted in green, and the L444 is highlighted in yellow. ( B ). The original and the established nucleotide sequence of the mutated amino acids. Highlighted in red are the mutated nucleotides. ( C ). Shown in red is the exon localization of the mutated amino acids. ( D ). Comparison between Gba1b fragments containing either the mutant (M) or the normal (N) sequence based on non-lethal genotyping. Boxed in blue are the nucleotide changes introduced to obtain the D370S (D415S) mutation, and in red are the nucleotide changes introduced to obtain the L444P (L494P) mutation. ( E ). Schematic representation of the Gba1b region on chromosome 3 of the homozygous Gba1b D370S/D370S line generated. ( F ). Schematic representation of the Gba1b region on chromosome 3 of the homozygous Gba1b L444P/L444P line generated.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Sequencing, Comparison, Mutagenesis, Generated

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Decreased GCase activity and substrate accumulation in the mutant fly lines. ( A ). GCase activity was measured in 50 µg protein lysates prepared from 2-day-old Gba1b L444P/L444P , Gba1b D370/D370SS (homozygous—H) lines, and Gba1b D370/+ , Gba1b L444P/+ (heterozygous—T) flies, as well as from Gba1b m/+ (T), Gba1b m/m (H), and w1118 lines, as detailed in the Methods section. The activity level of w1118 was considered 1. ( B ). The quantification of ( A ) is shown as the average ± standard error. ( C ). TLC analysis of GCase activity of the four selected homozygous lines (D370S- Gba1b D370/D370SS ; L444P- Gba1b L444P/L444P ) . ( D ). The quantification of ( C ) is shown as the average ± standard error. One-way ANOVA was used to calculate the significance of the results. ( E ). TLC plate showing substrate accumulation in lipid extracts prepared from 22-day-old homozygous flies (D370S- Gba1b D370/D370SS ; L444P- Gba1b L444P/L444P ). ( F ). Quantification of results as shown in ( E ). The results are presented as average ± standard error. One-way ANOVA was used to calculate the significance of the results. ** p < 0.01, *** p < 0.001, **** p < 0.0001. SEM—standard error. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Activity Assay, Mutagenesis

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Lysosomal abnormalities in the homozygous mutant flies. ( A ). Confocal images of the suboesophageal ganglion region in the brains of control w1118, Gba1b L444P/L444P lines 1-1 and 3-2, and Gba1b D370S/D370S lines 6-1 and 11-1 flies, at 15 days post-eclosion. Red—LysoTracker, Blue—DAPI. ( B ). Graphical presentation of the Drosophila brain was created using BioRender. MB—mushroom body, AL—anntenal lobe, SOG—suboesophageal ganglion, A—anntena. The imaged region is circled in red. ( C ). Quantification of LysoTracker intensity in images like the one shown in ( A ). The results are presented as an average ± standard error for 25 different brains for each line. Significance was calculated using one-way ANOVA. ( D ). Confocal images of the gut region of w1118, Gba1b L444P/L444P lines 1-1 and 3-2, and Gba1b D370S/D370S lines 6-1 and 11-1 flies at 15 days post-eclosion. Red—LysoTracker, Blue—DAPI. ( E ). Graphical presentation of the Drosophila gut. The image was taken from BioRender, and the imaged region is boxed in red. ( F ). Quantification of LysoTracker intensity in images like the one shown in ( D ). The results are presented as an average ± standard error for 7 different guts for each line. Significance was calculated using one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Mutagenesis, Control

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: ERAD of the mutant Gba1b variants. ( A ). A total of 60 µg of protein lysates, prepared from 2-day-old flies expressing the WT- Gba1b (UAS-WT Gba1b ), D370S (UAS-D370S), and L444P (UAS-L444P) mutants under the Da-GAL4 driver, were electrophoresed through SDS-PAGE and the corresponding blots were interacted with anti-myc antibody to visualize the GCase proteins and with anti-actin antibody, as a loading control. ( B ). Quantification of results as presented in ( A ). The results are presented as average ± standard error. One-way ANOVA was used to determine the statistical significance of the results. ( C ). Protein lysates (60 µg), prepared from 22-day-old mutant flies described in ( A ), were processed as specified in ( A ). ( D ). Quantification of results as presented in ( C ). The results are presented as average ± standard error. Analysis was performed as explained in ( B ). ( E ). Protein lysates (60 µg), prepared as in ( A ) and treated with endo-H were subjected to electrophoresis and blotting as in ( C ). The blots interacted with anti-myc antibody to visualize the GCase proteins and with anti-actin antibody as a loading control. * p < 0.05, ** p < 0.01. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Mutagenesis, Expressing, SDS Page, Control, Electrophoresis

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: UPR activation in the mutant flies. ( A ). mRNA levels of UPR markers: HSc-70-3, Atf4, Atf6, and sXbp1 were tested in the bodies ( A ) and heads ( B ) of 22-day-old homozygous Gba1b L444P/L444P fly lines 3-2 and 1-1 and homozygous Gba1b D370S/D370S fly lines 6-1 and 11-1. The results are presented as average ± standard error. Each dot represents a triplicate of an independent experiment. One-way ANOVA was used to determine the statistical significance of the results. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns—non-significant. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Activation Assay, Mutagenesis

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Inflammation and neuroinflammation in the mutant flies. ( A ). The inflammatory pathways in Drosophila (created with BioRender). ( B ). mRNA levels of inflammatory markers: AttC, Cec, Drs, and Mtk were tested in the bodies ( B ) and heads ( C ) of 22-day-old Gba1b L444P/L444P fly lines 3-2 and 1-1 and Gba1b D370S/D370S lines 6-1 and 11-1. The results are presented as average ± standard error. One-way ANOVA was used to determine the statistical significance of the results. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Mutagenesis

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Neuropathology and survival of the mutant flies. ( A ). Thirty flies from Gba1b L444P/L444P lines 3-2 and 1-1 and Gba1b D370S/D370S lines 6-1 and 11-1 were tested for their locomotion abilities at 2, 12, and 22 days post-eclosion. Results are presented as average ± standard error. Two-way ANOVA was used to determine the statistical significance of the results. ( B ) Kaplan–Meier curve presenting the survival of 100 control (w1118), homozygous Gba1b L444P lines 3-2 and 1-1, and Gba1b D370S/D370S lines 6-1 and 11-1 flies. Below is a table showing the significance measured by Kaplan–Meier’s multiple comparisons. * p < 0.05, ** p < 0.01. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Mutagenesis, Control

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: A table showing the amino acid positions of the three loops that are stabilized by ambroxol in human GCase and the parallel positions in the Drosophila Gba1b -encoded enzyme.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques:

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Molecular dynamic simulation of Gba1b -encoded GCase with ambroxol. ( A ). X-ray structure of human WT GCase with ambroxol (depicted in cyan) (based on B from JBC 2009 ). The loops that are stabilized upon formation of ambroxol–GCase complex are pink (loop A), green (loop B), and blue (loop C). ( B ). The predicted Gba1b WT model with ambroxol (16 ns stimulation). Ambroxol is painted in dark pink. Loops A, B, and C are colored as in ( B ). ( C ). RMSD stimulation of Gba1b GCase with (16 nanoseconds) and without (10 nanoseconds) ambroxol. Each graph shows the RMSD status of a different loop in the Gba1b -encoded GCase. Loops A and C are stabilized upon ambroxol binding. ( D ). GCase activity of homozygous Gba1b L444P/L444P flies (lines 1-1 and 3-2), grown for 22 days with and without ambroxol. GCase activity level of w1118 was considered 1. Results are presented as the average ± standard error. One-way ANOVA was used to calculate the statistical significance. ( E ). GCase activity of the homozygous Gba1b D370S/D370S flies (lines 6-1 and 11-1), grown for 22 days with and without ambroxol. Activity levels of w1118 with ambroxol were considered 1. Results are represented as the average ± standard error. One-way ANOVA was used to calculate the statistical significance. *** p < 0.005. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Binding Assay, Activity Assay

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Change in UPR parameters upon ambroxol treatment. ( A ). mRNA levels of UPR markers: HSc-70-3, Atf4, Atf6, and sXbp1 were tested in the bodies ( A ) and heads ( B ) of Gba1b L444P/L444P lines 3-2 and 1-1 and Gba1b D370S/D370S lines 6-1 and 11-1 flies that were grown for 22 days with and without ambroxol. The results are presented as average ± standard error. Relative mRNA expression level was calculated using the 2 −ΔΔCT method. Two-way ANOVA was used to calculate the statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Expressing

Journal: Cells

Article Title: Functional Analysis of Human GBA1 Missense Mutations in Drosophila : Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences

doi: 10.3390/cells13191619

Figure Lengend Snippet: Effect of ambroxol on inflammation/neuroinflammation. ( A ). mRNA levels of inflammatory markers: AttC, Cec, Drs, and Mtk were tested in the bodies ( A ) and heads ( B ) of homozygous Gba1b L444P/L444P lines 3-2 and 1-1 and Gba1b D370S/D370S 6-1 and 11-1 flies that were grown for 22 days with and without ambroxol. The results are presented as average ± standard error. Relative mRNA expression level was calculated using the 2 −ΔΔCT method. Two-way ANOVA was used to calculate the statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Each dot denotes an independent experiment.

Article Snippet: Gba1b m/m flies were described elsewhere [ ]. pUAST-L444P Gba1b -mycHis and pUAST-D370S Gba1b -mycHis (see plasmid construction) were used to establish transgenic lines by BestGene (Chino Hills, CA, USA).

Techniques: Expressing

Journal: NPJ Parkinson's Disease

Article Title: GBA -AAV mitigates sleep disruptions and motor deficits in mice with REM sleep behavior disorder

doi: 10.1038/s41531-024-00756-5

Figure Lengend Snippet: a–c Time spent in each stage of Wake, REM, and NREM sleep in WT and L444P mutant mice over a 24 h light–dark cycle ( n = 7). d–f Total amount of time in each stage of Wake, REM, and NREM states during the light and dark phase ( n = 7). g Statistical analysis of the ratio of EMG REM /EMG SWS between WT or L444P mutant mice ( n = 7). h and i EEG power spectrum of REM and NREM sleep were both not altered in L444P mice ( n = 7) compared to WT mice ( n = 6). j–l Statistical analysis of relative EEG power in WT ( n = 6) and L444P mice ( n = 7). Statistical significance was determined using two-way ANOVA followed by Bonferroni’s multiple comparison test. m–p The Rota-rod test, pole test, and open field test were performed between WT ( n = 5–7) and L444P mice ( n = 7–15). All data points represent the mean ± SEM. Statistical significance was determined using the unpaired two-tailed Student’s t -test.

Article Snippet: Human GBA L444P mutant mice were obtained from the Jackson Laboratory (Stock No. 050598).

Techniques: Mutagenesis, Comparison, Two Tailed Test

Journal: NPJ Parkinson's Disease

Article Title: GBA -AAV mitigates sleep disruptions and motor deficits in mice with REM sleep behavior disorder

doi: 10.1038/s41531-024-00756-5

Figure Lengend Snippet: a–c Time spent in each stage of Wake, REM, and NREM sleep after infection PFF at 2 months in WT ( n = 6) and GBA L444P mutant mice ( n = 8) over a 24 h light–dark cycle. d Schematic of EGFP-AAV and PFF or PBS, a bilateral viral infection of SLD regions in young WT and GBA L444P mutant mice. Scale bars, 100 μm. e–g Total amount of time in each stage (7:00–13:00). ( n = 6–8). Statistical significance was determined using the unpaired two-tailed Student’s t -test. h and i Typical EEG power spectrograms (light period, 14:00–14:30; top), EMG waveforms (middle), and Stage (bottom) of WT and L444P mutant mouse after PFF injection for 2 months. j–l EEG power spectrum of Wake was not altered, but REM and NREM sleep were both altered after PFF injection of L444P mice ( n = 7) compared to WT mice ( n = 8). Red dots indicate frequency bins that are significantly different between the two groups ( P < 0.05, two-sided paired t -test with Bonferroni multiple comparison correction). m–o Normalized EEG power spectra in qREM and aREM. And aREM (8.2–10 Hz) was elevated in L444P mice compared to WT mice injected with PFF, but qREM (6.5–7.5 Hz) showed no difference between the two groups. During the REM sleep phase, theta was elevated in the L444P mice group ( P = 0.054), and delta was decreased during the NREM sleep phase, suggesting a shallower sleep depth ( n = 7–8). Statistical significance was determined using two-way ANOVA followed by Bonferroni’s multiple comparison test. All data are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human GBA L444P mutant mice were obtained from the Jackson Laboratory (Stock No. 050598).

Techniques: Infection, Mutagenesis, Two Tailed Test, Injection, Comparison

Journal: NPJ Parkinson's Disease

Article Title: GBA -AAV mitigates sleep disruptions and motor deficits in mice with REM sleep behavior disorder

doi: 10.1038/s41531-024-00756-5

Figure Lengend Snippet: a–c Time spent in each stage of Wake, REM, and NREM sleep after infection PFF or PFF with GBA -AAV at 2 months over a 24 h light–dark cycle in WT mice ( n = 8 vs. 7). d–f Total amount of time in each stage, Wake, REM, and NREM states. Statistical significance was determined using two-way ANOVA followed by Bonferroni’s multiple comparison test. g Immunofluorescent co-labeling of the GBA protein with the neuronal-specific marker NeuN. Scale bars, 50 μm. h–j Time spent in each stage of Wake, REM, and NREM sleep after PFF or PFF with GBA -AAV infection at 2 months over a 24 h light–dark cycle in GBA L444P mice ( n = 7). k–m Amount of time in each stage, Wake, REM, and NREM states during the following 6 h (13:00–19:00). Statistical significance was determined using the unpaired two-tailed Student’s t -test. n Total amount of time in REM sleep during 24 h ( n = 5–6). Statistical significance was determined using two-way ANOVA followed by Bonferroni’s multiple comparison test. o–q EEG power spectrum of Wake, REM, and NREM sleep were both not altered after GBA -AAV over-expression ( n = 5–6). All data are shown as mean ± SEM. *P < 0.05, * *P < 0.01.

Article Snippet: Human GBA L444P mutant mice were obtained from the Jackson Laboratory (Stock No. 050598).

Techniques: Infection, Comparison, Labeling, Marker, Two Tailed Test, Over Expression

Journal: NPJ Parkinson's Disease

Article Title: GBA -AAV mitigates sleep disruptions and motor deficits in mice with REM sleep behavior disorder

doi: 10.1038/s41531-024-00756-5

Figure Lengend Snippet: a A schematic of the protocol and timeline used for the experiment. WT and GBA L444P mice were injected with PBS or PFF or PFF + GBA to induce the RBD animal model. b and c Assessment and comparison of the motor dysfunction ( b Rota-rod test and c Pole test) between the PBS, PFF, and PFF + GBA mice groups after 2 months. d Effect of PFF plus GBA -AAV on the exploration of a novel and a familiar object in the retention trial in NOR test between different groups. e and f Correlation analysis between REM sleep volume and latency to fall in the rota-rod test or climbing time in the pole test. g–i Animals were sacrificed 5 months later and analyzed by immunohistochemistry ( n > 10). Scale bars, 100 μm. Data are shown as mean ± SEM. Two-way ANOVA followed by Bonferroni’s post-tests. *P < 0.05, * *P < 0.01, *** *P < 0.0001.

Article Snippet: Human GBA L444P mutant mice were obtained from the Jackson Laboratory (Stock No. 050598).

Techniques: Injection, Animal Model, Comparison, Immunohistochemistry