Journal: The Journal of Neuroscience

Article Title: Neuronopathic GBA1L444P Mutation Accelerates Glucosylsphingosine Levels and Formation of Hippocampal Alpha-Synuclein Inclusions

doi: 10.1523/jneurosci.0680-22.2022

Figure Lengend Snippet: Figure 1. Primary hippocampal neurons from GBA11/L444P mice show increased a-syn inclusions 14d after exposure to fibrils and eliglustat, and reduced lysosome activity compared with GBA11/1 mice. A, Representative images of neurofilament-H (magenta, left) and p-a-syn (green, right) in hippocampal primary cultures at DIV21. There were also no differences at DIV14 (Extended Data Fig. 1-1). Top row of panels shows neurons treated with equivalent volumes of DMSO as eliglustat (100 nM, bottom row of panels). GlcCer levels were reduced with 100 nM of eliglustat treatment (Extended Data Fig. 1-2). Scale bars:100 mm; zoom,50 mm. B, Quantification of the percent area of NF (left; nested two-way ANOVA; Interaction: Time genotype: F(1,49) = 1.365, p=0.2484, Drug treatment: F(1,49) = 0.03061, p=0.8618, Genotype: F(1,49) = 1.004, p= 0.3212) and p-a-syn (right, nested two-way ANOVA; Time genotype: F(1,48) = 1.838, p= 0.1815, Drug treatment: F(1,48) = 0.8887, p= 0.3506, Genotype: F(1,48) = 20.29, ****p, 0.0001, N=9 for GBA11/1 and GBA11/L444P DMSO-treated neuron groups, N=15 GBA11/1 eliglu- stat-treated neurons, N=20 GBA11/L444P eliglustat-treated neurons). One GBA11/1 DMSO and one GBA11/L444P DMSO outlier were removed for both NF and p-a-syn analysis based on Grubbs’ test. Internalization of fibrils or a-syn expression was not altered by eliglustat treatment (Extended Data Figs. 1-3, 1-4). a-Syn expression was not changed at DIV7 or DIV21 (Extended Data Fig. 1-4). C, Representative images for DQ-BSA assay showing DQ Red BSA (visualized in black and white). Top row of panels shows neurons treated with equivalent volumes of DMSO as eliglustat (100 nM, bottom row of panels) between GBA11/1 and GBA11/L444P primary hippocampal neurons. D, Quantification of background corrected total cell fluorescence (three- way ANOVA; interaction: F(1,18) = 3.621, p = 0.0732, Drug treatment a-syn treatment: F(1,18) = 7.092, *p = 0.0158, Drug treatment genotype: F(1,18) = 9.580, **p = 0.0062, a-syn treatment genotype: F(1,18) = 2.512, p = 0.1304, Drug treatment: F(1,18) = 10.41, **p = 0.0047, a-syn treatment: F(1,18) = 1.966, p = 0.1778, Genotype: F(1,18) = 6.234, *p = 0.0225; N = 3 for GBA11/1 monomer DMSO and eliglustat-treated neuron groups, N=2 for GBA11/L444P monomer DMSO and eliglustat-treated neuron groups, N=4 for GBA11/1 and GBA11/L444P fibril DMSO and eliglustat-treated neurons groups). Eliglustat increased CatB activity only in GBA11/L444P hippocampal neurons (Extended Data Fig. 1-5). For all graphs, error bars indicate SEM. Scale bar, 100 mm. *p , 0.05. **p , 0.01. ***p , 0.001. ****p , 0.0001.

Article Snippet: 13C6 GlcSph and d5 GlcCer d18:1/18:0 (Avanti) internal standards were prepared as a DMSO stock solution at 2 mg/ml and 0.02 mg/ml, respectively; 25 ml of the internal standard stock solution was added to 100 ml of homogenate before the addition of 400ml of acetone/MeOH buffer (1:3).

Techniques: Activity Assay, Expressing, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Neuronopathic GBA1L444P Mutation Accelerates Glucosylsphingosine Levels and Formation of Hippocampal Alpha-Synuclein Inclusions

doi: 10.1523/jneurosci.0680-22.2022

Figure Lengend Snippet: Figure 4. Lipid analysis in GBA11/1 and GBA11/L444P mice. GBA11/1 and GBA11/L444P mice forebrains underwent mass spectrometry for lipid quantification for (A) GlcSph (two-way ANOVA; Interaction: F(3,50) = 2.270, p = 0.0918, Age: F(3,50) = 23.54, ****p , 0.0001, and Genotype: F(1,50) = 64.23, ****p , 0.0001) and (B) total GlcCer (Interaction: F(3,50) = 1.454, p = 0.2384, Age: F(3,50) = 3.340, *p = 0.0265, Genotype: F(1,50) = 2.493, p = 0.1206) at 3, 6, 9, and 12 months. C, Mice plasma was collected and underwent mass spectrometry for lipid quan- tification for C. GlcSph (independent t test, t(9) = 2.810, *p = 0.0204, N = 6 for GBA11/1, and N = 5 GBA11/L444P) and (D) GlcCer (t(10) = 1.015, p = 0.3338, N = 7 for GBA11/1 and N = 5 GBA11/L444P). Only GlcCer d18:1_18:0 isoform was reduced in aged GBA11/1 mice with no changes between GBA1L444P expression (Extended Data Fig. 4-1). For all graphs, error bars indicate SEM. *p , 0.05. **p , 0.01. ***p , 0.001. ****p , 0.0001.

Article Snippet: 13C6 GlcSph and d5 GlcCer d18:1/18:0 (Avanti) internal standards were prepared as a DMSO stock solution at 2 mg/ml and 0.02 mg/ml, respectively; 25 ml of the internal standard stock solution was added to 100 ml of homogenate before the addition of 400ml of acetone/MeOH buffer (1:3).

Techniques: Mass Spectrometry, Clinical Proteomics, Expressing

Journal: The Journal of Neuroscience

Article Title: Neuronopathic GBA1L444P Mutation Accelerates Glucosylsphingosine Levels and Formation of Hippocampal Alpha-Synuclein Inclusions

doi: 10.1523/jneurosci.0680-22.2022

Figure Lengend Snippet: Figure 5. p-a-syn pathology burden is increased in the hippocampal formation. A, Ten months after bilateral striatal injections, IF for p-a-syn was performed. Representative images of GBA11/1 and GBA11/L444P fibril-injected mice fed control or venglustat chow of the dmPFC were captured using confocal microscopy. Venglustat chow reduced GlcCer (Extended Data Fig. 5-2). Scale bars: 100 mm; zoom, 50 mm. B, Quantification of p-a-syn in the dmPFC (N = 6 for both control chow groups, N = 4 for venglustat chow groups). Two-way ANOVA: Interaction: F(1,16) = 6.890, *p = 0.0184, Drug treatment: F(1,16) = 13.95, **p = 0.0018, Genotype: F(1,16) = 1.842, p = 0.1936. Error bars indicate SEM. Changes in p-a-syn inclusions in the PFC was not because of changes in cell quantity (Extended Data Fig. 5-3). C, Representative images of GBA11/1 and GBA11/L444P fibril-injected mice fed control or venglustat chow of the hippocampus were captured using confocal microscopy. Scale bars: 100 mm; zoomed, 50 mm. D, Quantification of p-a-syn in the granule cell layer of the dentate gyrus area of the hippocampus (N = 6 for both control chow groups, N = 4 for venglustat chow groups, two outliers removed (one GBA11/1 and one GBA11/L444P). Interaction: F(1,16) = 7.484, *p = 0.0147, Drug treatment: F(1,16) = 0.05575, p = 0.8163, Genotype: F(1,16) = 23.18, ***p = 0.0002. Quantification of CA1-CA3 pyramidal cell layer with control chow (N = 7 GBA11/1 and N = 8 GBA11/L444P: t test: CA1 pyramidal cell layer: t(11) = 2.580; *p = 0.0256, CA2-CA3 pyramidal cell layer: t(13) = 1.997; p = 0.0672). Error bars indicate SEM. E, Representative images of GBA11/1 and GBA11/L444P fibril-injected mice fed control or venglustat chow of the SNc were captured using confocal microscopy. Scale bars: 100 mm; zoom, 50 mm. F, Quantification of p-a-syn in the SNc (N = 7 for GBA11/1 and N = 8 for GBA11/L444P control chow groups, N = 4 for GBA11/1 and GBA11/L444P venglustat chow groups).

Article Snippet: 13C6 GlcSph and d5 GlcCer d18:1/18:0 (Avanti) internal standards were prepared as a DMSO stock solution at 2 mg/ml and 0.02 mg/ml, respectively; 25 ml of the internal standard stock solution was added to 100 ml of homogenate before the addition of 400ml of acetone/MeOH buffer (1:3).

Techniques: Injection, Control, Confocal Microscopy