Neuroinflammation in neuronopathic Gaucher disease: Role of microglia and NK cells, biomarkers, and response to substrate reduction therapy
- Department of Internal Medicine, Yale School of Medicine, United States
- Translational Sciences, Sanofi, United States
- Yale Center for Genome Analysis, Yale School of Medicine, United States
- Department of Neuroscience, Yale School of Medicine, United States
- Department of Molecular & Cellular Physiology, Yale School of Medicine, United States
Figures
Figure 1 with 2 supplements
Loss of Gba induces microglial activation and immune cell infiltration in neuronopathic Gaucher disease (nGD) brain.
(A) Fluorescence-activated cell sorting (FACS) analysis of the whole brain of Gbawt/wt, Gbalsl/wt, and nGD (Gbalsl/lsl) mice performed at indicated days. The gates indicate cell populations revealed …
Figure 1—figure supplement 1
Loss of Gba disrupts microglial homeostasis and induces damage-associated microglia (DAM) phenotype and immune cell infiltration in neuronopathic Gaucher disease (nGD) (Gbalsl/lsl) brain.
(A) Representative fluorescence-activated cell sorting (FACS) plot showing CCR2+ MFs, microglia, and immune infiltrates in the whole brain of nGD vs. control mice brain. (B) Bar graph shows the …
Figure 1—figure supplement 2
Total RNA-seq analysis performed on flow-sorted microglia in neuronopathic Gaucher disease (nGD) (Gbalsl/lsl) and wild-type mice.
(A) Principal component analysis of gene expression profiles of microglia isolated from nGD brain and their controls. Each point represents a single mouse. (B) A heat map showing the differential …
Figure 2 with 2 supplements
Gba deficiency in neurons aid in microglial activation and immune cell infiltration.
(A) Schematic showing overview of the mouse models and methods used in the study. (B) Kaplan–Meier survival analysis of neuronopathic Gaucher disease (nGD), nGD Cx3cr1Cre/+, and nGD NesCre/+ mice …
Figure 2—figure supplement 1
Restoring Gba function in microglia and neurons enhances neuronopathic Gaucher disease (nGD) mice survival.
(A) Splicing of mRNA causes GBA1 deficiency in nGD mice. K14-mediated expression of Cre enabled removal of the lsl (lox-stop-lox) cassette in the skin tissue of the nGD mice (left and bottom …
-
Figure 2—figure supplement 1—source data 1
Gel image showing genotype of neuronopathic Gaucher disease (nGD), Het, and wild-type (wt).
The lsl (lox-stop-lox) cassette in nGD mice (left panel) was removed through to Krt14cre expression (right panel).
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig2-figsupp1-data1-v3.pdf
-
Figure 2—figure supplement 1—source data 2
Gel image showing genotype of neuronopathic Gaucher disease (nGD) Cx3cr1Cre/+ and wild-type (wt) mice.
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig2-figsupp1-data2-v3.pdf
-
Figure 2—figure supplement 1—source data 3
Gel image showing genotype of nGD NesCre/+ mice and wild-type (wt) mice.
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig2-figsupp1-data3-v3.pdf
-
Figure 2—figure supplement 1—source data 4
Gel image showing removal of lsl (lox-stop-lox) cassette in neuronopathic Gaucher disease (nGD) mice in various tissues due to Krt14cre expression.
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig2-figsupp1-data4-v3.pdf
-
Figure 2—figure supplement 1—source data 5
Gel image showing Cx3cr1Cre enabled removal of the lsl (lox-stop-lox) cassette in different tissues of neuronopathic Gaucher disease (nGD) Cx3cr1Cre/+ mice.
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig2-figsupp1-data5-v3.pdf
-
Figure 2—figure supplement 1—source data 6
Gel image showing NesCre enabled removal of the lsl (lox-stop-lox) cassette in brain of neuronopathic Gaucher disease (nGD) NesCre/+ mice.
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig2-figsupp1-data6-v3.pdf
Figure 2—figure supplement 2
Analysis of lipids in neuronopathic Gaucher disease (nGD), nGD Cx3cr1Cre/+, and nGD NesCre/+ mice brain and behavioral studies.
(A) Fold change increase in different glucosylceramide (GluCer) and glucosylsphingosine (GlcSph) species in nGD, nGD Cx3cr1Cre/+, and nGD NesCre/+ mice over respective control mice brain. (B) …
Figure 3 with 1 supplement
Aged Gbafl/flCx3cr1Cre/+ mice brain show alteration of microglia subsets and neurodegeneration.
(A) Quantitative analysis of total glucosylceramide/glucosylsphingosine (GluCer/GlcSph) levels in Gbafl/flCx3cr1Cre/wt mice and control mice (n = 3 mice/group). Statistical significance was …
Figure 3—figure supplement 1
Changes in microglia subsets coupled with neurodegeneration are seen in aged Gbafl/fl Cx3cr1Cre/+ mice brain.
(A) Genotyping for Cx3cr1 cre and loxp alleles. (B) Quantitative analysis of total galactosylceramide (GalCer) species and galactosylsphingosine (GalSph) levels by LC-ESI-MS/MS in Gbafl/fl Cx3cr1Cre/…
-
Figure 3—figure supplement 1—source data 1
Gel image showing genotype of Gbafl/fl Cx3cr1Cre/+ mice having conditional deletion of the Gba gene through Cx3cr1Cre expression.
- https://cdn.elifesciences.org/articles/79830/elife-79830-fig3-figsupp1-data1-v3.pdf
Figure 4 with 1 supplement
NK cell infiltration into the brain of neuronopathic Gaucher disease (nGD), nGD Cx3cr1Cre/+ mice.
(A) CD45+ cells form the whole brain of nGD, nGD Cx3cr1Cre/+, and control mice were gated to analyze CD3-NK1.1+ NK cells. Bar graphs represent the percentage of NK cells in the immune infiltrates of …
Figure 4—figure supplement 1
NK cell infiltration into the brain of neuronopathic Gaucher disease (nGD), nGD Cx3cr1Cre/+ mice.
(A) Representative fluorescence-activated cell sorting (FACS) plot showing NK1.1+ NK cells and Gzm-A+ NK cells in the spleen of nGD, nGD Cx3cr1Cre/+, and control littermates. (B) Lipidomic analysis …
Figure 5 with 1 supplement
snRNA-seq reveals key role of microglia astrocytes and neurons in Gaucher disease (GD)-associated neuroinflammation.
(A) UMAP data from neuronopathic Gaucher disease (nGD) (2 weeks old), nGD Cx3cr1Cre/+ (2 and 6 weeks old, respectively), nGD NesCre/+ mice (n = 3), and corresponding control mice, colored by …
Figure 5—figure supplement 1
snRNA-seq analysis of the brain of neuronopathic Gaucher disease (nGD), nGD Cx3cr1Cre/+ mice.
(A) Analysis of snRNA-seq data of nGD (green), Gbawt/lwt (blue), nGD Cx3cr1Cre/+ 2 and 6 weeks old (maroon), Gbawt/wtCx3cr1Cre/+ 2 and 6 weeks old (black), nGD NesCre/+ mice (pink) (n = 3), and Gbawt…
Figure 6 with 1 supplement
Effects of glucosylceramide synthase (GCS) inhibitor (GZ-161) on microglia and GzmA+ cells.
(A) Kaplan–Meier survival analysis of neuronopathic Gaucher disease (nGD) and nGD Cx3cr1Cre/+ mice after GZ-161 treatment compared to vehicle-treated controls (n = 4–10 mice/group). (B) Quantitative …
Figure 6—figure supplement 1
Immune regulatory effects of glucosyl ceramide synthetase (GCS) inhibitor GZ-161 on microglia and GzmA+ cells.
(A) Schematic illustration of mode of action of GCS inhibitor. (B) Representative fluorescence-activated cell sorting (FACS) plot showing percentage of Pro-IL-1ß+ microglia cells (left) and GzmA+ …
Figure 7 with 1 supplement
Long-term treatment with glucosylceramide synthase (GCS) inhibitor, GZ-161, counteracts age-related microglial dysfunction and NK cell activation.
(A) Signal intensities of hexosylceramides (HexCer) species and lysophosphatidylcholine (LysoPC) identified by MALDI across Gbafl/flCx3cr1Cre/wt mice treated with either vehicle or GZ161 and control …
Figure 7—figure supplement 1
Long-term treatment with glucosylceramide synthase (GCS) inhibitor, GZ-161, counteracts glucosylceramide (GluCer) accumulation, improves microglial homeostasis, and abrogates NK cell activation in Gba-deficient microglia.
UMAP showing GzmA expression in the clusters from control and Gbafl/fl Cx3cr1Cre/wt mice treated with either vehicle or GZ161.
Figure 8 with 1 supplement
Clinical evaluation of ApoE and neurofilament light chain (Nf-L) as biomarkers of Gaucher disease (GD)-associated neurodegeneration.
(A) Quantitative analysis of serum Nf-L levels in neuronopathic Gaucher disease (nGD), nGD Cx3cr1Cre/+, and nGD NesCre/+ mice with and without GZ-161 treatment compared with the vehicle-treated …
Figure 8—figure supplement 1
Expression of ApoE and Abca1 in different brain cell types of neuronopathic Gaucher disease (nGD) mice.
(A) Histogram showing differential expression of ApoE in microglia, astrocyte, neuron, and oligodendrocyte cluster from nGD (2 weeks old), nGD Cx3cr1Cre/+ (2 and 6 weeks old, respectively), and …
Figure 9
Remark flowchart.
Flow diagram of the study cohort used in the study.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | CD11b | BD Biosciences | 583,553 | (1:1000) |
| Antibody | CD11b | BD Biosciences | 552,850 | (1:1000) |
| Antibody | ACSA-2 | Miltenyi Biotech | 130-123-284 | (1:100) |
| Antibody | CD45 | Biolegend | 103,133 | (1:1000) |
| Antibody | NK1.1 | BD Biosciences | 562,864 | (1:250) |
| Antibody | CCR2 | R and D | FAB5538F | (1:100) |
| Antibody | CD3 | Biolegend | 100,241 | (1:250) |
| Antibody | NK1.1 | Biolegend | 108,753 | (1:250) |
| Antibody | 1 A/1-E | Biolegend | 107,639 | (1:1000) |
| Antibody | CD4 | BD Biosciences | 563,790 | (1:500) |
| Antibody | CD4 | BD Biosciences | 558,107 | (1:1000) |
| Antibody | Gr-1 | Biolegend | 108,440 | (1:1000) |
| Antibody | Ly-6C | Invitrogen | 47-5932-82 | (1:1000) |
| Antibody | CD64 | Biolegend | 139,306 | (1:200) |
| Antibody | B220 | eBioscience | 47-0452-80 | (1:200) |
| Antibody | CD8a | eBioscience | 56-0081-82 | (1:1000) |
| Antibody | IL-1Beta | Invitrogen | 17-7114-80 | (1:1000) |
| Antibody | IFNγ | Biolegend | 505,808 | (1:250) |
| Antibody | IFNγ | BD Biosciences | 562,020 | (1:250) |
| Other | Brdu Flow kit | BD Pharmingen | 51-9000019AK | |
| Other | 5-Bromo-2'-deoxyuridine | Cayam Chemical Company | 15,580 | 180 µg/ml (injected) and 800 µg/ml (in water) |
| Other | Cell Rox Deep Red kit | Invitrogen | C10422 |
