Fig 1: 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 controls (n = 4–10 mice/group; two independent experiments). (B) Comparison of serum Nf-L levels between young and aged Gbafl/flCx3cr1Cre/wt mice along with control mice (n = 8–11 mice/group). (C) Correlation between serum GlcSph and Nf-L levels in nGD, nGD Cx3cr1Cre/+ mice, nGD NesCre/+, and control mice. The p-value obtained from Spearman’s rank correlation coefficient test was <0.0001 (n = 69 mice). (D) Quantitative analysis of serum Nf-L levels (log2 scale) in GD3 patients (n = 5) compared with age-matched GD1 patient (n = 6). (E) Quantitative analysis of serum levels glucosylsphingosine (GlcSph) in GD3 patients (n = 3) compared with age-matched GD1 patient (n = 6). (F) Quantitative analysis of serum Nf-L levels (log2 scale) in young GD1 patients (n = 12) compared with adult GD1 patient (n = 25) and adult healthy controls (n = 28). (G) Signal intensities of lysophosphatidylcholine (LysoPC) identified by MALDI across Gbafl/flCx3cr1Cre/wt mice treated with either vehicle or GZ161 and control mice brain. The color bars in MALDI images show signal intensity: blue to red indicates low to high levels. (H) Quantitative analysis of serum levels of LysoPC 16:1 species in young GD1 patients (n = 12) compared with adult GD1 patient (n = 26). (I) Graph represents ApE levels in the sera of untreated GD1 patients (n = 55) and healthy controls (n = 43). (J) Graph represents ApoE levels in the sera of untreated GD1 patients (n = 55) and after enzyme replacement therapy (ERT) (n = 55). (K) Correlation between serum GlcSph and ApoE levels in sera of GD1 patients. The p-value obtained from Pearson’s correlation test was <0.001 (n = 21 patients). (L) Receiver-operating characteristic (ROC) curves for serum ApoE expression in GD1 patients and area under the curve (AUC). Means ± SEM are shown. Differences between groups were analyzed using unpaired t-test. (A, B) Mann–Whitey test (C, E, H, I), two-tailed. *p<0.05, **p<0.001, and ***p<0.0001.
Fig 2: 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 genotype (top) and cell clusters (bottom). (B) AUC analysis for select lysosome, Interferon signature genes (ISG), chemokine, and Apoe gene sets significantly enriched (false discovery rate [FDR] < 0.05) in microglia, Purkinje, oligodendrocyte, astrocyte, and neuro clusters of nGD (2 weeks) vs. control mice; nGD Cx3cr1Cre/+ (2 and 6 weeks, respectively) vs. control mice; nGD NesCre/+ mice vs. control mice. Row side bar colors indicate mice genotype, age and cell clusters. (C) Pie chart displays the number of differentially expressed genes (DEGs) in neuronal clusters of nGD (2 weeks) vs. control mice (green); nGD Cx3cr1Cre/+ (2 weeks [orange] and 6 weeks [maroon], respectively); nGD NesCre/+ mice (pink) vs. control mice with log2(fold change) and adjusted p-value < 0.05. Total DEGs from each set of mice are stated in the middle of pie chart with number of DEGs and the neuronal cluster in brackets shown to the right of each pie chart. (D) Bar graph represents the number of DEGs in neuronal clusters of nGD 2 weeks (green) vs. nGD Cx3cr1Cre/+ 6 weeks (maroon) with log2(fold change) and adjusted p-value < 0.05. (E) Heat map of DEGs associated with disease-associated astrocytes (DAA) from nGD; nGD Cx3cr1Cre/+ (2 weeks and 6 weeks, respectively); nGD NesCre/+ mice vs. control mice. p<0.05 was considered significant (two-sided t-tests). All individual DAA genes with significant differential expression are listed on bottom and the astrocyte clusters are shown in right. Red, positive z-score; white, zero z-score; blue, negative z-score. (F) Ingenuity pathway analysis (IPA) from nGD vs. WT, nGD Cx3cr1Cre/+ vs. WT Cx3cr1Cre/+ at 2- and 6-week-old mice; nGD NesCre/+ mice vs. WT NesCre/+ in neuron cluster, microglia, and astrocytes. Orange, positive z-score; white, zero z-score; blue, negative z-score; gray dots are statistically insignificant. (G) Representative flow cytometry histogram (left) and quantification of CellROX fluorescence in astrocytes. (H) Histogram (left) and quantification (right) of BODIPY fluorescence in astrocytes of nGD mice. (I) Flow cytometry histogram (left) and quantification (right) of CellROX fluorescence in activated and homeostatic microglia from nGD mice. (J) Histogram (left) and quantification (right) of BODIPY fluorescence in activated and homeostatic microglia from nGD mice n = 3–4 mice per group. Data were replicated in at least two independent experiments. Unpaired t-test, two-tailed was used to test significance. *p<0.05, **p<0.001, and ***p<0.0001.
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