Neuronopathic Gaucher disease: dysregulated mRNAs and miRNAs in brain pathogenesis and effects of pharmacologic chaperone treatment in a mouse model

Abstract

Defective lysosomal acid β-glucosidase (GCase) in Gaucher disease causes accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) that distress cellular functions. To study novel pathological mechanisms in neuronopathic Gaucher disease (nGD), a mouse model (4L;C*), an analogue to subacute human nGD, was investigated for global profiles of differentially expressed brain mRNAs (DEGs) and miRNAs (DEmiRs). 4L;C* mice displayed accumulation of GC and GS, activated microglial cells, reduced number of neurons and aberrant mitochondrial function in the brain followed by deterioration in motor function. DEGs and DEmiRs were characterized from sequencing of mRNA and miRNA from cerebral cortex, brain stem, midbrain and cerebellum of 4L;C* mice. Gene ontology enrichment and pathway analysis showed preferential mitochondrial dysfunction in midbrain and uniform inflammatory response and identified novel pathways, axonal guidance signaling, synaptic transmission, eIF2 and mammalian target of rapamycin (mTOR) signaling potentially involved in nGD. Similar analyses were performed with mice treated with isofagomine (IFG), a pharmacologic chaperone for GCase. IFG treatment did not alter the GS and GC accumulation significantly but attenuated the progression of the disease and altered numerous DEmiRs and target DEGs to their respective normal levels in inflammation, mitochondrial function and axonal guidance pathways, suggesting its regulation on miRNA and the associated mRNA that underlie the neurodegeneration in nGD. These analyses demonstrate that the neurodegenerative phenotype in 4L;C* mice was associated with dysregulation of brain mRNAs and miRNAs in axonal guidance, synaptic plasticity, mitochondria function, eIF2 and mTOR signaling and inflammation and provides new insights for the nGD pathological mechanism.

Figure 1.

Phenotypes of 4L;C* mice. (A) Gait analysis. 4L;C* mice showed significant decrease on left and right strides and increase on base width at 35 days of age and older. The tests repeat two to three times (n = 4–7 mice). (B) Glucosylceramides (GC) and glucosylsphingosine (GS) quantitation by LC/MS. Both GC (left panel) and GS (right panel) levels were significantly increased in 4L;C* brain regions at 45 days of age relative to WT levels (n = 3 mice). (C) Mitochondrial function. Percentage of ATP production and oxygen consumption rate were decreased in 4L;C* brain compared with WT mice. The data are from duplicate experiments (n = 2 mice). (D) Augmented loss of neurons in 4L;C* mice. Single-cell suspensions prepared from whole brain of 4L;C* and WT mice were analyzed by FACS. These cells were first gated for negativity of antibodies to CD11b and CD45 and then stained for positivity of antibodies to Map2, a neuron marker. The histogram (left) and their corresponding columns (right) showed decreased percentages of MAP2⁺ cells in 4L;C* mice brain (blue) compared with WT mice (orange) (n = 10 mice). (E) Immunohistochemistry of Map2 antibody staining on 4L;C* and WT brain. Map2 signals (brown) in 4L;C* brain were significantly decreased compared with WT brain at age of 45 days. Signal intensity was quantitated by NIH image J software (n = 2 mice, 2 sections/mouse). (F–H) Increased incidence of inflammatory subsets of microglial cells in 4L;C* mice brain. Single cell suspensions from the whole brain of 4L;C* and WT mice were analyzed by FACS. FACS-sorted CD45int CD11b+ and CX3CR1+ microglial cells (F) shown as percentage were significantly increased in 4L;C* compared with WT brain (G). Microglial cells have CD45int CD11b+ and CX3CR1+ positivity stained for antibodies to CCR2, Ly6C and Ly6G to evaluate their mean fluorescence intensity (MFI). 4L;C* brain had increased signals for CCR2, Ly6C and Ly6G than WT brains (H). Two independent experiments were conducted (n = 10 mice). (I) Inflammation in 4L;C* brain was determined by anti-CD68 (a marker for activated microglia/macrophage) antibody staining (brown). WT mice brain showed background level. Hematoxylin stains cell nuclei (blue). The values are represented as mean ± S.E. Group comparison was done by Student's t-test (*P < 0.05; ***P < 0.001; ****P < 0.0001).

Figure 2.

Differentially expressed mRNA in 4L;C* brain. DEGs in 4L;C* brain regions, CO, BS, MID and CB, were determined by RNASeq analyses. (A) The number of DEGs with increased expression (dark gray bars) or decreased expression (light gray bars) is labeled on the top of bars, respectively. (B) Venn diagram analyses show the commonality of DEGs (in A) derived from four different brain regions as indicated, respectively.

Figure 3.

Category of inflammatory and non-inflammatory DEGs. (A) Proportion of inflammatory DEGs in each brain region. The number of inflammatory DEGs distributes uniformly across four brain regions in 4L;C* mice. (B) Reduction of inflammatory DEG numbers (%) in IFG-treated 4L;C* brain regions. (C) Reduction of non-inflammatory (other) DEG numbers (%) in IFG-treated 4L;C* brain regions.

Figure 4.

eIF2 and mTOR signaling pathways. (A) Number of eIF2 DEGs. DEGs were enriched in CO, BS and MID. Most of the DEGs had increased expression. (B) Functional category and their fraction of eIF2 DEGs. (C) Number of mTOR DEGs. DEGs were enriched in CO, BS and MID. Most of the DEGs had increased expression. (D) Functional category and their fraction of mTOR DEGs. The number of DEGs in eIF2 and mTOR pathways was reduced in IFG-treated 4L;C* brain regions. UT, untreated; T, IFG treated.

Figure 5.

Mitochondrial function. (A) Hierarchical cluster analyses of 106 mitochondrial DEGs in WT, treated (T) 4L;C* and untreated (UT) 4L;C brain in CO, BS, MID and CB region. Each row represents a single DEG across four brain regions. Hierarchical charts are clustered by normalized intensity values (green-to-red scale bar) representing the fold changes (−1.8 to +1.8) of DEG. (B) Number of mitochondrial DEGs. MID had greater number of DEG than that in CO, BS and CB. Most of the DEG had increased expression. (C) Reduction of DEG numbers in IFG-treated 4L;C* brain regions. Untreated (UT, black bars) and treated (T, open bars).

Figure 6.

Axonal guidance signaling. (A) Number of axonal guidance DEGs. Most of the DEGs in CO, BS and MID showed decreased expression. (B) Function of DEGs in axonal guidance. The DEGs involved were highlighted with dotted circles in the pathway. The axonal guidance graph adapted from KEGG (download of dated 9 June 2014). (C) IFG treatment on axonal guidance DEGs. The number of DEGs in 4L;C* brain regions was reduced in IFG-treated CO, BS and MID. Untreated (UT, black bars) and treated (T, open bars).

Figure 7.

DEGs for synaptic transmission and LTP. (A) Number of DEGs for synaptic transmission. (B) Number of DEGs for LTP. Number of DEGs in IFG treated (T) compared with untreated (UT) brain regions for synaptic transmission and LTP are shown in the table below (A or B), respectively. (C) Decreased expression of Ryrs in both untreated (UT) and IFG-treated (T) CO. (D) Decreased expression of calcium channels in both untreated (UT) and IFG-treated (T) CO. The expression levels are plotted as fold change relative to WT level in (C) and (D).

Figure 8.

Differentially expressed miRNA in 4L;C* brain. DEmiRs in 4LC brain regions (CO, BS, MID and CB) were determined by RNASeq analyses. (A) The number of DEmiRs with increased expression (dark gray bars) or decreased expression (light gray bars) is labeled on the top of bars, respectively. (B) Venn diagram analyses show the commonality DEmiRs (in A) derived from four different brain regions as indicated, respectively. (C) DEmiRs and predicted DEG targets in 4L;C* brain regions.

Figure 9.

Dysregulated miRNAs in 4L;C* brain. (A) Number of DEmiRs in IFG-treated (open bar, T) and untreated (gray bar, UT) 4L;C* brain regions. (B) Number of DEmiRs in functional pathways of IFG-treated (T) and untreated (UT) 4L;C* brain regions. (C) DEmiRs in inflammatory group and IFG effect. Decreased miR-181c-5p, miR-34b-5p and miR-490-3p correlated with increased expression of inflammatory DEG targets, Plau, Gfap or Fcgr2a, respectively, in untreated (UT) 4L;C* BS. IFG treatment (T) partially normalized their expression. (D) DEmiRs in axonal guidance signaling pathway and IFG effect. In IFG-treated (T) 4L;C* MID, both miR-10a-5p and its target DEG, Epha8 and Arhgef11, were changed to normal level compared with untreated (UT). (E) DEmiRs in mitochondrial group and IFG effect. Compared with untreated (UT), both miR-423-5p/Atp5g3 and miR-181c-5p/Prdx3 were changed to normal level in treated MID (T).