Single Cell Transcriptome Analysis of Niemann-Pick Disease, Type C1 Cerebella

Abstract

Niemann-Pick disease, type C1 (NPC1) is a lysosomal disease characterized by endolysosomal storage of unesterified cholesterol and decreased cellular cholesterol bioavailability. A cardinal symptom of NPC1 is cerebellar ataxia due to Purkinje neuron loss. To gain an understanding of the cerebellar neuropathology we obtained single cell transcriptome data from control (Npc1^+/+) and both three-week-old presymptomatic and seven-week-old symptomatic mutant (Npc1^-/-) mice. In seven-week-old Npc1^-/- mice, differential expression data was obtained for neuronal, glial, vascular, and myeloid cells. As anticipated, we observed microglial activation and increased expression of innate immunity genes. We also observed increased expression of innate immunity genes by other cerebellar cell types, including Purkinje neurons. Whereas neuroinflammation mediated by microglia may have both neuroprotective and neurotoxic components, the contribution of increased expression of these genes by non-immune cells to NPC1 pathology is not known. It is possible that dysregulated expression of innate immunity genes by non-immune cells is neurotoxic. We did not anticipate a general lack of transcriptomic changes in cells other than microglia from presymptomatic three-week-old Npc1^-/- mice. This observation suggests that microglia activation precedes neuronal dysfunction. The data presented in this paper will be useful for generating testable hypotheses related to disease progression and Purkinje neurons loss as well as providing insight into potential novel therapeutic interventions.

Keywords: NPC1; Niemann–Pick disease; cerebellar ataxia; cerebellum; single cell RNA sequencing; transcriptome; type C1.

Figure 1

Identification of cerebellar cell populations. (A) Automatic K-means clustering was used to generate a t-SNE plot of single cell transcriptomes from Npc1^+/+ and Npc1^−/− cerebellar tissue at seven weeks of age. (B) Number of cerebellar cell type specific transcriptomes obtained from Npc1^+/+ and Npc1^−/− seven-week-old mice. (C) Representative immunostaining using antibodies corresponding to signature transcripts in seven-week-old mouse cerebella. Calb (Calbindin, Purkinje neurons); CD117 (basket/stellate neurons); IBA1 (microglia); CD68 (myeloid cells); TBR2 (unipolar brush neurons); NEUROD1 (cerebellar granule neurons); AQP1 (ependymal secretory cells); OLIG2 (oligodendrocytes); GFAP (astrocytes); AQP4 (astrocytes); NEUN (neurons); Parv (Parvalbumin, interneurons). Scale bar = 50 µm. (D) Histopathological quantification of number of cerebellar granule neurons, oligodendrocytes, astrocytes, and unipolar brush cells in parasagittal sections of seven-week-old cerebella. N > 6.

Figure 2

Differentially expressed genes in microglia. (A) Principal component analysis (PCA) of gene expression in microglia from seven-week-old Npc1^+/+ (red) and Npc1^−/− (gray) cerebellar tissue. (B) Volcano plot of differential gene expression between control and Npc1 mutant mice. (C) PCA plots showing expression level of microglial activation markers (Apoe, Cd68, and Igax). Increasing red intensity corresponds to increasing unique molecular identifier (UMI) counts.

Figure 3

Differentially expressed genes in vascular cells. Tukey box plots of differentially expressed genes in vascular smooth muscle cells (A), endothelial cells (B), and vascular leptomeningeal cells (C).

Figure 4

Differentially expressed genes in glial cells. Tukey box plot of differentially expressed genes in astrocytes (A), oligodendrocyte precursors (B), differentiated oligodendrocytes (C), ependymal ciliated (D), and ependymal secretory cells (E) between genotype.

Figure 5

Differentially expressed genes in neuronal cells. Tukey box plots of differentially expressed genes in basket/stellate neurons (A), granule neurons (B), unipolar brush cells (C), and Purkinje neurons (D). In control cerebellum, anterior (A) and posterior (B) Purkinje neurons could be differentiated. In Npc1^−/− cerebellum from seven-week-old mice, only posterior Purkinje neurons were identified.

Figure 6

Protein validation of selected differentially expressed transcripts identified in 7-week old Npc1^−/− cerebella. Immunohistochemistry quantification of cathepsin D (A), lysozyme (B), DAP12 (C), and apolipoprotein E (APOE) (D). Representative examples of cerebellar staining are provided in Figure S6. β2 microglobulin (E) was quantified by ELISA. N > 6.

Figure 7

Differentially expressed genes at three weeks of age. (A) Tukey Box plot of genes showing differential expression Npc1+/+ and Npc1^−/− endothelial cells. (B) Volcano plot of significantly differentially expressed genes in microglia from Npc1^+/+ and Npc1^−/−. X axis Log2 fold change between Npc1^−/− and Npc1^+/+ microglia. Y axis is the -Log10 p-value. Black dots and clear circles indicate data from three- and seven-week-old mice, respectively.

Figure 8

Single cell expression data for 38 differentially expressed gene. Increased (red) and decreased (blue) at both three and seven weeks is indicated for specific cell types. A: astrocytes; ECC: ependymal ciliated cells; ESC: ependymal secretory cells; O: oligodendrocytes; OP: oligodendrocytes precursor; EC: endothelial cells; VLC: vascular leptomeningeal cells; VSM: vascular smooth muscle cells; B/S: basket/stellate cells; GC: granule cells; IN: interneurons; PN: Purkinje neurons; UBC: unipolar brush cells. Cellular location of the encoded proteins is from Uniprot. E/S: extracellular space or secreted; PM: plasma membrane; C: cytoplasm; N: nucleus; ER: endoplasmic reticulum; GA: Golgi apparatus; L: lysosome; En: endosome; Ex: exosome; M: mitochondrion; Cy: cytoskeleton. Sphingolipid binding is from Haberkant et al. [57]. Cholesterol binding is from Hulce et al. [63] and * [64].