Mapping the glial transcriptome in Huntington's disease using snRNAseq: selective disruption of glial signatures across brain regions

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease with a fatal outcome. There is accumulating evidence of a prominent role of glia in the pathology of HD, and we investigated this by conducting single nuclear RNA sequencing (snRNAseq) of human post mortem brain in four differentially affected regions; caudate nucleus, frontal cortex, hippocampus and cerebellum. Across 127,205 nuclei from donors with HD and age/sex matched controls, we found heterogeneity of glia which is altered in HD. We describe prominent changes in the abundance of certain subtypes of astrocytes, microglia, oligodendrocyte precursor cells and oligodendrocytes between HD and control samples, and these differences are widespread across brain regions. Furthermore, we highlight possible mechanisms that characterise the glial contribution to HD pathology including depletion of myelinating oligodendrocytes, an oligodendrocyte-specific upregulation of the calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1 A (PDE1A) and an upregulation of molecular chaperones as a cross-glial signature and a potential adaptive response to the accumulation of mutant huntingtin (mHTT). Our results support the hypothesis that glia have an important role in the pathology of HD, and show that all types of glia are affected in the disease.

Keywords: Chaperones; Glia; Huntington’s disease; Transcriptomics.

Fig. 1

snRNAseq of four regions of human post mortem brain from HD donors and controls. A. Sample summary. B. UMAP plot of all nuclei across brain regions, disease and control, showing all major cell types in the brain. C. The proportion of total nuclei identified as medium spiny neurons in the CN (PPP1R1B+) is reduced in HD compared to control (plot shows median and range). D Expression of canonical markers for cell lineages, across glial populations. E UMAP plots of astrocytes, microglia and oligodendroglia (L-R), showing annotated subclusters. BAM = border-associated macrophages

Fig. 2

Altered glial abundance in HD. A-C (left). Beeswarm plots showing differential abundance of subclusters of oligodendroglia (A), microglia (B), astrocytes (C), with markers of clusters that were significantly enriched (highlighted in blue) or depleted (highlighted in red) in HD compared to controls. A-C (right) Corresponding dot plots showing selected marker genes for the glial subclusters

Fig. 3

PDE1A upregulation in oligodendrocytes in HD (A) We identified upregulation of PDE1A across multiple clusters and regions. (B) Violin plots of expression of PDE1A in different regions and different clusters comparing HD and control donors. Stars represent adjusted p-values from MAST, *** p < 0.001. (C) Duplex RNAScope identification of PDE1A transcript (yellow) in MBP-expressing (red) oligodendroglia in human HD brain. DAPI-stained nuclei in blue. (D) BaseScope identification of PDE1A transcript (blue) in OLIG2 transcript-expressing (pink) oligodendroglia in human HD brain. (E) Dot plot showing expression of immune markers across microglial subclusters

Fig. 4

Differential expression analysis between HD and control donors for each glial type and region. Volcano plots showing differentially expressed genes between HD and controls in each glial type and each region. The top eight most differentially expressed genes in either direction are labelled (all in Tables S3-5). Venn diagrams show overlapping and differentially expressed genes between HD and controls per cell type and region

Fig. 5

Molecular chaperone genes are upregulated across glial clusters in HD. A. Venn Diagram showing overlap of cluster markers between Astro_Thyme, Mglia_Violet and OPC_Pine. B. PPI network of shared cluster markers, coloured to denote genes belonging to one of the top 5 GO terms shown below. C-E. Expression of shared molecular chaperone genes in microglia, marking Mglia Violet (C), oligodendroglia, marking OPC Pine (D) and in astrocytes, marking Astro Thyme (E). Dot plots are colour coded according to chaperone family (key below). F-H. Volcano plots showing differentially expressed genes in Mglia Violet (F), OPC Pine (G) and Astro Thyme (H), labelling altered molecular chaperone genes and regions. Log2FC/p-values from MAST comparing HD and control within each cluster

Fig. 6

The molecular co-chaperone BAG3 is upregulated in HD microglia in a separate cohort. A-B. Double in situ hybridisation (A) on a separate human brain tissue cohort showing an increase (B) in microglia (ITGAM+, red) expressing BAG3, green. Arrow for double-positive cell. Plot shows median and range. (Welch Two Sample t-test, p-value = 0.023). C-D. Double in situ hybridisation (C) showing OPCs (PDGFRa+, red) expressing BAG3, green (D). Plot shows median and range (Wilcoxon rank sum test, p-value = 0.1). DAPI-stained nuclei coloured blue in images