High resolution time-course mapping of early transcriptomic, molecular and cellular phenotypes in Huntington's disease CAG knock-in mice across multiple genetic backgrounds

Abstract

Huntington's disease is a dominantly inherited neurodegenerative disease caused by the expansion of a CAG repeat in the HTT gene. In addition to the length of the CAG expansion, factors such as genetic background have been shown to contribute to the age at onset of neurological symptoms. A central challenge in understanding the disease progression that leads from the HD mutation to massive cell death in the striatum is the ability to characterize the subtle and early functional consequences of the CAG expansion longitudinally. We used dense time course sampling between 4 and 20 postnatal weeks to characterize early transcriptomic, molecular and cellular phenotypes in the striatum of six distinct knock-in mouse models of the HD mutation. We studied the effects of the HttQ111 allele on the C57BL/6J, CD-1, FVB/NCr1, and 129S2/SvPasCrl genetic backgrounds, and of two additional alleles, HttQ92 and HttQ50, on the C57BL/6J background. We describe the emergence of a transcriptomic signature in HttQ111/+ mice involving hundreds of differentially expressed genes and changes in diverse molecular pathways. We also show that this time course spanned the onset of mutant huntingtin nuclear localization phenotypes and somatic CAG-length instability in the striatum. Genetic background strongly influenced the magnitude and age at onset of these effects. This work provides a foundation for understanding the earliest transcriptional and molecular changes contributing to HD pathogenesis.

Figure 1

Time course of mHTT nuclear localization and of striatal Htt CAG length expansion in 4- to 20-week-old HD knock-in mice. (A) Striatal tissue was collected from a total of 731 4- to 20-week-old mice in order to compare mice with heterozygous mutant Htt alleles (Q111, Q92, Q50) to mice with wild-type Htt alleles (WT) on four genetic backgrounds. (B) Inherited CAG repeat length of Htt alleles in Htt^Q111 mice varies with genetic background. The CAG repeat length in each mouse with a nominal Htt^Q111 allele was measured in a tail snip sample at weaning. (C) Nuclear mutant huntingtin immunofluorescence using mAb5374 (red) and DAPI (blue) counterstain in 20-week-old B6.Q111, CD1.Q111, FVB.Q111, 129.Q111 mice, along with negative control 20 week-old B6.WT. All images were acquired at the same sensitivity and displayed with equal adjustment of the levels to enhance the red signal relative to background and nuclear staining. To illustrate the pattern of the weak staining in 129.Q111 the cell in the inset was further enhanced; arrows indicate positive cells. The white scale bar represents 10 µm. (D) Quantitation of mHtt immunofluorescence intensity in 9- to 20-week-old Q111 mice from each background (n = 84 total mice, n = 20–25 mice per strain). Each point indicates the immunofluorescence intensity in a single cell. (E). Age- and background strain-dependent expansion of Htt CAG tracts in the striata of 4- to 20-week-old Q111 mice. Each point represents the ‘instability index’ for a single mouse, a quantitative metric representing the mean CAG length change in the population, relative to the modal allele.

Figure 2

Age- and strain-dependent effects of Htt alleles on gene expression. (A) Counts of up- and down-regulated genes (FDR q < 0.05) in each genotype at three time windows: ‘early’ (4- to 9-week-old), ‘middle’ (9- to 16-week-old), and ‘late’ (16- to 20-week-old). (B) Overlap between differentially expressed genes (DEGs) detected in the three conditions with >25 DEGs. (C) Overlap between DEGs in 16- to 20-week-old B6.Q111 mice (this study) and DEGs in 6-month-old B6.Q111 mice from Langfelder et al. (2016). (D) Overlap between DEGs in 16- to 20-week-old B6.Q111 mice (this study) and DEGs in post-mortem caudate nucleus from HD cases vs. controls (Hodges et al. 2006).

Figure 3

Expression patterns of the top 100 differentially expressed genes. The top 100 genes were defined by their P-values in 16- to 20-week-old B6.Q111 mice. The heatmap indicates the -log₁₀(P-values) of these genes in 18 conditions defined by genotype and age window. Orange gradient = up-regulated in Q111; blue gradient = down-regulated in Q111; color range, -7 < z-score <7.

Figure 4

Age, CAG length and genetic background influences the magnitude but not the direction of gene expression changes in HD knock-in mice. Each scatterplot compares the fold changes of the top 100 genes in 16- to 20-week-old B6.Q111 mice (x-axis) to the fold changes of these genes in another condition, defined by age and genotype (y-axis). Each point on the scatterplot indicates the fold change estimate of a single gene.

Figure 5

Effects of genetic background and of HD mutations on Mlh1 expression. Box plots represent mean expression of Mlh1 in each strain and genotype group.