Retinal dysfunction in Huntington's disease mouse models concurs with local gliosis and microglia activation

Abstract

Huntington's disease (HD) is caused by an aberrant expansion of CAG repeats in the HTT gene that mainly affects basal ganglia. Although striatal dysfunction has been widely studied in HD mouse models, other brain areas can also be relevant to the pathology. In this sense, we have special interest on the retina as this is the most exposed part of the central nervous system that enable health monitoring of patients using noninvasive techniques. To establish the retina as an appropriate tissue for HD studies, we need to correlate the retinal alterations with those in the inner brain, i.e., striatum. We confirmed the malfunction of the transgenic R6/1 retinas, which underwent a rearrangement of their transcriptome as extensive as in the striatum. Although tissue-enriched genes were downregulated in both areas, a neuroinflammation signature was only clearly induced in the R6/1 retina in which the observed glial activation was reminiscent of the situation in HD patient's brains. The retinal neuroinflammation was confirmed in the slow progressive knock-in zQ175 strain. Overall, these results demonstrated the suitability of the mouse retina as a research model for HD and its associated glial activation.

Figure 1

RNA-seq analysis in the retina and striatum of R6/1 mice. (A) Venn diagram showing the number of DEGs in pairwise comparisons between wt and R6/1 mice (adj. p-value < 0.05, n of pools = 3 per genotype). Downregulated genes were indicated as a (exclusive retinal), b (common to both tissues) and c (exclusive striatal), and upregulated genes as d (exclusive retinal), e (common) and f (exclusive striatal). (B) Absolute values of log₂ fold change (left) and − log adj. p-value (right) in the R6/1 retina and striatum: outliers > 5 and > 30 were removed from each panel respectively to enable visualization of medians and quartiles of the values. (C) Normalized basal expression of DEGs in wt retinas and striata. **p-value < 0.005, Student’s t-test between tissues. (D) GO enrichment analysis of DEGs in the retina and the striatum of R6/1 mice (p-value < 0.001 (DAVID)); not significant results were retrieved for subset e. Letters indicate the subsets of genes defined in (A). Data are expressed as mean ± s.e.m. Vm membrane potential, cAMP cyclic AMP, MSN medium spiny neurons, IFN interferon, Ag antigen. (E) Venn diagram showing the genes affected by differential alternative splicing (diffAS) in R6/1 retina and striatum compared to wt littermates. Pie charts indicate the type of aberrant splicing compiled in vast-tools, rMATS and SUPPA2: A5, alternative 5’ splice-site; A3, alternative 3’ splice-site; RI, retained intron; Excl, excluded exon (mutually exclusive exons, alternative first and last exon); SE, skipped exon. (F) GO enrichment analysis of genes with diffAS in the retina and the striatum of R6/1 mice (p-value < 0.001 (DAVID)). Numbers besides bars indicate the number of genes contained in GO categories. (G) Retinal DEGs were ranked according to their significance and direction of change, and divided in bins of 100 genes. On the left, percentage of retinal cell-specific markers counted per bin. For “Other neuronal” we represent the average of counts from amacrine, bipolar and retinal ganglion cells, for “Macroglia” the averaged counts from Müller glia and astrocytes, and for “Other non-neuronal” the averaged counts from pericytes and perivascular fibroblasts. On the right, the same data were represented as grouped percentages in a bar graph, besides the genes affected by aberrant alternative splicing (diffAS).

Figure 2

Time-course profiling of molecular alterations in the R6/1 retina and striatum. (A,B) Time-course analysis of the downregulation (A) and upregulation (B) of selected genes in the R6/1 retina compared to wild-type littermates. 7 weeks, n = 6 (wt) and n = 8 (R6/1); 13–15 weeks, n = 5 (wt) and n = 7 (R6/1); 25–28 weeks, n = 3 per genotype. (C,D) The same analysis in the R6/1 and wild-type striata. 7 weeks, n = 7 (wt) and n = 8 (R6/1); 13–15 weeks, n = 7 (wt) and n = 9 (R6/1); 25–28 weeks, n = 8 per genotype. Data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.005, genotype effect; ^§p < 0.05, ^§§p < 0.005, age effect; ^#p < 0.05, ^##p < 0.005, interaction effect; ^~p < 0.1 in any effect, ANOVA test. (E,F) Protein extracts from the retina (E) and striatum (F) of R6/1 and wt littermates were analyzed by western blotting assays with antibodies against LC3II/I and α-tubulin as loading control. Retina early, 7/9/15-week-old, n = 4 (wt) and n = 6 (R6/1); Retina late, 21/25-week-old, n = 3 (wt) and n = 4 (R6/1); striatum early, 5/13-week-old, n = 6 (wt) and n = 9 (R6/1); striatum late, 25-week-old, n = 4 (wt) and n = 5 (R6/1). Data are expressed as mean ± s.e.m. *p < 0.05 genotype effect; ^§p < 0.05 age effect; ^#p < 0.05 interaction; ^~p < 0.1 in any effect, ANOVA test in R6/1 and wt littermates. More blots are shown in Supplementary Fig. S3.

Figure 3

A glial activation signature is only present in the R6/1 retina. (A) Plots showing the upregulated genes in 13–15 weeks-old R6/1 retina (top panels) and striatum (bottom panels) according to magnitude (log₂ fold change) and significance (− log adj. p-value) of change in the RNA-seq analysis that are markers for astrocytes (left panels) and microglia (right panels) at different stages of activation (see text for further details). For striatal genes no significance cut-off was applied to permit the analysis of the same number of genes as in retina to facilitate the comparison between both tissues. (B) Heatmap plot of the fold changes of DEGs belonging to the A1, A2 and panastrocytic signatures (see text). ¶, adjusted p-value < 0.05 from the RNA-seq analysis. *p-value < 0.05, Student’s t-test between the fold changes means of both tissues.

Figure 4

Morphological alterations associated with neuroinflammation in the R6/1 retinas. (A) Representative immunofluorescence stainings across the indicated time points of the gliosis marker GFAP in R6/1 and wt littermates. (B) Representative whole mounts of R6/1 and wt retinas with GFAP staining. (C) Upper panels, representative immunofluorescence staining of the microglia marker Iba-1 for R6/1 and wt littermates. Lower panel, quantification of the Iba-1⁺ cells distinguishing their morphology in ramified and amoeboid in R6/1 retinas compared to wt littermates. N = 2–4 per genotype, age and model; n = 3–5 slices per animal, n = 3 fields per slice; field area = 84,100 μm². Data are expressed as mean ± SD. *p-value < 0.05, Student’s t-test between Iba-1⁺ cell subtypes. Green, glial marker; blue, DAPI staining. Scale = 20 μm. ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, Ramif ramified, Amoeb amoeboid. (D) Representative SD-OCT images from the fundus of a R6/1 mouse and wt littermates. Red inset, magnified image indicating hyperreactive spots (*) and thicknesses of INL and ONL (yellow and green lines, respectively). Arrows denote the B-scanned transects for thickness calculations. Scale bars = 100 μm. (E) Quantifications of the retinal thickness in 25-week-old R6/1 (n = 5) and matched-age wt (n = 5). Data are shown as mean ± SD. *p < 0.05; **p < 0.005, Student’s t-test between genotypes. (F) Quantification of DAPI-stained cells in the retinal ONL of mutant mice compared to wt across different time points, N = 3–4 for each genotype, n = 2–5 slices per animal. Data are shown as mean ± SD. **p < 0.005 genotype effect; ^§§p < 0.005 age effect; ^##p < 0.005 interaction effect from ANOVA test in R6/1 and wt littermates.

Figure 5

Retinal neuroinflammation is also detected in the zQ175 retinas. (A,B) The same analysis as in Fig. 2A and B for zQ175 and wt littermates. At the early time point (7 months) we included homozygous mice for the CAG expansion to check whether there was a possible exacerbation of the transcriptional dysregulation that was only observed for downregulated striatal genes. 7 months in each tissue, n = 5 (wt), n = 4 (zQ175^+/−) and n = 4 (zQ175^+/+); 12 months in striatum, n = 6 per genotype; in retina, n = 5 (wt) and n = 7 (zQ175^+/−). Data are expressed as mean ± s.e.m. *p < 0.05, **p < 0.005, genotype effect; ^§p < 0.05, ^§§p < 0.005, age effect; ^##p < 0.005, interaction effect; ~ , p < 0.1 in any effect, ANOVA test. (C) Representative whole mounts of zQ175^+/− and wt retinas with GFAP staining. (D) The same analysis as in Fig. 4C for zQ175 and wt littermates. N = 2 per genotype, age and model; n = 3–5 slices per animal, n = 3 fields per slice; field area = 84,100 μm². Data are expressed as mean ± SD.*, p-value < 0.05, Student’s t-test between Iba-1⁺ cell subtypes. Green, microglial marker; blue, DAPI staining. Scale = 20 μm. ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer; GCL ganglion cell layer, Ramif ramified, Amoeb amoeboid. (E) Representative SD-OCT images from the fundus of a zQ175 mouse and wt littermate. Red inset, magnified image indicating hyperreactive spots (*) and thicknesses of INL and ONL (yellow and green lines, respectively). Arrows denote the B-scanned transects for thickness calculations. Scale bars = 100 μm. (F) Quantifications of the retinal thickness in 12-month-old zQ175 (n = 2) and matched-age wt (n = 4). Data are shown as mean ± SD. *p < 0.05; **p < 0.005, Student’s t-test between genotypes. (G) Quantification of DAPI-stained cells in the retinal ONL of 12-month-old mutant mice compared to wt. N = 3 for each genotype, n = 2–4 slices per animal. Data are shown as mean ± SD. **p < 0.005, Student’s t-test between wt and zQ175 mice.