Human herpesvirus-associated transposable element activation in human aging brains with Alzheimer's disease

Abstract

Introduction: Human herpesvirus (HHV) has been linked to Alzheimer's disease (AD), but the underlying mechanisms remain unknown.

Methods: We leveraged functional genomics data from Religious Orders Study or the Rush Memory and Aging Project (ROS/MAP) and Mount Sinai Brain Bank (MSBB) brain biobanks and single-cell RNA-sequencing data from HHV-infected forebrain organoids to investigate HHV-infection-associated transposable element (TE) dysregulation underlying AD etiologies.

Results: We identified widespread TE dysregulation in HHV-positive human AD brains, including an astrocyte-specific upregulation of LINE1 subfamily TEs in HHV-positive human AD brains. We further pinpointed astrocyte-specific LINE1 upregulation that could potentially regulate target gene NEAT1 expression via long-range enhancer-promoter chromatin interactions. This LINE1 dysregulation can be partially reversed by the usage of anti-HHV drugs (valacyclovir and acyclovir) in a virus-infected human brain organoid model. Finally, we demonstrated that valacyclovir rescued tau-associated neuropathology and alleviated LINE1 activation in an experimental tau aggregation model.

Discussion: Our analysis provides associations linking molecular, clinical, and neuropathological AD features with HHV infection, which warrants future clinical validation.

Highlights: Via analysis of bulk RNA-seq data in two large-scale human brain biobanks, ROS/MAP (n = 109 pathologically confirmed AD and n = 44 cognitively healthy controls) and MSBB (n = 284 AD and n = 150 cognitively healthy controls), we identified widespread TE activation in HHV-positive human AD brains and significantly positive associations of HHV RNA abundance with APOE4 genotype, Braak staging score, and CERAD score. We identified cell type-specific LINE1 upregulation in both microglia and astrocytes of human AD brains via long-range enhancer-promoter chromatin interactions on lncRNA nuclear enriched abundant transcript 1 (NEAT1). We determined that usage of valacyclovir and acyclovir was significantly associated with reduced incidence of AD in a large real-world patient database. Using the HEK293 tau P301S model and U2OS mt-Keima cell model, we determined that valacyclovir treatment rescued tau-associated neuropathology and alleviated activation of LINE1 with increased cellular autophagy-level mechanistically supported clinical benefits of valacyclovir in real-world patient data.

Keywords: Alzheimer's disease (AD); human herpesvirus (HHV); long interspersed nuclear element 2 (LINE1); neuroinflammation; transposable element (TE); valacyclovir.

FIGURE 1

TE dysregulation in HHV‐positive AD brains. (A) Bulk RNA‐seq datasets from two brain biobanks, including MSBB and ROS/MAP, were downloaded from the AD Knowledge Portal. RNA‐seq data from MSBB were derived from four brain BM, including BM10 (n = 126), BM22 (n = 95), BM36 (n = 128), and BM44 (n = 85). RNA‐seq data from ROS/MAP were derived from DLPFC of 109 AD and 44 cognitively healthy control subjects. We next quantified locus‐based TE expression in noncoding regions, including intronic and intergenic regions. We used HSV‐1 infected dorsal forebrain organoid model to validate our findings. (B) HHV RNA abundance associated with pathological parameters. Significant associations p < .05 were highlighted. (C) Volcano plots comparing HHV‐positive AD brains (n = 24) to HHV‐negative cognitive healthy controls (n = 24). TEs were colored by non‐differentially expressed (q > 0.05; gray), differentially expressed with modest effects (q < 0.05 && |log₂FC| < 1.0; light blue and orange), and differentially expressed with stronger effects (q < 0.05 && |log₂FC| $\ge$ 1.0; dark blue and red). We highlighted several differentially expressed TEs and their nearest genes in the ROS/MAP cohort. (D) Nearest genes of differentially expressed TEs in HHV‐positive brains were enriched in immune‐related pathways. Enriched terms were visualized using the cnetplot tool in the enrichplot package. (E) Expression correlation analysis between L2b subfamily TE (chr1: 226660160‐226660299) and its nearest gene ITPKB. The x‐axis represents expression level of TEs, and the y‐axis represents expression level of ITPKB. The p value was calculated using Spearman's correlation test. R² values show the strength of expression correlation by Spearman's correlation test. (F) Heatmap depicting the hierarchical clustering of the top 10 TEs that exhibited differentially expression between HHV‐positive AD brains and HHV‐negative cognitive healthy controls. AD, Alzheimer's disease; BM, Brodmann areas; DLPFC, dorsolateral prefrontal cortex; HHV, human herpesvirus; MSBB, Mount Sinai Brain Bank; ROS/MAP, Rush Memory and Aging Project; TE, transposable element.

FIGURE 2

Cell type‐specific TE dysregulation in HHV‐positive human AD brains. (A) Heatmap showing meta‐features in HHV‐positive subjects, including HHV RNA abundance in OC and OTC regions, diagnosis, APOE genotype, and sex. (B and C) t‐SNE plot of clustering of 117,362 cells in OC regions (B) and 130,888 cells in OTC regions (C). (D) Heatmap depicting expression changes of cell type‐specific marker genes and subfamily TEs in OC region. (E and F) Differentially expressed genes in microglia that co‐occurred with transcription factors IRF8 (E) and STAT6 (F) were enriched in immune‐related pathways. (G) Network depicting differentially expressed genes that co‐occurred with PPARA transcription factors in astrocytes. Diamonds: AD GWAS genes, triangles: genes associated with Aβ, and V: genes associated with neuroinflammation. (H) t‐SNE plot depicting expression of CLU gene per cluster in OC regions. (I) Heatmap depicting expression changes of cell type‐specific marker genes and differentially expressed subfamily TEs in HSV‐1 infected dorsal forebrain organoids. AD, Alzheimer's disease; APOE, apolipoprotein E; CLU, clusterin; HHV, human herpesvirus; OC, occipital cortex; PPARA, peroxisome proliferator activated receptor alpha; TE, transposable element; tSNE, t‐distributed stochastic neighbor embedding.

FIGURE 3

LINE1 activation triggers neuroinflammation in astrocytes. (A) Module–trait relationships from the weighted gene co‐expression network analysis. The left panel shows the 12 modules. The color scale on the right panel shows correlation coefficients from −1 (blue) to 1 (red). Significant correlations with p < .05 are highlighted. (B) Upset plot showing enriched AD‐related molecular signatures among genes co‐expressed with TEs. Dots with connecting lines indicate signature combinations. (C) Boxplot of expression levels of L1M4c subfamily TE (chr11: 65499661‐65499925) in HSV‐1‐infected and uninfected dorsal forebrain organoids. q was calculated using Benjamini–Hochberg procedure in DEseq2. (D) UCSC genome browser showing cell type‐specific enrichment of H3K27ac, ATAC, H3K4me3, and 3D chromatin interactions in differentially expressed locus TE, including L2b subfamily TE (chr1: 226660160‐226660299) and L1M4c subfamily TE (chr11: 65499661‐65499925). The locus‐based TE region is highlighted in blue. (E and F) Expression correlation between differentially expressed L1M4c subfamily TE (chr11: 65499661‐65499925) and its nearest gene NEAT1 (E) and between differentially expressed L1M4c subfamily TE (chr11: 65499661‐65499925) and Braak staging score (F). P value was calculated using Spearman's correlation test. R ² values show strength of expression correlation by Spearman's correlation test. (G and H) t‐SNE plot depicting expression of NEAT1 gene per cluster in OC and OTC tissues. (I) Network depicting functional enrichment of lncRNA NEAT1 target genes. Hexagon representing AD‐related pathways. AD, Alzheimer's disease; HSV‐1, human simplex virus type 1; L1M4c, LINE1 subfamily; OC, occipital cortex; TE, transposable element; t‐SNE, t‐distributed stochastic neighbor embedding.

FIGURE 4

Antiviral drug use associated with reduced AD incidence in real‐world data. We performed hazard ratio analysis of antiviral drug usage in all subjects of Optum Clinical Informatics data (n = 604,026) (A) and in gender‐specific (B and C) and age‐specific subgroup analysis (D and E) after controlling for covariables. AD, Alzheimer's disease.

FIGURE 5

Valacyclovir treatment resulted in decreased tau aggregation and attenuated LINE1 ORF1p activation. (A) Confocal images showing both tau puncta and LINE‐ORF1p puncta changes in HEK293 tau‐Venus cells with and without tau assemblies (10 nM, 48 h). Scale bars: 10 µm. (B) Valacyclovir treatment on degradation of aggregated endogenous tau in HEK293 P301S tau‐Venus cells. (C) Western blotting showing protein‐level changes with and without valacyclovir treatment, including total tau, designated phosphorylated tau sites (Thr181, Ser022/Thr205), LINE‐ORF1p, NIX, and LC3B II. (D) Valacyclovir treatment attenuated activation of LINE1 ORF1p in HEK293 P301S tau‐Venus cells. All quantitative data were shown in mean ± SEM. *p < .05, **p < .01, ***p < .001, ****p < .0001.

FIGURE 6

TE dysregulation in HSV‐1‐infected human brain organoid and its reversibility by usage of anti‐HHV medicines. (A and B) t‐SNE plots of single‐cell gene expression of HSV‐1 uninfected (A) and infected dorsal forebrain organoids plus ACV treatment at 3 days (B). (C) Highlighted pathways enriched by reversed nearby genes of differentially expressed subfamily TEs after acyclovir treatment. (D) Venn diagram showing overlapping differentially expressed subfamily TEs before and after acyclovir treatment. (E) Heatmap depicting expression changes of L1M4c subfamily TE (chr11: 65499661‐65499925) and its nearest gene NEAT1 in HSV‐1 infected dorsal forebrain organoids with and without acyclovir treatment. (F) ACV treatment rescued dysregulated subfamily TE induced TE‐TF‐gene regulatory network by HSV‐1 infection. Diamonds: reversed subfamily TEs, circles: enriched TFs, and hexagons: co‐occurring genes with enriched TFs. ACV, acyclovir; HHV, human herpesvirus; HSV‐1, human simplex virus type 1; TE, transposable element; t‐SNE, t‐distributed stochastic neighbor embedding.

FIGURE 7

Proposed transposable element upregulation‐associated AD‐like mechanisms in human AD brains. We found cell type‐specific LINE1 expression upregulation in HHV‐positive human AD brains, including upregulated LINE1 subfamily TE (L1MB3 and L1MEd) and LINE1 subfamily TE (L1MA9 and L1MA2) in microglia and astrocytes, respectively. The upregulated LINE1 subfamily TE might contribute to microglia into activated microglia and transform astrocytes into reactive astrocytes, which resulting in the generation of proinflammatory cytokines. Among the perturbed genes regulated by elevated LINE1, we found that upregulated ITPKB and NEAT1 could potentially be involved in both amyloid beta and tau pathology. The upregulated LINE1 and AD pathologies can be partially reversed using anti‐HHV medicines valacyclovir and acyclovir. AD, Alzheimer's disease; HHV, human herpesvirus; TE, transposable element.