Activation of endogenous retroviruses during brain development causes an inflammatory response

Abstract Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9‐based gene disruption of the epigenetic co‐repressor protein Trim28, we found a dynamic H3K9me3‐dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV‐derived proteins in aggregate‐like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.

A Differential expression analysis of individual TEs in mouse NPCs upon CRISPR/Cas9-mediatied disruption of Trim28 was performed with DESeq2 (described in Materials and Methods). B The upregulated transcription of MMERVK10C elements was validated by qRT-PCR. Columns show an average of the individual data points. C Genes located in the close vicinity of upregulated MMERVK10C elements were significantly upregulated. X-axis indicate the window-of-inclusion for genes located close to an MMERVK10C. Boxplot hinges represent first and third quartile, and the median is indicated by the central band. Whiskers extend to 1.5 times the interquartile range. D RNA-seq differential expression analysis of protein coding genes performed with DESeq2 (described in Materials and Methods) in mouse NPCs upon CRISPR/Cas9mediatied disruption of Trim28. E, F PCA analysis of gene expression was unable to distinguish the Trim28-KO cells from Ctl, while PCA analysis of TE expression was. The spread of the groups is visualized with opaque ellipses.
Source data are available online for this figure.
▸ Figure EV2. Deletion of Trim28 in adult neurons in vivo does not result in an upregulation of ERVs.
A A schematic of the workflow targeting Trim28 in the forebrain of Stop-Cas9-GFP knock-in mice using AAV vectors expressing the gRNA and a nuclear RFP reporter as well as an AAV vector expressing Cre, specifically in neurons by the Synapsin promoter. 8 weeks after injection, the animals were analyzed either by IHC or by DNA/RNA-sequencing following nuclei isolation by FACS. B Gene editing efficiency was evaluated by amplicon sequencing of the respective targeted sequences. DNA was isolated from 50,000 RFP + nuclei per animal, one animal per group was analyzed. Black bars indicate % of frameshift mutations. C, D Neuron-specific editing of the Trim28-loci resulted in a robust loss of the Trim28 protein in neurons, as evaluated by IHC where the expression of Trim28 in RFP + cells was quantified and is displayed as mean AE SEM. Approximately 550 RFP + /GFP + cells per animal and group was evaluated. Scale bar 30 µm. E Differential expression analysis of TEs in adult neurons from the Stop-Cas9-GFP knock-in mice upon Trim28-KO was done by using TEtranscripts and DESeq2 (described in Materials and Methods). F A schematic of the workflow targeting Trim28 in adult neurons in the forebrain of Trim28-flox mice (+/À and +/+) by co-injecting AAV vectors expressing Cre or nuclear GFP under the control of the Synapsin promoter. 8 weeks after injection, the animals were analyzed either by IHC or by RNA-sequencing following nuclei isolation by FACS. G RNA-seq analysis of the isolated GFP + nuclei revealed a loss of the Trim28 transcript. Boxplot hinges represent first and third quartile, and the median is indicated by the central band. Whiskers extend to 1.5 times the interquartile range. H TE differential expression analysis on the adult neurons in the Trim28-flox animals was done using TEtranscripts and DESeq2 (described in Materials and Methods). I IHC for Trim28, GFP, and the neuronal marker NeuN revealed a complete neuronal loss of the protein Trim28 in the Trim28-KO animals (Trim28-flox mice (+/+)). Scale bar 75 µm. A RNA-seq analysis of Trim28 expression in the adult cortex of Emx1-Cre/ Trim28-KO animals (Emx1-Cre(+/À), Trim28-flox(+/+)). Boxplot hinges represent first and third quartile, and the median is indicated by the central band. Whiskers extend to 1.5 times the interquartile range. B Differential TE expression analysis for individual TEs in the adult cortex of Emx1-Cre/Trim28-KO animals compared to their Cre-negative control litter mates was performed with DESeq2 (described in Materials and Methods). C Expression of genes nearby upregulated full length MMERVK10Cs were not upregulated in the Emx1-Cre/Trim28-KO animals. X-axis indicate the window-of-inclusion for genes located close to an MMERVK10C (test performed with DESeq2, Log2FC > 0). Boxplot hinges represent first and third quartile, and the median is indicated by the central band. Whiskers extend to 1.5 times the interquartile range. D No transcriptional readthrough into nearby genes from upregulated MMERVK10Cs were observed, exemplified with an UCSC screenshot of the same location in the genome as shown in Fig 1G. ◀ Figure EV5. Iba1 + densities and inflammatory/viral defense genes upon Trim28-KO. ▸ A The number of Iba1 + cells did not differ in the cortex of Emx1-Cre/Trim28-KO animals and controls, here shown as mean AE SEM, unpaired t-test. Iba1 + cells were counted in 20 photographs (20x objective) per animal, control n = 3 and KO n = 2. B Immunohistochemical analysis for the microglia marker upon Trim28-KO in mature neurons in vivo (AAV.Syn-Cre/Trim28-KO animals) revealed no difference in Iba1 morphology between Trim28-KO animals and controls. Scale bar 75 lm. C Inflammatory genes were chosen from 216 genes annotated in the immune response GO term (GO0006954) if none of the confidence intervals overlapped zero Three genes were significantly downregulated upon the Emx1-Cre/Trim28-KO and are labeled in red: Bmp6, Cd163, and Ptgdr (P-adj < 0.05), n = 3 per condition. Error lines showing 95% confidence intervals. The list of viral defense genes was retrieved from (Liu et al, 2018). None of these were significantly different upon the Emx1-Cre/ Trim28-KO.

GFP/RFP/
Source data are available online for this figure.