Repetitive element transcripts are elevated in the brain of C9orf72 ALS/FTLD patients

Abstract

Significant transcriptome alterations are detected in the brain of patients with amyotrophic lateral sclerosis (ALS), including carriers of the C9orf72 repeat expansion and C9orf72-negative sporadic cases. Recently, the expression of repetitive element transcripts has been associated with toxicity and, while increased repetitive element expression has been observed in several neurodegenerative diseases, little is known about their contribution to ALS. To assess whether aberrant expression of repetitive element sequences are observed in ALS, we analysed RNA sequencing data from C9orf72-positive and sporadic ALS cases, as well as healthy controls. Transcripts from multiple classes and subclasses of repetitive elements (LINEs, endogenous retroviruses, DNA transposons, simple repeats, etc.) were significantly increased in the frontal cortex of C9orf72 ALS patients. A large collection of patient samples, representing both C9orf72 positive and negative ALS, ALS/FTLD, and FTLD cases, was used to validate the levels of several repetitive element transcripts. These analyses confirmed that repetitive element expression was significantly increased in C9orf72-positive compared to C9orf72-negative or control cases. While previous studies suggest an important link between TDP-43 and repetitive element biology, our data indicate that TDP-43 pathology alone is insufficient to account for the observed changes in repetitive elements in ALS/FTLD. Instead, we found that repetitive element expression positively correlated with RNA polymerase II activity in postmortem brain, and pharmacologic modulation of RNA polymerase II activity altered repetitive element expression in vitro. We conclude that increased RNA polymerase II activity in ALS/FTLD may lead to increased repetitive element transcript expression, a novel pathological feature of ALS/FTLD.

Figure 1

Repetitive element transcripts are enriched in the frontal cortex of c9ALS cases. Using RNAseq data from 9 healthy controls, 10 sALS and 8 c9ALS cases (17), we evaluated changes in the expression of repetitive elements. (A) MA plot showing the Log2 fold change and mean repetitive element expression levels in the human cortex tissue of c9ALS brains compared to sALS. Note the 300 significant changes are depicted in red (FDR < 0.1). (B) Main class distribution of the repetitive elements significantly altered in the frontal cortex of c9ALS vs. sALS cases.

Figure 2

Increased repetitive element accumulation in the frontal cortex of c9ALS/FTLD cases. A series of repetitive elements identified to be upregulated in c9ALS vs. sALS cases in our RNAseq analyses were validated in a large cohort of cases (Supplementary Material, Table S1) by qRT-PCR: 56 C9+, 46 C9-, 9 healthy controls. Validations were done for elements belonging to LTR (A), SINE (B) and LINE (C) repetitive element classes. Note, three of the repetitive elements shown (LTR70, MER21B, AluYa5) did not reach statistical significance from the RNAseq data (FDR > 0.1) but we observed significant changes by qRT-PCR. Statistical analyses were performed to compare all groups using a One-way ANOVA followed by Dunn’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Inhibition of CDK9 leads to a decrease in RNApol II activity and repetitive element expression. HEK293T cells were treated with DMSO control and the indicated amounts of LDC000067, a CDK9 inhibitor, for a total of 3 h. (A) Immunoblots showing the levels of phosphorylated RNApol II largest subunit (pRPB1), total RPB1 and GAPDH, in response to different doses of CDK9 inhibitor. (B) Quantification of immunoblots from 2 independent experiments in which each condition was performed in duplicates, and in which pRPB1 and total RPB1 protein levels were normalized to GAPDH or as pRPB1/Total RPB1 ratio. (C, D) qRT-PCR analyses show a significant response to CDK9 inhibition of repetitive elements belonging to the LTR (C) and SINE (D) classes. Statistical analyses were performed to compare all groups using a One-way ANOVA followed by Bonforreoni’s multiple comparison test, *P < 0.05, **P < 0.01, ^#P < 0.0001.

Figure 4

CDK9 and SUPT4H RNA levels are elevated in C9orf72-positive cases. The levels of CDK9 (A) and SUPT4H1 (B) RNA were assessed by qRT-PCR as described in methods for our large cohort of cases (Supplementary Material, Table S1). Statistical analyses were performed to compare all groups using a One-way ANOVA followed by Dunn’s multiple comparison test, **P < 0.01, ***P < 0.001. Of note, results from a subsequent linear regression model were also significant (C9+ vs. C9-: SUPT4H, P = 0.0288; CDK9, P = 0.0059), while correcting for disease duration, disease subtype, and age at death.

Figure 5

The levels of phosphorylated RPB1 protein are elevated in C9orf72-positive cases. The levels of phosphorylated RPB1 (pRPB1, (A) and total RPB1 (B) proteins were assessed by immunoblotting as described in methods for our large cohort of cases (Supplementary Material, Table S1) and normalized to GAPDH protein levels. Note the levels of pRPB1 are also presented normalized to total RPB1 levels (C). Statistical analyses were performed to compare all groups using a One-way ANOVA followed by Dunn’s multiple comparison test, *P < 0.05.