Messenger RNA processing is altered in autosomal dominant leukodystrophy

Abstract

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.

Figure 1.

Transcriptional signature of ADLD. (A and B) LMNB1 mRNA quantification in ADLD fibroblasts (A) and whole blood (B) relative to control samples (CTR). LMNB1 mRNA levels were normalized to HMBS mRNA (hydroxymethylbilane synthase gene) expression. Bars represent the mean ± SD of values obtained from fibroblasts (ADLD, n = 4; CTR, n = 6) or blood (ADLD, n = 4; CTR, n = 8); *P < 0.05, Student's t-test. (C and D) Validation of gene expression obtained from microarray data by RT-qPCR. Gene expression in ADLD (n = 4) fibroblasts (C) or blood (D). Results from microarray and RT-qPCR are shown. Bars represent the average fold change over CTR ± SD. Six/eight CTRs were used for analyses on fibroblasts and blood, respectively.

Figure 2.

RAVER2 expression varies as a function of lamin B1 levels. (A) HeatMap of DEGs with statistically significant changes (FDR-adjusted P-value smaller than 0.05) in fibroblasts (ADLD, n = 4; CTR, n = 5) and blood (ADLD, n = 4; CTR, n = 8). The GENE-E software (http://www.broadinstitute.org/cancer/software/GENE-E/) was used to generate the heatmaps. (B) Quantitative analysis of RAVER2 mRNA in vADLD and CTR fibroblast samples. The RAVER2 mRNA levels were normalized to HMBS mRNA expression. Bars represent the mean relative expression ± SD of one vADLD (two different batches of cells from the same donor) versus fibroblasts from six CTRs. *P < 0.05, Student's t-test. (C) Representative western blot of lamin B1 and raver-2 in fibroblasts from two ADLD patients and two CTRs. γ-Tubulin was used as loading control. (D) Quantitative analysis of raver-2 protein levels in fibroblasts from ADLD patients (n = 4) and age-matched CTR subjects (n = 4). The data are normalized to γ-tubulin and expressed as percentages of CTR levels. Bars represent average ± SEM. *P < 0.05, Mann–Whitney Rank Sum test. (E) Representative confocal images of primary fibroblasts from one ADLD patient and one age-matched CTR immunodecorated against raver-2 (green). Nuclei are counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride. Scale bars: 10 µm. (F) Representative western blot of lamin B1 protein expression in brain of Lmnb1+/+ and Lmnb1Δ/Δ E18.5 embryos. (G) Raver2 mRNA expression in brain of E18.5 mouse embryos analyzed by RT-qPCR. *P < 0.05, Student's t-test.

Figure 3.

Abnormal lamin B1 expression is associated with altered splicing of PTB-target genes. Top significantly altered PTB-regulated alternative splicing identified in ADLD blood (A) and fibroblasts (B). Alternative splicing was analyzed by RT-PCR and dHPLC. For each gene, a representative RT-PCR is shown above the dHPLC data graph. Bars represent the average percentage of each splicing event ± SD. *P < 0.05, Student's t test, n = 4. EX, exon; IN, exon inclusion; SK, exon skipping; F, fibroblasts; B, blood. The splicing pattern of other PTB-regulated genes analyzed in blood and fibroblasts are shown, respectively, in Supplementary Material, Figures S1 and S2. (C) Significantly altered PTB-regulated alternative splicing events identified in brains of Lmnb1+/+ (n = 4) and Lmnb1Δ/Δ (n = 3) E18.5 embryos. Bars represent the average percentage of each splicing event ± SD. *P < 0.05, Student's t test. The splicing pattern of Fam38a, that was not significantly altered, is shown in Supplementary Material, Figure S3.

Figure 4.

Abnormal levels of lamin B1 alter PLP1 splicing. (A) Analysis of embryonic (DM20) and adult (PLP) isoforms of PLP1 gene in ADLD (n = 4) and CTR (n = 5) fibroblasts analyzed through RT-PCR and dHPLC run. Bars represent the mean ± SD. *P < 0.05, Student's t-test, n = 5. (B) Analysis of Plp1 splicing pattern in brain of Lmnb1+/+ (n = 4) and Lmnb1Δ/Δ (n = 3) embryos. *P < 0.05, Student's t-test. Representative gels of mRNA splicing isoforms are shown above dHPLC data graphs.