Molecular effect on pre-mRNA tau alternative splicing of two novel intronic MAPT gene mutations associated to a sporadic case of frontotemporal dementia

Abstract

Frontotemporal lobar degeneration (FTLD) is a heterogeneous syndrome encompassing different nosological entities characterized by behavioural and personality change, accompanied by deterioration of executive function, language and movement. Clinically FTLD results in at least three distinct syndromes: Frontotemporal dementia (FTD), Semantic dementia (SD) and Primary progressive aphasia (PPA), while the pathological classification is based on histopathological presence or absence of neuronal inclusions of tau and/or ubiquitin proteins accumulating in the neuronal/glial inclusions, being forms of FTLD differentiated in tau-positive, ubiquitin-positive and tau-negative. The most common clinical manifestation of FTLD is FTD, characterized by atrophy of the frontal and temporal lobes, with neuronal loss, gliosis and spongiosis of the superficial layers. FTD is mostly a presenile disorder showing changes in personality, impaired social conduct, emotional blunting, loss of insight, disinhibition, perseverative behaviour and hyperorality; cognitive deterioration, especially in language and in executive functions, appear later. Despite most cases of FTD are sporadic, approximately 10%-50% of FTD patients have a positive family history for dementia. Familial FTDL was associated to mutations in four genes: Microtubules associated protein tau (MAPT) and Progranulin (PGRN) genes that are responsible for the most genetic forms of FTD; instead, Valosin containing protein (VCP) gene is involved in rare forms of FTD with inclusion body myopathy and Paget’s disease of the bone and Charged multivescicolar body protein 2B (CHMP2B) is mutated in some families with a combination of FTD and Amyotrophic lateral sclerosis (ALS). Mutations in MAPT gene are responsible for 10%-20% of familial FTD. Alternative splicing of exons 2, 3 and 10 in MAPT pre m-RNA results in the expression of six isoforms. Exclusion or inclusion of Exon 10 gives rise to tau isoforms with three (tau3R, E10-) o four (tau4R, E10+) microtubule-binding repeats. In normal adult human brain the overall ratio of 3R to 4R tau is generally 1, whereas in fetal brain only the shortest tau isoform with 3R is expressed, indicating that tau expression is developmentally regulated.To date, 44 different potential pathogenic MAPT mutations have been reported, divided into two groups depending on the primary molecular mechanism involved: missense or deletion mutations that commonly modify tau interaction with microtubules and splicing mutations that affect the alternative splicing of exon 10, leading to changes of the ratio of 3R-tau/4R-tau. However, a third group of mutations exists that might have effects at protein and RNA levels. In the present study we report the molecular effect of two novel heterozygous MAPT gene mutations, a T to C transition at position -15 of intron 9 [T(-15)C] and an A to C transversion at position +4 of intron 10 (E10+4), identified in a patient with sporadic FTD, clinically and neuropathologically ascertained. Considering that both mutations are located in the splicing regulatory regions surrounding Exon 10, we analyzed their molecular effect on the alternative splicing of MAPT pre-mRNA in a minigene model system and in brain tissue. Semi-quantitative RT-PCR analyses, in minigene costructs and in brain tissue, have shown that the two novel mutations cause a novel Exon 10 splicing effect giving rise to a higher increase of mRNAs transcripts lacking Exon 10 (E10- or Tau3R) when compared with FTD-Ub+ control. Immunohistochemical and biochemical analyses on brain tissue evidenced neuronal and oligodendroglial tau deposits mostly made of Tau3R isoforms and an increased increased availability of shorter Tau3R isoform respectively. Data obtained with minigenes derived by the phenotipically healthy patient’s parents demonstrate that when the mutations are inherited in a non compound heterozygous condition the ratio of E10 including/E10 excluding transcripts is quite normal. Although the molecular mechanism underlying exon 10 splicing regulation remain to be completely elucidated, the exon 10 splice donor site is predicted to give rise to a RNA stem loop structure considered crucial for the quantitative regulation of exon 10 alternative splicing. Most of previously characterized mutations identified in the upper part of the stem loop strongly alter mRNA splicing by destabilizing the secondary structure, with a corresponding increase of E10 inclusion and 4RTau expression. Considering that the E10+4 mutation is located into the exon 10 splice donor site, we also investigated the effect of the E10+4 mutation on the thermodynamic stability of the RNA stem loop structure. Our data, based on bioinformatic prediction of the stem loop sequence thermostability and Ultraviolet Melting experiments demonstrated a strong increasing of stability in the stem-loop structure carrying the E10+4 mutation. This higher stability could be important for the skipping of exon 10, even though the E10+4 mutation alone is not able to give rise to a pathologic phenotype. We cannot exclude that the T to C transition, localized in a regulatory region upstream of exon 10, could also alter the binding of specific trans-splicing factors increasing the effect of the E10+4 mutation, giving rise when both mutation are present in the compound heterozygous condition (namely the FTD patient) to the E10 exclusion and the altered 4R/3R tau ratio observed. Thus, we can hypothesize a trans-acting regulatory effect of both mutations with known, or unknown splicing factors, which might have contributed to the very atypical clinical and pathological FTD phenotype of the patient.