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. 2021 Jul;142(1):159-177.
doi: 10.1007/s00401-021-02317-z. Epub 2021 May 2.

A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease

Vega García-Escudero #  1   2   3 , Daniel Ruiz-Gabarre #  1   2   3 , Ricardo Gargini  3   4 , Mar Pérez  1   3 , Esther García  3 , Raquel Cuadros  3 , Ivó H Hernández  3 , Jorge R Cabrera  5 , Ramón García-Escudero  6   7   8 , José J Lucas  3   9 , Félix Hernández  3   9 , Jesús Ávila  10   11
Affiliations

A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease

Vega García-Escudero et al. Acta Neuropathol. 2021 Jul.

Abstract

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer's patients' brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3β, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer's disease and other tauopathies.

Keywords: Alternative splicing; Alzheimer’s disease; Intron retention; Tau; Tauopathies; Truncation.

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Conflict of interest statement

The authors declare that they have no competing interests to disclose.

Figures

Fig. 1
Fig. 1
TIR-MAPT RNA expression. a Schematic representation of the MAPT gene and MAPT and TIR-MAPT mRNAs generated from it. A, C, D and E represent the hybridization sites of the primers designed for semi-quantitative PCR employed for the PCRs in b (Supplementary Table 2, online resource). The fragment of intron 12 that would remain upon retention is represented with colored stripes. b Representative images of agarose gels showing of semi-quantitative PCR results using total or cytoplasmic-enriched RNA of SH-SY5Y cells. Results showed the existence of RNA species from exon 11 to intron 12 where intron 11 was spliced out (PCR 5 and 6). Controls of the addition (RT+) or no addition of retrotranscriptase (RT−) were included. PCR 9 shows MAPT in which both intron 11 and 12 are spliced out. Detailed information of all semi-quantitative PCR combinations and amplicon sizes is provided in Supplementary Table 5, online resource. c Schematic representation of the MAPT gene including the hybridization sites of the oligos used for quantitative PCR. d TIR-MAPT RNA levels by qPCR in cytoplasmic-enriched fraction or whole extracts (total) of SH-SY5Y cells. e Comparison of TIR-MAPT level in SH-SY5Y cells and hippocampus and frontal lateral cortex of human brain. Graphs show means and SE of technical triplicates. f Percentage of brain samples having expression of MAPT or TIR-MAPT genes. Data are shown for three different regions within brains (see Supplementary Table 3, online resource). g Scatter dot blot of expression values of MAPT and TIR-MAPT genes in brain regions having TPM (transcripts per kilobase million) > 0. Number of samples f, g: cortex = 122; frontal cortex = 113; hippocampus = 98. Graphs represent mean and SEM. p values were calculated using a T test, ****p value ≤ 0.0001
Fig. 2
Fig. 2
W-Tau antibody validation and protein expression in human brain. a Immunoprecipitation assay of HEK239T cells overexpressing W-T30 using W-Tau (Abyntek) or total Tau (NOVUSBIO, NB100-82247) antibodies and Western blot detection of immunoprecipitated Tau using 7.51 antibody. b Immunoprecipitation of the same cells overexpressing W-T30 and control untransfected cells using W-Tau antibody and detection of immunoprecipitated Tau using total Tau (Tau 7.51) antibody. c HEK293T cells were transfected with eukaryotic expression vectors empty or encoding different MAPT isoforms (T42, T30, W-T42, W-T30, ET-42 and ET-T30). Representative Western Blot for Tau 7.51 and W-Tau antibodies proving W-Tau specificity. d Western blot detection of W-Tau in frontal lateral cortex and hippocampus of one human subject that show bands at 50 kDa (W-T42), 38 KDa (W-T30), and 31 KDa (W-Tau truncated). e Representative Western blot of frontal lateral cortex and hippocampus samples of the same three human subjects (a, b, c) showing the presence of W-Tau and total Tau (Tau5)
Fig. 3
Fig. 3
W-Tau phosphorylation pattern. a Schematic representation of different Tau protein isoforms: full-length isoform with four repeats (R) and two insertions (N) (T42), full-length isoform with three repeats and no insertions (T30), the truncated by intron retention isoforms W-Tau with four repeats, two insertions and an extra peptide (W-T42) or with three repeats, no insertions and the extra peptide (W-T30); and the correspondent asparagine-endopeptidase-truncated isoforms (ET-T42 and ET-T30). Representation of the antibodies recognizing the Tau molecule at their corresponding epitopes: Antibodies recognize all isoforms of Tau (Tau12 on amino acids 6–18; Tau 5 on amino acids 210–241 and Tau 7.51 on amino acids 315–376) or specific against dephosphorylated Tau in residues Ser195, 198, 199 and 202 (Tau1), and phospho-Tau in residues Ser202/Thr205 (AT8), Thr231 (AT180), Ser404 (Tau404), Ser396 (Tau396) and Ser396/Ser404 (PHF1). W-Tau antibody recognizes the unique peptide present on W-Tau isoforms. b Samples from HEK293T cells transfected with the different isoforms were probed with different antibodies for phosphorylated and non-phosphorylated Tau. c Quantification of the data of different Tau epitopes with respect to total Tau measured with Tau 7.51 antibody, showing mean and SEM (n = 4). One-way ANOVA for multiple comparisons followed by a Kruskal–Wallis test was performed to compare each isoform and T42 full-length level of phosphorylation
Fig. 4
Fig. 4
W-Tau aggregation capacity. a Representative Western blot of the presence of Tau in 1% sarkosyl-soluble and insoluble cell fractions of HEK293T cell overexpressing different Tau isoforms (T42, T30, W-T42, W-T30, ET-42 and ET-T30) detected with Tau 5 antibody. b, c Quantification of the signal obtained from Tau 5 showing sarkosyl-soluble vs insoluble fractions (b) or soluble/insoluble ratio (c). Graphs show means and SEM (N = 3). One-way ANOVA for multiple comparisons followed by a Kruskal–Wallis test was performed to compare each isoform with the correspondent full-length isoform, *p ≤ 0.05. d Representative electron microscopy images of Tau aggregates obtained upon in vitro incubation from partially purified T42, W-T42, and ET-42 extracts from bacteria in the presence of heparin to prompt aggregates formation. Scale bars show 500 nm wide. e Western blot showing soluble (supernatant) and aggregated (pellet) protein from the incubation in d, upon centrifugation to separate both fractions. Quantification shows the ratio between soluble and aggregated protein
Fig. 5
Fig. 5
Modulation of TIR-MAPT and W-Tau by GSK3. a. Detection of mature cytoplasmic RNA levels of TIR-MAPT, total MAPT and the ratio TIR-MAPT/total MAPT in SK-N-MC human neuroblastoma cell line in the absence or presence of 5 µg/ml amyloid-β1–42 and the GSK3 inhibitor AR-014418 (10 µM) for 24 h. Graphs show mean and SEM. One-way ANOVA and Dunnett’s multiple comparisons test were performed and statistical significance between untreated and cells treated with either Aβ1–42 and AR-014418 was given. ***p ≤ 0.001; ****p ≤ 0.0001. b Western blot analysis of the protein levels of W-Tau (W-Tau antibody) in SK-N-MC cells treated with SB216763 (25 µM), Aβ1–42 (1.1 µM) or AR-14418 (10 µM). Right panel shows the quantification of the signal of complete lanes obtained with W-Tau antibody. Graphs show mean and SEM. One-way ANOVA for multiple comparisons followed by a Kruskal–Wallis test was performed to compare treated and untreated cells. *p ≤ 0.05. c Schematic representation of the modulation of TIR-MAPT by splicing factor SC35, regulated by GSK3-mediated phosphorylation. Blue arrows represent activation; red, truncated arrows represent inhibition
Fig. 6
Fig. 6
TIR-MAPT RNA expression in the brains of AD patients. a Measurement of TIR-MAPT, b total MAPT and c TIR-MAPT/total MAPT ratio of RNA levels by RT-qPCR of non-demented (n = 8) and AD hippocampal samples (n = 16) classified according to their Braak stage (Braak II n = 3, Braak III n = 2, Braak V n = 7 and Braak VI n = 4), and healthy vs. AD patients. Graphs show means and SE. One-way ANOVA and Dunnett’s multiple comparisons test were performed and statistical significance of each group with respect to non-demented control individuals was given. For comparisons between non-demented individuals and AD patients as groups, p values were calculated using a T test (*p ≤ 0.05; ***p ≤ 0.001, †p = 0,078)
Fig. 7
Fig. 7
Tau protein determination in AD patients’ brain. a Immunodetection of the presence of W-Tau isoforms using W-Tau antibody in two different frontal lateral cortex brain extracts derived from AD patients’ brains (Braak V and VI, respectively). The immunoprecipitate performed with W-Tau antibody was characterized by Westernblotting. Left panel shows the blot developed using W-Tau antibody; right panel using Tau 7.51 antibody. b Western blot analysis of the levels of W-Tau and total Tau in frontal lateral cortical samples of non-demented (n = 9) and AD patients classified according to their Braak stage (Braak I = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8). c Quantification of W-Tau and total Tau protein levels as well as W-Tau/total Tau ratio of each group. One-way ANOVA and Dunnett’s multiple comparisons test were performed and statistical significance of each group with respect to non-demented control individuals was given (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). A.U. arbitrary units
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