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. 2016 Apr;9(4):451-62.
doi: 10.1242/dmm.022590. Epub 2016 Jan 14.

Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

Naoto Ito  1 , William T Hendriks  1 , Jyotsna Dhakal  1 , Christine A Vaine  1 , Christina Liu  1 , David Shin  1 , Kyle Shin  1 , Noriko Wakabayashi-Ito  1 , Marisela Dy  2 , Trisha Multhaupt-Buell  2 , Nutan Sharma  2 , Xandra O Breakefield  3 , D Cristopher Bragg  4
Affiliations

Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

Naoto Ito et al. Dis Model Mech. 2016 Apr.

Abstract

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells, XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34'), was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration.

Keywords: Induced pluripotent stem cells; TAF1; X-linked dystonia-parkinsonism.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H2O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts (n=5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC. Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t-test. *P<0.05; **P<0.01; N, not detected. (C) Schematic depicting approximate position of the DSCs and SVA relative to TAF1 (gray) and MTS exons (red). N-TAF1 incorporates the alternative exon, 34′ (blue). Below the graph are shown the relative positions of primer sets used for qRT-PCR. Numbers in parentheses indicate exons targeted by forward and reverse primers. Primers MTS-32′/34′, MTS-37/1 and MTS-37/3 amplify fragments in which the indicated TAF1 exons are reportedly spliced to MTS exons.
Fig. 2.
Fig. 2.
Derivation of XDP and control induced pluripotent stem cells. (A) Expression of pluripotency-related genes Dmnt3b, hTERT, Nanog, Oct4, Rex1 and Sox2 in each reprogrammed XDP and control iPSC clone compared with the parent fibroblast lines determined by qRT-PCR. Heatmap based on ΔCt values. Red, high expression; blue, low expression. (B) Pluripotency marker protein expression in two representative iPSC clones (A and I) derived from XDP fibroblast line 32517. Both clones expressed alkaline phosphatase (AP), Oct4, Nanog, SSEA-3, SSEA-4 and TRA-1-60. Immunostaining was visualized using secondary antibodies conjugated to Alexa Fluor 488 (green; Oct4, Nanog and SSEA-4) or Alexa Fluor 594 (red: SSEA-3 and Tra-1-60) with DAPI (blue) to visualize nuclei. Images shown represent overlays. Scale bars: 10 µm.
Fig. 3.
Fig. 3.
Pluripotency analyses. (A) Trilineage potential of each XDP and iPSC clone determined by Taqman Scorecard Assay analysis of RNA from embryoid bodies differentiated in culture from each cell line. Heatmaps depicting expression of 94 marker genes related to self-renewal, primitive mesendoderm, mature ectoderm, mesoderm and endoderm. Values represent mean fold changes (FC) for each gene relative to Scorecard reference pluripotent cell lines, calculated by PSC Scorecard analysis software. Red, high expression; blue, low expression. (B-G) Representative XDP (33363-C; upper panel) and control (33362-C; lower panel) teratomas produced in mice following cell implantation. Arrows denote ectodermal neuroepithelium (B) and hair follicle cells (E); mesodermal cartilage (C,F); endodermal mucous gland (D) and gut epithelium (G). Scale bars: 100 µm.
Fig. 4.
Fig. 4.
Generation and characterization of neural stem cells. (A) Brightfield micrograph depicting characteristic morphology of a representative iPSC colony on Geltrex prior to neural conversion. (B) Epifluorescent image of neural stem cells (NSCs) generated by conversion of iPSCs in PSC Neural Induction Medium, labeled with calcein-AM (Life Technologies). NSCs on Geltrex formed discrete clusters of cells with occasional fine processes. Images in A and B captured at final magnification of 20×. (C-E) NSCs were also evaluated by immunofluorescence for expression of (C) nestin; (D) Sox1; and (E) musashi. Staining for each target was detected by a secondary antibody coupled to Alexa Fluor 594 (red), along with counterstaining to visualize cytoplasm (Wheat germ agglutinin-Alexa Fluor 488; green) and nuclei (Topro-3-iodide; blue). Images represent overlays of all three channels captured by laser confocal microscopy at final magnification of 100×. Immunoreactivities for nestin and musashi seem predominantly cytoplasmic, whereas Sox1 labeling was observed primarily within the nucleus. Scale bars: 10 µm. (F) Heatmap of ΔCt values depicting comparative expression of 22 neural marker genes in XDP and control NSCs vs corresponding iPSCs from which they were differentiated. Red, high expression; blue, low expression.
Fig. 5.
Fig. 5.
TAF1 and MTS transcript expression levels in NSCs. Quantitative expression analysis of TAF1 transcript fragments in XDP (n=7) vs control (n=6) NSC clones based on comparative Ct method with targets normalized to the geometric mean of housekeeping genes HPRT1 and TFRC. (A) N-TAF1, amplified by primer set TA14-391, was detected in NSCs at levels approximately threefold lower in XDP vs control cells. Data represent mean fold changes±standard errors, analyzed by Student's t-test. *P<0.05; N, not detected. (B) Amino acids 1556-1874 of N-TAF1 highlighted to indicate unique alanine (A) and lysine (K) residues relative to: C-terminal segment of second bromodomain motif (gray); acidic glutamate (E) and aspartate (D) residues (pink); prolines (P; blue); and a threonine at position 1643 (T1643; green) that is a potential phosphorylation site.
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