Wren et al. Molecular Neurodegeneration (2015) 10:46 DOI 10.1186/s13024-015-0042-7 RESEARCH ARTICLE Open Access Frontotemporal dementia-associated N279K tau mutant disrupts subcellular vesicle trafficking and induces cellular stress in iPSC-derived neural stem cells Melissa C. Wren1, Jing Zhao1, Chia-Chen Liu1, Melissa E. Murray1, Yuka Atagi1, Mary D. Davis1, Yuan Fu1, Hirotaka J. Okano2, Kotaro Ogaki1, Audrey J. Strongosky3, Pawel Tacik3, Rosa Rademakers1, Owen A. Ross1, Dennis W. Dickson1, Zbigniew K. Wszolek3, Takahisa Kanekiyo1* and Guojun Bu1* Abstract Background: Pallido-ponto-nigral degeneration (PPND), a major subtype of frontotemporal dementia with parkinsonism related to chromosome 17 (FTDP-17), is a progressive and terminal neurodegenerative disease caused by c.837 T > G mutation in the MAPT gene encoding microtubule-associated protein tau (rs63750756; N279K). This MAPT mutation induces alternative splicing of exon 10, resulting in a modification of microtubule-binding region of tau. Although mutations in the MAPT gene have been linked to multiple tauopathies including Alzheimer’s disease, frontotemporal dementia and progressive supranuclear palsy, knowledge regarding how tau N279K mutation causes PPND/FTDP-17 is limited. Results: We investigated the underlying disease mechanism associated with the N279K tau mutation using PPND/ FTDP-17 patient-derived induced pluripotent stem cells (iPSCs) and autopsy brains. In iPSC-derived neural stem cells (NSCs), the N279K tau mutation induced an increased ratio of 4-repeat to 3-repeat tau and accumulation of stress granules indicating elevated cellular stress. More significant, NSCs derived from patients with the N279K tau mutation displayed impaired endocytic trafficking as evidenced by accumulation of endosomes and exosomes, and a reduction of lysosomes. Since there were no significant differences in cellular stress and distribution of subcellular organelles between control and N279K skin fibroblasts, N279K-related vesicle trafficking defects are likely specific to the neuronal lineage. Consistently, the levels of intracellular/luminal vesicle and exosome marker flotillin-1 were significantly increased in frontal and temporal cortices of PPND/FTDP-17 patients with the N279K tau mutation, events that were not seen in the occipital cortex which is the most spared cortical region in the patients. Conclusion: Together, our results demonstrate that alterations of intracellular vesicle trafficking in NSCs/neurons likely contribute to neurodegeneration as an important disease mechanism underlying the N279K tau mutation in PPND/FTDP-17. Keywords: FTDP-17, iPSCs, N279K, Neural stem cells, PPND, Tau * Correspondence: [email protected]; [email protected] Melissa C. Wren and Jing Zhao are co-first authors 1Department of Neuroscience, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA Full list of author information is available at the end of the article © 2015 Wren et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wren et al. Molecular Neurodegeneration (2015) 10:46 Page 2 of 13 Background induced pluripotent stem cell (iPSC) technology provides Frontotemporal dementia with parkinsonism related to a novel and unparalleled approach to aid the study of mo- chromosome 17 (FTDP-17) prevails as one of the most lecular and cellular dynamics of disease pathogenesis by common form of early-onset dementia [1]. FTDP-17 pre- utilizing patient-specific cells with pathologically linked sents as a fulminant progressive neurodegenerative de- genetic mutations on the inherent genetic background mentia, with no curative treatment or effective palliative [15], which has facilitated its exponential use as a novel relief. Clinically, FTDP-17 displays a triad of symptoms, tool to understand the pathogenesis of multiple neurode- with parkinsonism, behavioral changes and personality generative diseases [16]. Herein, we investigated the dysfunctions, and later cognitive impairments [1, 2]. Two pathogenic mechanism underlying FTDP-17T with mu- genetic causes have been identified that lead to distinct tant N279K tau by using cells derived from two patients forms of FTDP-17; mutations in progranulin (GRN)cause from the PPND family who are both clinically affected. FTDP-17U (ubiquitin) with TDP-43 pathology and muta- Neural stem cells (NSCs) derived from the patient-specific tions in the microtubule-associated protein tau (MAPT) iPSCs revealed abnormal amounts of cellular vesicle com- gene cause FTDP-17T (tau) [3]. Approximately one-half ponents in the presence of aberrantly spliced tau, accom- of all FTDP-17 cases are caused by autosomal dominant panied by increases in several components of cellular mutations in MAPT. The pallido-ponto-nigral degener- stress. Correspondingly, altered cellular markers reflecting ation (PPND) family is the largest and most comprehen- abnormal vesicle trafficking were observed in the autopsy sively studied kindred of tau N279K mutant carriers and brains of patients carrying the N279K mutation, support- of all FTDP-17 families, containing to date, 59 affected in- ing the results from the iPSC-based approach. Our find- dividuals [4]. ings demonstrate that patient-specific iPSCs can be used The N279K substitution is encoded within exon 10 of to model neurodegenerative diseases and uncover cellular the MAPT gene locus and is one of the most frequent mu- dysfunctions associated with a disease-causing mutation. tations in FTDP-17T patients [2]; together with muta- tions P301L and intron 10 + 16. Together these three Results mutations account for up to 60 % of FTDP-17T cases [5]. Generation and characterization of iPSCs Multiple MAPT transcripts exist due to the alternative Two PPND family members known as N279K tau mutant splicing of the MAPT gene, and transcripts of MAPT can carriers were recruited into this study. Both were de- be classified based on the inclusion/exclusion of exon 10 scribed as clinically affected at the time of dermal extrac- [6]. Most MAPT mutations including the N279K muta- tion. Skin biopsy specimens were immediately processed tion increase the transcript containing exon 10 resulting to generate primary fibroblasts. Commercially available in the overproduction of tau protein isoforms containing human dermal fibroblasts, documented to be clinically four tandem microtubule-binding domain repeats (4R- normal, were used as a control. Genomic sequencing of tau); whereas levels of the other major isoform in human exon 10 of the MAPT locus confirmed the retention of brain containing only three repeats (3R-tau) is believed to the c.837 T > G mutation (rs63750756; N279K) in fibro- be unaffected [7]. Although it is not fully understood how blasts from the PPND/FTDP-17T patients (Fig. 1a). The the increase in 4R-tau contributes to disease pathogenesis, fibroblasts were transfected with three episomal vectors MAPT mutations have been shown to cause multiple containing five transcription factors (OCT 3/4, SOX2, L- tauopathies [8–10]. It is believed that tauopathies are MYC, KLF4, LIN28) and p53 shRNA for the conversion caused by aberrant hyperphosphorylation of tau, leading to iPSCs, as previously described [17]. After 3-4 weeks of to the assembly of variable neurotoxic tau aggregates and the transfection, approximately 30 iPSC colonies per sub- deposition of insoluble tau fibers in both neurons and glia ject were selected and expanded. We first confirmed that [11]. Progressive tau deposition in FTDP-17T patients is the iPSCs expressed several pluripotent stem cell specific associated with severe neocortical atrophy of the frontal markers, including Nanog, TRA-1-81 and TRA-1-60 by and temporal lobes, in association with the degeneration immunostaining (Fig. 1b). In addition, all iPSC lines could of medial temporal structures [12]. Destruction of the spontaneously differentiate into cell types of all three germ basal ganglia and depigmentation of the substantia nigra layers in vitro when they were stained for an endoderm may also be present to some degree in MAPT mutant car- marker α-fetoprotein (AFP), a mesoderm marker α- riers, but are documented to be a consistent pathological smooth muscle actin (SMA) and an ectoderm markers hallmark in N279K kindreds [13, 14]. Tuj1 (β III tubulin) and Nestin (Fig. 1c). The exact mechanism owing to the cell death of specific We examined mRNA expression of pluripotent markers neuronal populations in FTDP-17T remains to be identi- Oct3/4, Sox 2, Klf4, c-Myc, Nanog, REX, GDF3, FGF-4, fied; however, the characteristic dominant penetrance of and ESG1 by RT-PCR in the iPSC lines. While skin fibro- the N279K tau mutant suggests a gain-of-toxic-function blasts expressed very low levels of these mRNAs except that results in specific subsets of degenerating cells. The Klf4 and c-Myc, all markers were up-regulated and/or Wren et al. Molecular Neurodegeneration (2015) 10:46 Page 3 of 13 Fig. 1 Generation and characterization of patient-specific iPSCs with N279K tau mutation. a Genomic DNA sequencing of the heterozygous MAPT missense mutation c.837 T > G (p.N279K) in fibroblasts from the PPND/FTDP-17 patients. b Immunostaining of pluripotency markers (Nanog, TRA- 1-81 and TRA-1-60) in a control iPSC and two N279K iPSC lines. Scale bar, 50 μm. c In vitro differentiation of a control iPSC and two N279K iPSC lines into cells of all three germ layers. Cells were immunostained for AFP (endoderm), SMA (mesoderm), Tuj1/Nestin (ectoderm), and DAPI (nucleus). Scale bar, 50 μm. d-e Total mRNA levels of the reprogramming factors in a human ES cell line, a commercial iPSC line, a control iPSC line and two N279K iPSC lines relative to the values in a control fibroblast were assessed by qRT-PCR.