- Open Access
A novel form of necrosis, TRIAD, occurs in human Huntington’s disease
- Emiko Yamanishi†1,
- Kazuko Hasegawa†2,
- Kyota Fujita†1,
- Shizuko Ichinose†3,
- Saburo Yagishita2,
- Miho Murata4,
- Kazuhiko Tagawa1,
- Takumi Akashi5,
- Yoshinobu Eishi5 and
- Hitoshi Okazawa1, 6Email author
© The Author(s). 2017
Received: 12 January 2017
Accepted: 22 February 2017
Published: 8 March 2017
We previously reported transcriptional repression-induced atypical cell death of neuron (TRIAD), a new type of necrosis that is mainly regulated by Hippo pathway signaling and distinct from necroptosis regulated by RIP1/3 pathway. Here, we examined the ultrastructural and biochemical features of neuronal cell death in the brains of human HD patients in parallel with the similar analyses using mutant Htt-knock-in (Htt-KI) mice. LATS1 kinase, the critical regulator and marker of TRIAD, is actually activated in cortical neurons of postmortem human HD and of Htt-KI mouse brains, while apoptosis promoter kinase Plk1 was inactivated in human HD brains. Expression levels of YAP/YAPdeltaC were decreased in cortical neurons of human HD brains. Ultra-structural analyses revealed extreme enlargement of endoplasmic reticulum (ER), which characterizes TRIAD, in cortical neurons of human HD and those of Htt-KI mice. These biochemical and morphological results support that TRIAD occurs in human and mouse neurons under the HD pathology.
The nature of cell death in neurodegenerative diseases remains obscure. A number of clinical trials against neurodegenerative diseases using anti-apoptosis or anti-necroptosis chemicals were so far unsuccessful. The therapeutic effect of rapamycin on mouse model of amyotrophic lateral sclerosis (ALS) is controversial [16, 18]. Minomycin, which has anti-apoptosis and anti-inflammatory effects and whose therapeutic effect on ALS mouse model was reported , caused notable deterioration instead of amelioration in human clinical trial of ALS patients . Though necrosis, apoptosis or autophagic cell death has been implicated in neurodegeneration, actual phenotype of neuronal death in vivo, actual molecular mechanisms to explain the cell death in vivo, and relative contribution of different prototypes of cell death to neurodegeneration in vivo are still largely unknown.
We proposed previously that the atypical necrosis induced by transcriptional repression (TRIAD) defined by extremely enlarged and unstable ER with intact mitochondria and nuclei, could be a prototype of cell death in the HD pathology . The necrotic cell death (or Type III cell death in the category by Schweichel and Merker)  was induced by the RNA-polymerase inhibitor, α-amanitin, and suppressed by new isoforms of YAP [3, 8] that interacts with transcription factor TEAD or p73 as a critical mediator of Hippo signaling pathway.
The molecules involved in TRIAD were comprehensively analyzed using Drosophila genetic screen, and the identified genes were integrated to the network executing TRIAD . The analysis newly identified that splicing disturbance caused by decreased expression of multiple hnRNPs additively enhanced TRIAD . Moreover, we revealed that mutant Htt-Exon1 expression at a physiological level induces TRIAD in primary cortical neurons and that targeting of TEAD/YAP-dependent TRIAD recovers HD mouse models .
In the previous works, we also revealed that two kinases, LATS and Plk1, switch apoptosis and necrosis (TEAD/YAP-dependent necrosis TRIAD) in neurons through the balance of cytoplasmic and nuclear YAP and the switch of transcription factors interacting with YAP . Activation of Plk1 increases the ratio of apoptosis in relevance to necrosis, while activation of LATS increases the ratio of necrosis but suppresses apoptosis in neurons in which proliferative cell-specific Plk1 is usually inactive . In this scheme, single activation of LATS without Plk1 more strongly promotes necrotic cell death TRIAD .
However, the questions remain on how activities of these kinases are actually changed in vivo in human HD and whether TRIAD-specific morphological changes actually occur in vivo in human HD brains. Although we previously reported aberrant expression of YAPdeltaC in motor neurons of ALS model mice  while we could not directly indicate the existence of TRIAD by ultra-structural analysis of the cell death or by new markers of TRIAD such as LATS1 and Plk1 that had been reported later.
In this study we employed these new tools and addressed whether TRIAD occurs in the brains of human HD patients and mutant Htt-KI mice. The results obviously supported that TRIAD actually occurs in human HD brains.
Materials and methods
HD model mice
Mutant Htt-KI mice are a generous gift from Prof. Marcy MacDonald (Massachusetts General Hospital, Harvard Medical School)  in which human mutant Htt carrying 111CAG repeats is integrated. Their original genetic background was 129SvEv/CD1 (mixed background by crossing 129SvEv male and CD1 female) . However, their genetic background had been changed to C57BL/6 when we received mutant Htt-KI mice. Furthermore, we crossed the male mutant Htt-KI mice with female C57BL/6 mice for more than 5 generations before this study. Accordingly, C57BL/6 mice were used as negative controls in this study.
We obtained informed consent and ethics committee approval (Sagamihara National Hospital, NCNP and TMDU) to examine autopsy specimens from three HD patients and three control patients without neurological disorders (lung cancer, leukemia, and cholangiocarcinoma). The diagnosis of HD was confirmed by genetic analysis of CAG repeat of Htt gene. Frontal cortex from five HD patients and two PSP patients were used for ultra-structural analyses. Frontal and parietal cortex tissues of three HD patients and five non-neurological disease patients were used for immunohistochemistry.
Brain tissues were dissected from Htt-KI mice or littermate control mice and washed three times with ice-cold PBS and dissolved in lysis buffer containing 62.5 mM Tris–HCl (pH 8.0), 2% (w/v) SDS, 2.5% (v/v) 2-mercaptoethanol and 5% (v/v) glycerol. The protein concentration was quantified using the BCA method (Micro BCA Protein Assay Reagent Kit, Thermo Fisher Scientific, MA, USA). Primary and secondary antibodies were diluted for immunoblotting as follows: rabbit anti-LATS1 (1:2000, Cell Signaling Technology, MA, USA, #3477), rabbit anti-phospho-LATS1 (Ser909, 1:5000, Cell Signaling Technology, MA, USA, #9157), mouse anti-PLK1 (1:2000, Invitrogen, MA, USA, #37-7000), anti-phospho-PLK1 (Thr210, 1:30000, Abcam, Cambridge, UK, #ab155095); HRP-conjugated anti-mouse IgG (NA931VA) and anti-rabbit IgG (NA934VS) (both of them, 1:3000, GE Healthcare, Buckinghamshire, UK). Antibodies were diluted in Can Get Signal (TOYOBO, Osaka, Japan). ECL prime (GE Healthcare, Buckinghamshire, UK) was used to detect the bands using LAS4000 (GE Healthcare, Buckinghamshire, UK) .
Immunohistochemistry was performed as previously described with minor modifications . After deparaffinization, rehydration, and antigen retrieval (microwaved in 10 mM citrate buffer, pH 6.0, at 100 °C, 5 min, three times), the sections were incubated sequentially with 0.5% TritonX-100 in PBS for 30 min at room temperature (RT) to membrane permeation, with 10% FBS for 60 min at RT, with primary antibodies: rabbit anti-phospho-LATS1 (Ser909, 1:100, Cell Signaling Technology, MA, USA, #9157), rabbit anti-phospho-PLK1 (Thr210, 1:100, Abcam, Cambridge, UK, #ab155095), mouse anti-MAP2 (1:200, Santa Cruz, TX, USA, #sc-32791) and mouse anti-NeuN (1:100, Abcam, Cambridge, UK, ab104224) one or two overnight, and finally with secondary antibodies: Alexa Flour 488-labeled anti-mouse IgG (1:1000, Invitrogen, MA, USA) and Cy3-labeled anti-rabbit IgG (1:500, Jackson ImmunoResearch, PA, USA) for 1 h at RT. Images were acquired by confocal microscopy: Olympus FV1200 (Olympus, Tokyo, Japan) and LSM710 (Carl Zeiss, Oberkochen, Germany).
Signal acquisition from immunohistochemistry
Immunohistochemistry images obtained by Olympus FV1200 were next analyzed by Image-J (NIH, MD USA: https://imagej.nih.gov/ij/). Signal intensities (AU/pixel) of YAP, YAPdeltaC, phospho-LATS1 and phospho-PLK1 in each neuron (NeuN-positive or MAP2-positive cell) were quantified by free-hand-surrounding the shape of neuron with Image-J. From human immunohistochemistry images 4 visual fields were randomly selected, and 100 neurons in total were analyzed. Background signals were collected from 8 areas that did not include cells, and the signal intensity of each neuron was subtracted with the mean value of the background signals. The mean value of the signal intensities of 100 neurons after subtraction of the background signals was used as the representative value for a patient or a control, and statistical analysis was performed between 3 patients and 3 controls.
Electron microscopic observation was performed basically following the method described previously . After deparaffinization and rehydration, tissues were washed with PBS three time, fixed in 2.5% glutaraldehyde/0.1 M phosphate buffer (PB) (pH7.4), and treated with 1% OsO4/0.1 M PB for 2 h. Fixed tissues were dehydrated through a graded ethanol series and embedded in epoxyresin. Ultrathin sections were stained with uranyl acetate and lead citrate. Data acquisition was performed with a transmission electron microscope (H-9000, H7600 or H-7100, Hitachi, Tokyo, Japan).
Regarding human brain samples, frontal cortex were fixed in 2.5% glutaraldehyde/0.1 M cacodylate buffer for 2 h and treated with 1% OsO4/0.1 M cacodylate buffer between 90 min and 2 h, within 5 h after death.
Statistical analyses were performed with Student’s t-test.
Activation of LATS1 and Plk1 in Htt-KI mice
Activation of LATS1 in human HD brains
On the other hand, activation of Plk1, which induces apoptosis rather than TRIAD , was not obvious. At low magnification, the basal level of phosphorylated Plk1 was already high in the control, and it was almost similar or slight lower in HD (Fig. 3b, upper panel). At high magnification, phospho-Plk1 stains were found in the nucleus of a part of neurons (Fig. 3b, lower panel), while the intensity in the cytoplasm and the number of stain positive neurons were decreased (Fig. 3b, lower graph). The decrease of phospho-Plk1 in human cortical neurons might be consistent with the late-stage reduction of phospho-Plk1 in striatal neurons of mutant Htt-KI mice. The morphology of Plk1-positive neurons in the control brains was normal at least at the level of fluorescent microscopy.
Decrease of YAP/YAPdeltaC in human HD brains
TRIAD occurs in mouse HD model brains
Such morphological changes were not dominant in ultra-structural analyses of cortical neurons in Htt-KI mice at 39 weeks before the onset (the onset is around 50 weeks) (Fig. 5e). However, we found that some neurons showed mild dilatation of ER, loss of our nuclear membrane connected to ER and/or nuclear membrane invagination (Fig. 5f–h), which might suggest initiation of morphological change of TRIAD. No definite nuclear chromatin condensation or fragmentation was observed. In addition, we found that a few neurons possessed intranuclear fibrils that might correspond to intranuclear Htt inclusion bodies (Fig. 5i). In the background mice (C57BL/6) at 68 weeks, we found no such TRIAD-related morphological changes in the cortex and the striatum (Fig. 5j).
TRIAD occurs in human HD brains
We also analyzed two cases of progressive supranuclear palsy (PSP) as disease controls. The fixation of brain tissues for ultrastructural analysis was completed within 5 h after death of the HD and PSP patients. On the other hand we could not find brain samples of non-neurological disease patients in bio-resource of our hospitals or in the other brain banks in Japan that could be used as control. Unexpectedly, the expansion of ER was also detected in PSP although the extent of ER expansion was less prominent than in HD patients (Additional file 1: Figure S1, Additional file 2: Figure S2 and Additional file 4: Table S1). Lipofuscin granules were frequently observed in neurons of HD and PSP patients.
In our previous studies, we proposed TRIAD as an atypical cell death that can be categorized to Type 3 necrotic cell death and might be relevant to the cell death in neurodegenerative diseases . Simultaneously, we identified YAP isoforms (full-length YAP2 and YAPdeltaC) as molecules regulating TRIAD . We next analyzed the feature of cell death in the cell model of HD, and found that overexpression of Htt-Exon1 induced Type 3 necrotic cell death that is morphologically and biochemically identical to TRIAD . Moreover, we revealed that ER instability was enhanced in living neurons of two HD model mice (mutant Htt-Exon1-transgenic R6/2 mice  and mutant Htt-knock-in mice ) and showed that targeting the TEAD/YAP-mediated transcription or the Hippo pathway ameliorated the cell death and symptoms of HD model mice .
However, the evidences were insufficient to prove that the new type of necrosis actually occurs in human HD pathology. To address the question, we directly investigated cerebral neurons in human HD. Consequently we confirmed activation of the TRIAD-linked kinase LATS1 , inactivation of apoptosis promoting kinase Plk1 , and ultra-structural changes of ER actually in human HD brains that was morphologically identical to TRIAD.
In EM analysis of human HD brains, we found mitochondrial enlargement (Additional file 4: Table S1) that had been unusual at the early stage of TRIAD in primary neurons . This finding might suggest the possibility that TRIAD partially shares apoptotic signaling in vivo. Our previous screening by fly model that revealed partial share of signaling molecules in TRIAD and apoptosis, might support this idea . Given that Plk1 was reported essential for recovering mitochondrial dysfunction , the decrease of Plk1 at the late stage of human and mouse HD pathologies might affect mitochondrial integrity and induce morphological changes of mitochondria in human postmortem HD brains. However, detailed mechanisms underlying the mitochondrial changes in TRIAD need further investigation. Especially, it would be of significance to analyze chronologically YAP phosphorylation at Thr77 and Ser127 that shifts the balance between apoptosis and TRIAD necrosis , in parallel with YAP phosphorylation at Tyr357 by c-Abl, a DNA damage signal mediator, that switches on/off apoptosis . These analyses might elucidate factors that modify the TRIAD prototype in vivo and in human, and should be performed in the future.
Interestingly, necrotic cell death such as “ballooned neuron” or “neuronal achromasia” has been described in tauopathy like corticobasal degeneration [1, 13], Pick’s disease (a form of frontotemporal dementia)  and progressive supranuclear palsy (PSP) . As achromasia is based on ER staining, it is highly possible that these forms of cell death are similar to TRIAD from the aspect of the extreme ER expansion. Moreover, achromasia is found in Alzheimer’s disease, motor neuron disease and Creutzfeldt-Jacob disease [6, 14].
Assuming that this type of cell death in multiple neurodegenerative diseases and TRIAD are identical, we might be able to unite cell deaths in various neurodegenerative diseases to a single prototype, and the model might enable us to generally discuss cell death of neurodegenerative diseases. The hypothesis should be further tested in the future in other neurodegenerative diseases than ALS and HD that we have analyzed.
Finally, considering with such evidences of TRIAD in HD (this study) and ALS , application of anti-Hippo pathway drugs such as S1P agonists should be considered positively to clinical trials against these diseases.
In this study, we examined whether TRIAD, a new type of necrosis dependent on YAP and Hippo pathway, occurs in human HD brains. Our results showed activation of LATS1, suppression of Plk1, and decrease of YAP/YAPdeltaC. EM analysis also revealed typical morphological features of TRIAD. These data collectively supported that TRIAD occurs in human HD brains in vivo. In addition, biochemical and EM analyses revealed the chronological shift from early-phase to late-phase TRIAD changes, supporting the existence of TRIAD in the HD pathology in vivo.
This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (16H02655), a Grant-in-Aid for Scientific Research (A) and Brain Mapping by Integrated Neurotechnologies for Disease Studies from Japan agency for Medical research and Development (AMED) to Hitoshi Okazawa. We thank Drs. Hidenori Homma, Toshikazu Sasabe, Tayoko Tajima, Noriko Katsuta (TMDU) and Ms. Miyako Ishiyama (Sagamihara National Hospital) for technical support and data acquisition. We thank Prof. Marcy MacDonald (Massachusetts General Hospital, Harvard Medical School) for mutant Htt-KI mice. Hitoshi Okazawa deeply thanks continuous encouragement provided by Prof. Ichiro Kanazawa (The University of Tokyo, National Center for Neurology and Psychiatry) who passed away in 2016, for our Huntington’s disease research studies.
EY, KH, KF, and SI carried out experiments, analyzed the data, and wrote the paper. SY, MM, TA, YE, and TT carried out experiments and analyzed the data. HO designed the whole research and wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
The experiments with human samples were approved by the Ethics Committees of the Tokyo Medical and Dental University (2014-5-4). All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (0170032A, 2016-007C).
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- Gibb WR, Luthert PJ, Marsden CD (1989) Corticobasal degeneration. Brain 112(Pt 5):1171–1192View ArticlePubMedGoogle Scholar
- Gordon PH, Moore DH, Miller RG, Florence JM, Verheijde JL, Doorish C, Hilton JF, Spitalny GM, MacArthur RB, Mitsumoto H et al (2007) Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol 6:1045–1053View ArticlePubMedGoogle Scholar
- Hoshino M, Qi ML, Yoshimura N, Miyashita T, Tagawa K, Wada Y, Enokido Y, Marubuchi S, Harjes P, Arai N et al (2006) Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73. J Cell Biol 172:589–604View ArticlePubMedPubMed CentralGoogle Scholar
- Keshet R, Adler J, Ricardo Lax I, Shanzer M, Porat Z, Reuven N, Shaul Y (2015) c-Abl antagonizes the YAP oncogenic function. Cell Death Differ 22:935–945View ArticlePubMedGoogle Scholar
- Lippa CF, Smith TW, DeGirolami U (1990) Lobar atrophy with pontine neuronal chromatolysis (“ballooned” neurons). Hum Pathol 21:1076–1079View ArticlePubMedGoogle Scholar
- Lowe J, Errington DR, Lennox G, Pike I, Spendlove I, Landon M, Mayer RJ (1992) Ballooned neurons in several neurodegenerative diseases and stroke contain alpha B crystallin. Neuropathol Appl Neurobiol 18:341–350View ArticlePubMedGoogle Scholar
- Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, Lawton M, Trottier Y, Lehrach H, Davies SW et al (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87:493–506View ArticlePubMedGoogle Scholar
- Mao Y, Chen X, Xu M, Fujita K, Motoki K, Sasabe T, Homma H, Murata M, Tagawa K, Tamura T et al. (2016) Targeting TEAD/YAP-transcription-dependent necrosis, TRIAD, ameliorates Huntington’s disease pathology. Hum Mol Genet. doi:10.1093/hmg/ddw303
- Mao Y, Tamura T, Yuki Y, Abe D, Tamada Y, Imoto S, Tanaka H, Homma H, Tagawa K, Miyano S et al (2016) The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing. Cell Death Dis 7:e2207View ArticlePubMedPubMed CentralGoogle Scholar
- Matsumoto T, Wang PY, Ma W, Sung HJ, Matoba S, Hwang PM (2009) Polo-like kinases mediate cell survival in mitochondrial dysfunction. Proc Natl Acad Sci U S A 106:14542–14546View ArticlePubMedPubMed CentralGoogle Scholar
- Mori H, Oda M (1997) Ballooned neurons in corticobasal degeneration and progressive supranuclear palsy. Neuropathology 17:248–252View ArticleGoogle Scholar
- Morimoto N, Nagai M, Miyazaki K, Kurata T, Takehisa Y, Ikeda Y, Kamiya T, Okazawa H, Abe K (2009) Progressive decrease in the level of YAPdeltaCs, prosurvival isoforms of YAP, in the spinal cord of transgenic mouse carrying a mutant SOD1 gene. J Neurosci Res 87:928–936View ArticlePubMedGoogle Scholar
- Rebeiz JJ, Kolodny EH, Richardson EP Jr (1968) Corticodentatonigral degeneration with neuronal achromasia. Arch Neurol 18:20–33View ArticlePubMedGoogle Scholar
- Sakurai A, Okamoto K, Fujita Y, Nakazato Y, Wakabayashi K, Takahashi H, Gonatas NK (2000) Fragmentation of the Golgi apparatus of the ballooned neurons in patients with corticobasal degeneration and Creutzfeldt-Jakob disease. Acta Neuropathol 100:270–274View ArticlePubMedGoogle Scholar
- Schweichel JU, Merker HJ (1973) The morphology of various types of cell death in prenatal tissues. Teratology 7:253–266View ArticlePubMedGoogle Scholar
- Staats KA, Hernandez S, Schonefeldt S, Bento-Abreu A, Dooley J, Van Damme P, Liston A, Robberecht W, Van Den Bosch L (2013) Rapamycin increases survival in ALS mice lacking mature lymphocytes. Mol Neurodegener 8:31View ArticlePubMedPubMed CentralGoogle Scholar
- Wheeler VC, Auerbach W, White JK, Srinidhi J, Auerbach A, Ryan A, Duyao MP, Vrbanac V, Weaver M, Gusella JF et al (1999) Length-dependent gametic CAG repeat instability in the Huntington’s disease knock-in mouse. Hum Mol Genet 8:115–122View ArticlePubMedGoogle Scholar
- Zhang X, Li L, Chen S, Yang D, Wang Y, Zhang X, Wang Z, Le W (2011) Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Autophagy 7:412–425View ArticlePubMedGoogle Scholar
- Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu DC et al (2002) Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 417:74–78View ArticlePubMedGoogle Scholar