HD model mice
Mutant Htt-KI mice are a generous gift from Prof. Marcy MacDonald (Massachusetts General Hospital, Harvard Medical School) [17] 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) [17]. 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.
Human brains
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.
Western blotting
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) [8].
Immunohistochemistry
Immunohistochemistry was performed as previously described with minor modifications [8]. 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 microscopy
Electron microscopic observation was performed basically following the method described previously [8]. 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 analysis
Statistical analyses were performed with Student’s t-test.