Extracts from Artemisia annua preparation
Dried extracts from artemisia annua were obtained from Nanjing Puyi Biotechnology company. The extraction process was performed as follows: Artemisia annua water-soluble Ex1 extracts were obtained by soaking artemisia annua dried powder in distilled water (1:10) wt./vol for 2 hours and then heated and boiled for additional 2 hours (primary extraction). The extract was then filtered, soaked again in distilled water (1:8) wt./vol and left boiling for 1.5 hours (secondary extraction). After filtration, the extracting solutions were evaporated at 65°C under reduced pressure (-0.08 MPa), followed by lyophilization to obtain a relative density of 1.12. Artemisia annua Ex2 and Ex3 extracts were obtained, following the same methodology, by soaking artemisia annua dried powder in 55% and 75% ethanol, respectively.
Animals and treatment
3xTg transgenic mice (APPSWE, TauP301 L and PS1M146V) were obtained from the Jackson Laboratory and bred in the animal facility of the University of Macau. All animal experiments were approved by the University of Macau Animal Ethics Committee (protocol No. UMAEC-001-2020). The animals were housed in 24-26°C room, 12 hours/dark-light cycle and food and water were available ad libitum. Animals were randomly divided into four groups: Wild-type (WD, Ctrl), 3xTg (Ctrl), 3xTg+6.7 mg/ml artemisia annua and 3xTg+20 mg/ml artemisia annua groups (n=10 animals per group, female, ~30g, aged 9 months). Animals from 3xTg+6.7 mg/ml group and 3xTg+20 mg/ml group were treated with artemisia annua extracts dissolved in the drinking water. Animals from the WT and 3xTg groups received equal amounts of water. After 3 months of treatment the behavioral performance of all mice was examined by Morris water maze.
Morris water maze (MWM) test was used to analyze the effect of artemisia annua extracts on the memory and learning abilities of mice according to previously described methods [39-41]. Briefly, the animals were submitted to a place navigation test during 5 consecutive days, followed by a spatial probe trial on the sixth day. During the place navigation tests the platform was placed in the middle of one of the quadrants and 1 cm above the water surface. The path distance and latency of finding the platform were tested and used as indicators of the mice learning ability. On the day of spatial probe trial, the mice searched the platform for 60 s, which had been previously removed. The time spent in the target quadrant and the number of crossings was measured. All data acquisition and processing were done by image analyzing software (ANY-maze; Stoelting). Before the Morris water maze test, the swimming abilities of all mice were assessed with mice unable to swim being excluded from the study.
Tissue samples preparation
All mice were euthanized using chloral hydrate (0.25 mg/ml) and the brains were dissected and fixed in 4% paraformaldehyde (4% PFA) for 48 h at 4℃. After fixation, some of the samples were dehydrated and embedded in OCT, and kept at -20 °C until further analysis. Other samples were put into 75% alcohol, dehydrated and embedded with paraffin (Leica, EG1150), and kept at 4 °C until further analysis.
For immunofluorescence, the brains were cut into 20 μm slices using a low temperature thermostat. After incubation with 0.2% Triton X-100 (Gibco) for 30 mins, the sections were washed with PBS and incubated with blocking buffer composed of 3% BSA (Sigma-Aldrich, A9647) in PBS for 30 min at room temperature. The samples were then incubated with primary antibodies overnight at 4°C: SOX2 (Millipore, MAB5603, 1:400), BrdU (Bu20a) mouse mAb (CST, 5292, 1:500 ), NESTIN (Millipore, MAB5922, 1:400), MAP2 (Millipore, MAB1637,1:400), GFAP (CST, G9269, 1:600), Iba1 (GeneTex, GTX10042, 1:400), Phospho-Tau (Thr181) (CST, D9F4G, 1:500), Anti-Tau (phospho S396) (Abcam, ab109390, 1:400), β-Amyloid, Mouse (6E10) (BioLegend, 803004, 1:500), YAP (Santa cruz, SC-101199, 1:1000), p-YAP(Ser127) (CST, 4911s, 1:1000), TAZ (CST, 83669, 1:1000), Bcl2 (CST, 32012, 1:1000), Bax (CST, 34260-2, 1:1000), caspase 3 (CST, 1:1000), cleaved caspase 3 (CST, 1:1000), MST1(SCT, 3682,1:1000); Mst1 (Phospho-Thr183) (ASB, 12144, 1:1000); LATS1(CST，3477,1:1000), LATS1 (Phospho-Ser909) (SAB, 13033,1：500)，MOB1(CST, 13730,1；1000), Phospho-MOB1 (Thr35) (CST, 8699,:1000), Survivin (SAB, 24092,1:1000 ),PML(SAB, 32211,1:1000), and GAPDH (CST, 3683s, 1:1000). In the next day, the samples were rinsed with PBS and incubated for one hour at room temperature with the corresponding secondary antibody (Alexa Fluor 488 anti-mouse or 594 anti-rabbit (Invitrogen, 1: 500)). The sections were blocked by anti-fluorescence quenching blocking solution with DAPI, then analyzed and photographed using a microscope (Carl Zeiss Confocal, LSM710). All studies were performed three times, with 10 animals in each group.
Cells culture and treatments
PC12 and SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin/streptomycin and incubated at 37 °C with 5% CO2 humidified atmosphere. The medium was changed every 2–3 days, and the cells were sub-cultured when reaching 80–90% confluency using 0.25% trypsin. After digestion, the cells were centrifuged at 1000 rpm for 3 min and resuspended in fresh medium.
Primary neuronal cells were isolated from C57BL/6 mice brains and cultured in neuronal culture medium, as previously described . Briefly, newborn C57BL/6 mice, obtained from the animal facility of the University of Macau, were sacrificed and the brain was surgically removed and washed with cold PBS to remove all the blood. Brain homogenates were digested with 0.25% trypsin for 10 min at 37℃. Obtained cell suspension was filtered through a 0.45μm pore size filter unit, centrifuged at 1000xg for 5 min, and the supernatant removed. Obtained cells were seeded in poly-D-lysine plates with neurobasal medium containing 1% B27, 1% N2, 1% NEAA and 50μM glutamine.
CRISPR/Cas9 genome editing
CRISPR/Cas9 sgRNA targeting YAP (ATACCCTTACCTGTCGCGAG) was designed using Crispr design online tool (http://www.genome-engineering.org/crispr/). The sgRNA oligo primer was synthesised by BGI, China. After that, sgRNA was cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 sequence (Addgene plasmid #62988) from Feng Zhang lab. The presence of the insert was verified by isolating the plasmid DNA from several bacterial colonies and performing sequencing from the U6 promoter (human) (CCGTAACTTGAAAGTATTTCG). Isolated plasmid from positive colonies was transfected into PC12 cells using Lipofectamine 3000 according to the manufacturer's instructions (Thermo Fisher). The cells were sorted out using 2.5ug/ml puromycin and cultured into single colonies in 96-well plates. YAP expression in WT and KO cells was assessed by western blot.
The cell viability was assessed using MTT assay as previously described [44, 45]. Briefly, the cells were seeded in 96-well plates at a density of 5×103 cells/well in complete medium. In the following day, seeded cells were incubated for 24 hours with: (1) different concentrations of artemisia annua extracts; (2) different concentrations of Aβ1-42 and (3) Aβ1-42 with or without different concentrations of artemisia annua extracts. After the treatment, cells were further incubated with MTT (0.5 mg/ml) for additional 3-4 hours, and the medium was replaced with 100 μl DMSO to dissolve the blue formazan crystals formed by live cells. The absorbance was measured at 570 nm using a microplate reader (SpectraMax 250, Molecular Device, Sunnyvale, CA, USA). Cell viability was calculated as a percentage of the control group.
Measurement of reactive oxygen species (ROS)
The levels of intracellular ROS were tested using Cell ROXs Deep Red Reagent (Thermo Fisher Scientific, USA), according to the protocol provided by the manufacturer. After appropriate treatment, the cells were kept in the dark with CellROXs Deep Red Reagent (5 mM) in DMEM (without FBS) for 30 min and washed twice with phosphate buffered saline (PBS) solution. The ﬂuorescence was measured with an Inﬁnite M200 PRO Multimode Microplate using an emission wavelength of 665 nm and an excitation wavelength of 640 nm.
Measurement of mitochondrial membrane potential (△ψm)
The mitochondrial membrane potential (△ψm) was measured by JC-1 assay, according to the protocol provided by the manufacturer. After appropriate treatment, the cells were incubated with 1x JC-1 (10 μg/ml in medium without FBS) at 37°C for 30 min and washed two times with PBS solution. The intensities of red ﬂuorescence (excitation 560 nm, emission 595 nm) and green ﬂuorescence (excitation 485 nm, emission 535 nm) were measured using an Inﬁnite M200 PRO Multimode Microplate. The ratio of JC-1 red/green ﬂuorescence intensity was used to calculate the △ψm. All the values were normalized to the control group.
Caspase 3 activity assay
The levels of caspase 3 were measured using a caspase 3 activity assay kit (C1115, Beyotime Institute of Biotechnology, Shanghai, China). After 24 h of treatment, the cells were digested with 0.25% trypsin at 37℃ for 1 min, collected and centrifuged at 500x g for 5 min at 4 °C. The supernatant was then removed and the cells were washed once with PBS solution. Following the manufacturer’s instructions, 100 μL of lysis buffer were added per two million cells. Cells were then lysed for 15 min on ice followed by centrifugation at 12000xg for 15 min at 4°C. Afterwards, 40 μL of the detection buffer and 10 μL of Ac-DEVD-pNA (2 mM) were added to 50 μL of each sample. Obtained solutions were mixed carefully avoiding the production of bubbles and incubated for 60-120 min at 37°C. The production of pNA was measured by the absorbance values at 405 nm using an Infinite M200 PRO Multimode Microplate. Caspase-3 activity results were normalized to the control group.
TUNEL staining was used to test cellular apoptosis, according to the instructions provided by the manufacturer (C1090, Beyotime, Shanghai, China). Briefly, after appropriate treatment, the cells were washed two times with PBS, and fixed in 4% paraformaldehyde (PFA) for 30 min. The cells were then washed one time with PBS, incubated with 0.3 % Triton X-100 in PBS for 10 min at room temperature, rinsed once with PBS, followed by incubation with 0.3 % H2O2 in PBS for 30min. After this period the cells were incubated with 50 μl of TUNEL reaction mixture (5 μl of TdT enzyme and 45 μl of fluorescent labeling solution) for 60 min at 37℃ protected from light. TUNEL-positive cells (green fluorescence) were observed under a fluorescent microscope and counted. The apoptosis was calculated as a percentage of the total number of cells. For tissue samples the same methodology was used.
Flow cytometric assay was performed following the instructions provided by the Sangon Biotech manufacturer (REF:E606336-0100). Briefly, after appropriate treatment the cells were harvested and centrifuged at 1000 rpm for 5 min. The cells were rinsed twice with ice-cold PBS and resuspended in Annexin V-FITC/PI binding buffer (195 μL). Annexin V-FITC (5 μL) was added and the cells were kept in the dark at room temperature for 30 min. Cells were then centrifuged at 1000 rpm for 5 min and re-suspended in Annexin V-FITC/PI binding buffer (190 μL). Propidium iodide (PI) (10 μL) was further added and allowed to incubate in the dark for 5 mins. The quantification of apoptotic cells was performed using flow cytometry analysis.
Western blot was performed to assess the expression levels of molecules or enzymes involved in Aβ production and degradation, Tau phosphorylation, oxidative stress, synapse-related proteins, apoptosis-related proteins, and hippo pathway-associated proteins. Protein samples from cultured cells and brain homogenates were successfully extracted with RIPA buffer and quantified using the BCA assay kit (Thermo Fisher, 23225). After electrophoresis, the proteins were transferred to 0.22 μm PVDF membranes for 1.5 hours. The blotted PVDF membranes were blocked for 2 h with 5% BSA (TBST) and incubated with the primary antibodies (1:1000) at 4°C overnight. After washing with TBST (3 times, 10 min each), the membranes were incubated for 2 h with the secondary antibody (1:5000) at room temperature. Enhanced chemiluminescent was used to detect the blots.
Statistical analysis was performed using GraphPad Prism 5 software. All results are expressed as the mean ± SEM from three experiments. The statistical significance between multiple groups was determined using one or two-way ANOVA followed by Tukey’s post-hoc test. p < 0.05 was considered statistically significant.