p47phox deficiency improves cognitive impairment and attenuates tau hyperphosphorylation in mouse models of AD

Dulbecco’s modified Eagle’s medium (DMEM), neurobasal-A, B-27^®, and trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) were purchased from Gibco (Invitrogen, Carlsbad, CA). The BCA protein assay kit, normal goat serum (NGS), 4,6-diamidino-2-phenylindole (DAPI), and phenylmethanesulfonyl fluoride (PMSF) were obtained from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Okadaic acid (OA) and glucose were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Mouse anti-total tau (Tau5) and phospho-tau (AT8) antibodies and rabbit anti-phospho-tau pSer199, pThr205, pSer396, and pSer404 antibodies were obtained from Invitrogen (Carlsbad, CA). Mouse anti-non-phospho-tau (Tau1) and anti-glial fibrillary acidic protein (GFAP) antibodies were obtained from Merck (Darmstadt, Germany). Rabbit anti-β-Amyloid and mouse anti-β-Amyloid antibodies were purchased from Cell Signaling Technology (Danvers, MA) and Biolegend (San Diego, CA), respectively. Rabbit anti-MAP2 and mouse anti-GFAP Cy3™ antibodies were from Sigma-Aldrich (St. Louis, MO). Mouse anti-p47^phox antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas). Rabbit antibodies against p67^phox, NOX2/gp91^phox, and amyloid precursor protein (APP) were obtained from Abcam (Cambridge, MA). Rabbit anti-Iba1 antibody was obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rabbit anti-β-Actin antibodies were from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 488-conjugated anti-mouse IgG, Alexa Fluor 568-conjugated anti-rabbit IgG, and Alexa Fluor 568-conjugated anti-mouse IgG secondary antibodies were from Invitrogen. IRDye® 800CW and IMDye® 800CW secondary antibodies were from LI-COR, Inc. (Lincoln, NE). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

The p47^phox-deficient (Ncf1 knock out, Ncf1^−/−) mice in C57BL/6 J background were kindly provided by Dr. Steven M Holland (National Institutes of Health, Bethesda, MD), as reported previously [20]. Mouse genotypes were determined by PCR. The following primers were used: Ncf1 F: 5′-ACA TCA CAG GCC CCA TCA TCC TCC-3′; Ncf1 R: 5′-GGA GAG CCC CCT TTC TCT CCC TCA-3′; Ncf1 neo: 5′-CAA CGT CGA GCA CAG CTG CGC AAG-3′. The expected size of the PCR product using Ncf1 F and R primers is 650 bp, and the product using Ncf1 F and neo is 900 bp. The mutant mice were identified by the presence of a 900 bp PCR product, and the WT mice were identified by the presence of a 650 bp PCR product. Heterozygous mice contained both of the products. APP/PS1 transgenic mice in C57BL/6 J background (APP_SWE/PS1ΔE9^+/−, stock number 005864) were obtained from the Jackson Laboratory (Bar Harbor, ME). p47^phox-deficient mice were crossed to APP/PS1 mice to generate APP/PS1-Ncf1^+/− mice, and then the latter were further crossed with Ncf1^+/− mice to create the following four groups: WT (APP/PS1^−/−-Ncf1^+/+), APP/PS1 (APP/PS1^+/−-Ncf1^+/+), Ncf1^−/− (APP/PS1^−/−-Ncf1^−/−), and APP/PS1-Ncf1^−/− (APP/PS1^+/−-Ncf1^−/−). Mouse genotypes were determined by PCR. The mice of different genotypes were kept in separate cages. Mice of the same genotype were housed (3–5 mice per cage) with a 12/12 h light/dark cycle with ad libitum access to food and water. The housing, breeding, and animal experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with procedures approved by the Biological Research Ethics Committee, Shanghai Jiao Tong University. All four groups of male and female mice with 9 months of age (WT, 6 males and 4 females; APP/PS1, 4 males and 4 females; Ncf1^−/−, 5 males and 5 females; APP/PS1-Ncf1^−/−, 5 males and 5 females) and 12 months of age (WT, 10 males and 10 females; APP/PS1, 10 males and 1 female; Ncf1^−/−, 5 males and 4 females; APP/PS1-Ncf1^−/−, 4 males and 3 females) were subjected to a battery of behavioral tests. The behavioral tests were performed by two independent investigators blinded to the genotype of the mice.

Spatial reference learning and memory were evaluated in a water maze [23]. The test was performed in a white pool of 150 cm in diameter filled with water tinted with non-toxic white paint and maintained at room temperature. During training, an 8-cm diameter platform was submerged 1 cm below the water surface. All mice were given four trials per day for five consecutive days. The starting position was randomized among four quadrants of the pool every day. For each trial, the animal was given 90 s to locate the hidden platform. If a mouse failed to find the platform within 90 s, it was gently guided to it. At the end of each trial, the mouse was left on the platform for 20 s, then dried and returned to its home cage until the next trial. The probe trial was given 24 h after the last day of training. During probe trial, mice were allowed to swim in the pool without the platform for 60 s. The latency to reach the platform site (s) and swim distance (cm) were recorded using an automated tracking system (Smart video tracking system, Shanghai Mobile Datum, Shanghai, China).

Motor coordination and balance of mice were tested by using a Rotarod test. Test on an accelerating Rotarod was conducted by three trials on a rotating cylinder. The speed increased steadily from 5 to 30 rpm over a 90-s period. The latency to fall off the Rotarod was recorded. Inter-trial intervals were 10–15 min for each mouse.

Anxiety and exploratory activities were detected by an open field test. The testing apparatus was an open field (a polyvinyl chloride square arena, 80 × 80 cm, with walls 30 cm high), surmounted by a video camera connected to a computer. The arena was lit from the ceiling of the room with incandescent light, with a measurement inside the apparatus of 60 lx. Each mouse was placed individually in the corner and allowed to freely explore for 15 min. The 40-cm-diameter square defined the inner zone. The distance traveled in the arena, and the time and distance traveled in the center area (inner zone) were recorded and analyzed by Smart video tracking system (Shanghai Mobile Datum).

Elevated plus maze was used to test anxiety/emotionality of the mice. It consisted of four arms (30 × 5 cm) connected by a common 5 × 5 cm center area. Two opposite facing arms were open (open arms), whereas the other two facing arms were enclosed by 20-cm high walls (closed arms). The entire plus maze was elevated on a pedestal to a height of 82 cm above floor level in a separate room from the investigator. The mouse was placed onto the central area facing an open arm and allowed to freely explore the maze for a single 8-min session. Between each session, feces were cleared from the maze, and the maze floor was cleaned with 75% alcohol to remove any urine or scent cues. The time spent in open arms and closed arms were recorded by Smart video tracking system (Shanghai Mobile Datum).

After behavioral tests, all mice were sacrificed by decapitation and their brains removed immediately. The hippocampi and cerebral cortices of the left hemisphere of the brain were dissected, flash frozen in dry ice, and stored at − 80 °C for biochemical analyses later. The right hemispheres of the brain were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered (PBS) for 24 h, followed by cryoprotection in 30% sucrose. Sagittal sections of 30 μm thickness were cut using a freezing sliding microtome. The sections were stored in glycol anti-freeze solution (ethylene glycol, glycerol, and 0.1 M PBS in 3:3:4 ratio) at − 20 °C until immunofluorescence staining. In this study, we included 3–6 mice/group for biochemical analyses and immunofluorescence staining.

Mouse brain tissue was homogenized in lysis buffer containing 50 mM Tris-HCL (pH 7.4), 100 mM NaF, 2 mM EDTA, 10 mM β-mercaptoethanol, 2 mM NaVanadate, 8.5% sucrose, 5 μg/ml aprotinin, 100 μg/ml leupeptin, and 5 μg/ml pepstatin. Protein concentrations were determined by using BCA Kits according to the manufacturer’s protocol. Quantitative homogenates were added 5 × sodium dodecyl sulfate (SDS) to heat for 10 min at 99 °C and resolved in 10% SDS-PAGE for separation. After separated, samples were transferred onto nitrocellulose membranes (GE Healthcare, Wauwatosa, WI), and the membranes were blocked with 5% non-fat milk for 1 h at room temperature. Then, the membranes were incubated with primary antibodies including Tau5 (1:1000), Tau1 (1:1000), the anti-phospho-tau antibodies pS199 (1:1000), pT205 (1:1000), pS396 (1:1000), pS404 (1:1000), anti-β-Amyloid (6E10, 1:2000), anti-APP (1:20000), anti-p47^phox (1:250), anti-p67^phox (1:1000), anti-NOX2/gp91^phox (1:1000), anti-GFAP (1:1000), anti-GAPDH (1:20000), and anti-β-Actin (1:20000), followed by the respective IRDye® 800CW or IMDye® 800CW secondary antibodies. The membranes were scanned using an Odyssey P140-CLx Infrared Imaging System (LI-COR, Inc.). Densitometric quantification of protein bands was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD).

Free-floating sections were processed for standard immunofluorescence staining [24]. Briefly, the sections were washed three times for 5 min with pre-cold 0.05 M Tris-buffered saline (TBS, containing 0.05 M Tris buffer and 9 g/L NaCl, pH 7.4), followed by blocked with 5% NGS in 0.05 M TBS (0.1% Tween-20) for 1 h at room temperature. Then, the sections were incubated with anti-β-amyloid antibody (1:200), anti-AT8 antibody (1:500), or anti-Iba1 antibody (1:500) in TBS at 4 °C overnight. After rinsing with TBS, the sections were further incubated with Alexa Fluor 488-conjugated anti-rabbit or Alexa Fluor 568-conjugated anti-mouse (1:500) secondary antibodies for 1 h at room temperature. After three washes in TBS, the sections were stained for nuclei with 100 ng/ml DAPI for 10 min and mounted on glass slides.

For double immunofluorescence staining of AT8 and thioflavin-S (Thio-S), the staining of AT8 was performed as described above. Then, the sections were rinsed in TBS and stained with 1% Thio-S for 10 min at room temperature. The sections were washed with 50% ethanol for two times. After three washes in TBS, the sections were stained for nuclei with 100 ng/ml DAPI for 10 min and mounted on glass slides. For double immunofluorescence staining of p47^phox and MAP2, the sections were first incubated with anti-p47^phox antibody (1:200) in TBS at 4 °C overnight and treated in Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:500) for 1 h. The sections were rinsed in TBS, stained with anti-MAP2 antibody (1:200) at 4 °C overnight, and incubated with Alexa Fluor 568-conjugated anti-rabbit secondary antibody (1:500) for 1 h. For double immunofluorescence staining of p47^phox and GFAP, the sections were first incubated with anti-p47^phox antibody (1:200) in TBS at 4 °C overnight and incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:500) for 1 h. Then, the sections were rinsed in TBS, stained with anti-GFAP-Cy3™ antibody (1:500) for 1 h. After double immunofluorescence staining, the sections were washed in TBS, stained for nuclei with 100 ng/ml DAPI for 10 min, and mounted on glass slides.

The fluorescent confocal images were taken on a laser-scanning confocal fluorescent microscope (TCS SP8, Leica Microsystems, Wetzlar, Germany). For quantification of the expression of p47^phox, Aβ deposits, phosphorylated tau (AT8), astrocytes (GFAP), and microglia (Iba1), the relative immunofluorescence intensity was quantified using the ImageJ software. For quantification of colocalization of p47^phox with MAP2 or GFAP, the number of colocalized cells per square millimeter of the sections was calculated. The results were expressed as the means ± SEM based on 3–5 mice each group, or a minimum of four viewing fields for each region, using at least two mice.

Mouse brain tissue was homogenized in lysis buffer containing 50 mM Tris-HCL (pH 7.4), 100 mM NaF, 2 mM EDTA, 10 mM β-mercaptoethanol, 2 mM sodium vanadate, 8.5% sucrose, 5 μg/ml aprotinin, 100 μg/ml leupeptin, and 5 μg/ml pepstatin. The homogenate was centrifuged at 14000 rpm for 15 min at 4 °C. The supernatant (soluble fraction) was collected. The pellets were dissolved in guanidine HCl solution (6 M guanidine hydrochloride, 50 mM Tris-HCl, 1% PMSF, pH 8.0), cooled for 20 min in an ice bath, and centrifuged at 14000 rpm for 15 min at 4 °C. The supernatant (insoluble fraction) was collected. Protein concentrations were determined by using BCA Kits according to the manufacturer’s protocol. Then, soluble and insoluble Aβ_1–40 and Aβ_1–42 were measured using commercially available ELISA kits from Shanghai Westang Bio-tech Co., LTD (Shanghai, China), according to the manufacturer’s instructions.

Neuronal cultures were prepared in cortices obtained from 1-day-old WT and Ncf1^−/− mice as described before [25]. In brief, cerebral cortices were removed from the brains of mice, the meanings and microvessels were removed, and tissues were minced with a sterile razor blade. Tissues were digested with 0.025% trypsin-EDTA at 37 °C for 10 min. The cell suspension was filtered through a 200-mesh sieve, and cells were plated on poly-D-lysine-coated 12-well plates at a density of 10 × 10⁵ cells per well. Four hours later, the DMEM medium (containing 10% FBS, 100 U/ml of penicillin, and 100 μg/ml streptomycin sulfate) were replaced with neurobasal medium containing 2% B-27® supplements for 2 days. Culture medium was changed to neurobasal with 10% FBS and 5 μg/ml cytosine-β-D-arabinofuranoside (Ara-C) in the following 1 days, and then again switched back to neurobasal medium containing 2% B-27^® supplements. Experiments were performed on days 7–8 after initiation of the culture.

Astrocyte cultures were prepared from 1-day-old WT and Ncf1^−/− mice as described before [26]. Specifically, separated cells were cultured in poly-D-lysine-coated 75 cm² flasks with DMEM medium (containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate). The medium was replenished on day 1 and day 3. On day 7, microglia cells in the culture flasks were shaken off at 260 × rpm for 2.5 h. Then, the remaining astrocytes were maintained in DMEM with 10% FBS until seeding into 24-well plates. Experiments were performed after one passage of the cells.

When astrocytes cultured in plates grew to 70–80% confluence, the culture medium was replaced with fresh DMEM without FBS, or with the above medium containing 75 mM or 150 mM glucose. The cells were incubated for another 24 h and 48 h. At the end of the incubation, conditioned medium (CM) was collected, and cells were lysed in 5 × SDS buffer for immunoblotting analysis. The astrocyte CM collected at 24 h or 48 h was added to the culture of neurons from WT mice for another 48 h. The neuronal cell lysate was prepared and analyzed by SDS-PAGE and Western blotting as detailed above.

All data are presented as the means ± SEM. Mean values of paired groups were analyzed using Student’s t test. Mean of multiple groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The survival rate of the mice was analyzed using Gehan-Breslow-Wilcoxon test. The bodyweight of the mice, Morris water maze, and Rotarod test were analyzed using two-way ANOVA. All analyses were performed with the statistical software GraphPad Prism 8 (San Diego, CA), and p value less than 0.05 was considered statistically significant.

To assess the potential role of p47^phox in AD, we generated the APP/PS1 transgenic and p47^phox-deficient (Ncf1 knockout, APP/PS1-Ncf1^−/−) mice. We crossed APP/PS1 transgenic mice on a C57BL/6 background to the Ncf1^−/− mice (also on a C57BL/6 background) to generate APP/PS1-Ncf1^+/− mice, then crossed APP/PS1-Ncf1^+/− mice with Ncf1^−/− mice to generate APP/PS1-Ncf1^−/− mice. We first observed the mortality in these mice. All four groups of mice (WT, APP/PS1, Ncf1^−/−, and APP/PS1-Ncf1^−/− mice) initially showed healthy. However, at the age of 3 months, there was a significant increase in the mortality rate of APP/PS1 mice compared with the other three groups of mice (Fig. 1a). The mortality rate of APP/PS1-Ncf1^−/− mice did not increase until the age of 5 months. By the age of 12 months, 30% of APP/PS1-Ncf1^−/− mice died, compared with 55% of APP/PS1 mice (p = 0.072, Fig. 1a). These data show that p47^phox deficiency decreases the mortality in APP/PS1 mice.

Next, we investigated the effect of p47^phox on cognitive performance. We tested all four groups of mice aged 9 and 12 months in Morris water maze (MWM). The result showed that APP/PS1 mice at both ages took a longer time (Fig. 1b, d) and swam a longer distance (Fig. 1c, e) than the WT mice to find the escape platform, indicating a learning impairment in APP/PS1 mice. However, in APP/PS1-Ncf1^−/− mice, a shorter escape latency and swimming distance on day 5 to reach the escape platform were observed compared with the APP/PS1 mice (Fig. 1b–e), suggesting that p47^phox deficiency improves learning of the APP/PS1 mice. After one day off, probe trials were performed on day 6 to evaluate the spatial reference memory of the mice. As expected, APP/PS1 mice aged 9 months and 12 months spent less time and traveled less distance in the target quadrant than WT mice, indicating an impairment of spatial reference memory (Fig. 1f–i). However, p47^phox deficiency increased the time spent and distance swam of APP/PS1-Ncf1^−/− mice in the target quadrant compared with APP/PS1 mice (Fig. 1f–i). All these findings suggest that p47^phox deficiency improves the spatial learning and memory of APP/PS1 mice.

Furthermore, we established another mouse model of AD to confirm the effect of p47^phox on cognitive function. The ICV injection of STZ mouse is widely recognized as a mouse model of sporadic AD [22]. STZ is a diabetogenic compound. In ICV-STZ mice, brain insulin resistance, impaired glucose metabolism, aggregation of tau, and learning and memory deficits have been reported [27, 28]. The ICV-STZ AD model was generated by ICV injection of STZ into Ncf1^−/− mice and WT littermates, using normal saline as a control (ICV-Saline) (Fig. 1j). As STZ-induced animal models are characterized by insulin deficiency accompanied by lower body weight, changes in the body weight after ICV injection were monitored. ICV-STZ mice had lower body weight compared with ICV-Saline mice (Fig. 1k), consistent with previous reports [29]. However, in the Ncf1^−/− group, ICV-STZ mice had similar body weight with ICV-Saline mice (Fig. 1l), suggesting that p47^phox deficiency attenuated the decrease in body weight of ICV-STZ mice. For the MWM test, the ICV-STZ mice took more time and swam a longer distance than ICV-saline mice to find the escape platform on day 5 (Fig. 1m, n). This result is consistent with what was reported previously [30]. In the Ncf1^−/− group, no difference was observed between the STZ and saline groups, and both of these groups showed less time spent and shorter distance swum to the escape platform on day 5, compared with the WT ICV-STZ group. Furthermore, probe trails test showed that p47^phox deficiency increased the time spent and distance swum in the target quadrant of mice receiving ICV-STZ, compared with WT ICV-STZ mice (Fig. 1o, p). Consistent with the results of APP/PS1 mouse model, all these findings suggest that p47^phox deficiency improves cognitive impairment in AD.

In addition to the cognitive functions, general behaviors were also evaluated in APP/PS1 mice. Motor coordination and balance ability were measured using Rotarod test. APP/PS1 mice aged 9 months and 12 months displayed a decreasing trend in fall latency compared with WT mice (Additional file 1: Fig. S1a and g). p47^phox deficiency did not change the fall latency of APP/PS1 mice (Additional file 1: Fig. S1a and g), indicating that p47^phox has no effect on the motor coordination and balance of AD mice. Then, we evaluated spontaneous exploratory activity of mice. We observed that no significant difference in the total distance explored between four groups in the open field test (Additional file 1: Fig. S1b and h), suggesting that p47^phox has no effect on spontaneous exploratory ability in mice. Next, we measured anxiety in mice using open field and elevated plus maze tests. We observed that APP/PS1 mice spent less time in the center area of the open field (Additional file 1: Fig. S1c, e, i, and k) and in the open arms of the elevated plus maze (Additional file 1: Fig. S1d, f, j, and l), compared with WT mice. Interestingly, similar to APP/PS1 mice, the p47^phox-deficient mice also showed shorter time staying in the center area and in the open arms in these two tests compared with WT mice (Additional file 1: Fig. S1c-f, i-l). However, p47^phox deficiency did not seem to affect the time APP/PS1 mice spent in the center area and in the open arms (Additional file 1: Fig. S1c-f, i-l). These results suggest that although p47^phox deficiency induces anxiety in mice, it does not aggravate the anxiety in APP/PS1 mice.

Then, we also analyzed tau phosphorylation in the brain of ICV-STZ injection mice. Similar to APP/PS1 mice, in the hippocampus and the cortex of ICV-STZ mice, tau phosphorylation at Ser199, Thr205, Ser396, and Ser404 was increased and non-phosphorylated tau was decreased when compared with ICV-saline mice (Fig. 3e, f; Additional file 1: Fig. S3e and f). In the Ncf1^−/− mice, however, no significant difference was found in the phosphorylated and non-phosphorylated tau in the brain of ICV-STZ mice compared with ICV-saline mice (Fig. 3e, f; Additional file 1: Fig. S3e and f). These results confirmed that p47^phox is involved in tau pathology in AD.

Since the neuronal p47^phox is involved in tau phosphorylation directly, we further investigate whether and how p47^phox expressed in astrocytes participates in regulating tau phosphorylation in neurons indirectly. We have shown that both activated astrocytes and p47^phox expressed in astrocytes were significantly increased in the brain of APP/PS1 mice (Fig. 4d, e). Then, we verified whether p47^phox deletion affected the activation of astrocytes in these mice. As shown in Additional file 1: Fig. S7a and b, the expression of GFAP was significantly increased in CA and DG areas of the hippocampus as well as in the cortex of APP/PS1 mice at the age of 9 and 12 months, compared with age-matched WT mice. However, p47^phox deficiency obviously inhibited the activation of astrocytes in APP/PS1 mice (Additional file 1: Fig. S7a and b). These results suggest that p47^phox participates in the activation of astrocytes in AD mice. In addition, studies have found that p47^phox is highly expressed in microglia and NADPH oxidase plays a vital role in microglia activation [37]. Thus, we detected the effect of p47^phox on microglia activation in APP/PS1 mice. We found that compared with the WT mice, microglia showed a certain degree of activation in the hippocampus and the cortex of 12-month-old APP/PS1 mice (Additional file 1: Fig. S8). p47^phox deficiency inhibited the activation of microglia in APP/PS1 mice (Additional file 1: Fig. S8), suggesting that p47^phox is involved in microglia activation in AD mice. However, the activation of astrocytes in aged APP/PS1 mice and the inhibition of p47^phox deletion on astrocyte activation seem to be more obvious than microglia. Therefore, we further examined whether p47^phox expressed in astrocytes could affect tau phosphorylation by activating astrocytes.

This study has several limitations. First, by the age of 12 months, APP/PS1-Ncf1^−/− mice displayed a decreasing trend in mortality rate compared with APP/PS1 mice, whereas there was no significant difference in statistical analysis (p = 0.072). The current sample size of mice is a bit small. In addition, whether there is a causal link between the decrease of tau hyperphosphorylation and the improvement in cognitive function observed in APP/PS1 mice lacking p47^phox has not been verified. Finally, we used mice of two sexes for experiments. We did not find that gender has a significant effect on the results of this study. Except for 12-month-old mice, the bodyweight of APP/PS1 male mice is slightly higher than that of APP/PS1-Ncf1^−/− male mice, while there is no difference in the bodyweight of female mice.

Additional file 1: Figure S1. The effect of p47^phox deficiency on the locomotor, spontaneous exploratory, and anxiety of APP/PS1 mice. The locomotion and spontaneous exploratory activity of mice aged 9 (a, b) and 12 (g, h) months was evaluated on Rotarod and open field tests, respectively. The anxiety of mice aged 9 months (c, d) and 12 months (i, j) was examined in open field and an elevated plus maze. Representative traces of a mouse’s movements during the open field test (e, k) and the elevated plus maze (f, l) are shown. Figure S2. p47^phox deficiency does not affect APP levels in APP/PS1 mice. Representative Western blots showing APP expression in the hippocampus and the cortex of WT, APP/PS1, and APP/PS1-Ncf1^−/− mice aged 9 months (a) and 12 months (c). b, d Quantification of immunoreactivity of Western blots, normalized against GAPDH. Figure S3. p47^phox deficiency attenuates tau hyperphosphorylation in the cortex of APP/PS1 mice and ICV-STZ mice. Representative Western blots showing tau phosphorylation at S199, T205, S396, and S404 in the cortex of WT, APP/PS1, Ncf1^−/−, and APP/PS1-Ncf1^−/− mice aged 9 months (a) and 12 months (c), and of WT and Ncf1^−/− mice receiving ICV injection of STZ or saline at the age of 6 months (e). (b, d, f) Quantifications of the immunoreactivity of Western blots are shown. Figure S4. p47^phox deficiency attenuates tau aggregation and NTs in the hippocampus and the cortex of APP/PS1 mice. (a) Representative immunofluorescence staining showing the decreased tau aggregation and NTs in APP/PS1-Ncf1^−/− mice aged 12 months, compared with age-matched APP/PS1 mice. Quantification of the fluorescence of AT8 is shown. Figure S5. p47^phox deficiency attenuates NP tau in the hippocampus and the cortex of APP/PS1 mice. Representative immunofluorescence staining of AT8 and thioflavin-S (Thio-S) showing the decreased NP tau in APP/PS1-Ncf1^−/− mice aged 12 months, compared with age-matched APP/PS1 mice. Figure S6. The expression of NOX subunits (p47^phox, p67^phox, and NOX2/gp91^phox) in APP/PS1 mice. Representative Western blots showing p47^phox, p67^phox, and gp91^phox expression in the brain of WT and APP/PS1 mice at the age of 9 months (a) and 12 months (c). (b, d) Quantifications of the immunoreactivity of Western bolts are shown. Figure S7. p47^phox deficiency inhibits the activation of astrocytes in APP/PS1 mice. (a) Representative immunofluorescence staining showing the decreased expression of GFAP in APP/PS1-Ncf1^−/− mice aged 9 months and 12 months, compared with age-matched APP/PS1 mice. (b) Quantification of the fluorescence of GFAP is shown. Figure S8. p47^phox deficiency inhibits the activation of microglia in APP/PS1 mice. (a) Representative immunofluorescence staining showing the decreased expression of Iba1 in APP/PS1-Ncf1^−/− mice aged 12 months, compared with age-matched APP/PS1 mice. (b) Quantification of the fluorescence of Iba1 is shown. Figure S9. p47^phox deficiency inhibits high glucose-induced activation of primary astrocytes. Primary cultures of astrocytes from WT and Ncf1^−/− newborn mice were treated with DMEM with or without high glucose (75 mM and 150 mM) for 24 h and 48 h. (a) Representative Western blots showing the expression of GFAP. (b, c) Quantification of the immunoreactivity of Western blots is shown.