Omipalisib may lower harmful tau protein by affecting cell cleanup and growth pathways

Omipalisib (C₂₅H₁₇F₂N₅O₃S, HY-10297, > 99% purity, Fig 1A) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Paraformaldehyde (PFA), phosphate-buffered saline (PBS, pH 7.4), Triton X-100, dimethyl sulfoxide (DMSO), and Tween 80 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roswell Park Memorial Institute media (RPMI) was purchased from Welgene (Gyeongsan, Republic of Korea). Fetal bovine serum (FBS), GlutaMAX, and penicillin-streptomycin (P/S) were purchased from Gibco (Waltham, MA, USA).

P301L (0N4R) mutant tau-expressing SH-SY5Y cells (SH-Tau; P30705↗) were purchased from Innoprot (Ibaizabal Bidea, Spain). Cells were cultured in RPMI containing 10% FBS and 1% P/S, GlutaMAX, and 125 μg/mL G418 at 37 °C in a humidified atmosphere of 5% CO₂. After stabilization for 24 hr, cells were treated with 0.1, 0.3, or 1 nM omipalisib.

A water-soluble tetrazolium (WST-1) assay was performed to assess omipalisib cytotoxicity. SH-Tau cells were seeded at a density of 8 × 10⁴ cells/well in 96-well plates. Subsequently, the cells were treated with 0.1, 0.3, 1, 3, and 10 nM omipalisib for 24 hr. WST-1 solution was added to each well and incubated for 2 hr. The absorbance of the cells was measured at 430 nm using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

We conducted a pilot tolerability study evaluating omipalisib at 1 and 3 mg/kg and monitored body weight, and clinical condition. Animals were monitored at least once daily, and predefined humane endpoints included severe lethargy, inability to access food or water, marked weight loss (>20% of baseline body weight), persistent hunched posture, or severe respiratory distress. Animals meeting these criteria were immediately removed from the study and euthanized. In the 3 mg/kg group, 3 of 7 mice reached humane endpoint criteria and were euthanized, whereas no animals in the 1 mg/kg group reached humane endpoints or died (). Based on these observations, 1 mg/kg was selected for the main experiment. All animal procedures were performed by trained personnel experienced in laboratory animal care and handling. S1 Fig

Wild-type (WT) and tau P301S (1N4R) mutant (PS19) male mice (aged 6-month-old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Experimental mice (n = 40) were divided into four groups based on the following treatment: WT+vehicle (10% DMSO and 5% Tween-80 in saline-treated WT mice), WT+omipalisib (omipalisib 1 mg/kg-treated WT mice), PS19 + vehicle (vehicle-treated PS19 mice), and PS19 + omipalisib (omipalisib 1 mg/kg-treated PS19 mice). Vehicle or omipalisib (1 mg/kg) was intraperitoneally injected into the mice once a day for 2 months (S2 Fig). Body weight of the mice was measured weekly throughout the treatment period to monitor any potential changes (S3 Fig). During the 2-month treatment period, no animals reached humane endpoint criteria. The animals were housed in plastic containers (2–4 mice per container), with controlled temperature (23 ± 1 °C), humidity (60 ± 10%), a 12 hr light/dark cycle, and ad libitum access to food and water. Mice were assigned to experimental groups using a block randomization method. To minimize bias, all behavioral testing and quantitative analyses were conducted under blind conditions. The mice were anesthetized by intraperitoneal injection of avertin. For western blot analysis, euthanasia was performed by cervical dislocation under deep anesthesia. For immunofluorescence, mice were euthanized by transcardial perfusion with PBS followed by 4% PFA fixation. All procedures were carried out under anesthesia to minimize pain and distress. All animal studies were performed in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th edition” (National Institutes of Health, 2011) and the ARRIVE guidelines. The study was approved by Kyung Hee University on August 9, 2023 (approval number: KHUASP(SE)-23-523).

To evaluate spatial learning, the Morris water maze (MWM) test was performed, as previously described [13]. The MWM test was conducted in a white tank (1.0-m diameter, 30-cm height) filled with water to a depth of 20 cm (22–24 °C). A white, opaque powder was added to conceal the platform in water. The mice were acclimatized one day before training, followed by testing three times per day, in one of four quadrants. The time taken by the mouse to swim to the hidden platform was recorded, up to a maximum of 60 s. If the mouse did not mount onto the platform within 60 s, it was guided to the platform and allowed to remain there for 10 s. After 7 days of training, the platform was removed, and a probe test was performed. Mice were allowed to swim freely for 60 s. Data was collected for the time the mouse required to traverse the cleared platform area and the time spent in each quadrant. All tests were recorded using a video monitor and a camera connected to a computer.

The brains of the mice were harvested, weighed, and suspended in a 12 × volume of radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher, Waltham, MA, USA) and protease/phosphatase inhibitor cocktail. The brains were homogenized and incubated on ice for 30 min. Subsequently, the homogenates were centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatants were collected as RIPA-soluble fractions and stored at −80 °C for further analysis. After centrifugation for 20 min, the pellets were washed once with RIPA buffer and incubated with 2% sodium dodecyl sulfate (SDS) solution at room temperature (RT) for 1 hr followed by centrifugation at 15,000 rpm for 1 min. The collected supernatant was categorized as RIPA-insoluble fractions.

Western blotting was performed as previously described [14]. An equal amount of protein lysates (10 or 20 µg) was quantified by Bradford assay. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were washed five times with 0.1% Tween 20 in Tris-buffered saline (TBST) for 5 min each. The membranes were first blocked by incubating in a blocking solution (5% skim milk in 0.1% TBST) for 1 hr at RT and then treated with primary antibodies in the blocking solution overnight at 4 °C. The primary antibodies used were as follows: Ser262 (Thermo Fisher, 44-750G, 1:1,000), Ser404 (Thermo Fisher, 44-758G, 1:1,000), Ser396 (Abcam, ab109390, 1:1,000), AT8 (Thermo Fisher, MN1020, 1:1,000), Tau5 (Thermo Fisher, AHB0042, 1:2,000), GAPDH (Santa Cruz, sc-32233, 1:5,000), phospho-mTOR (Cell Signaling, 2971s, 1:1,000), mTOR (Cell Signaling, 2983s, 1:1,000), p62 (Cell Signaling, 5114s, 1:1,000), LC3B (Abcam, ab48394, 1:1,000).

The next day, the membranes were washed three times with TBST for 5 min each and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) against mouse or rabbit IgG in the blocking solution for 1 hr at RT. The membranes were washed five times with TBST 5 min each. Protein expression was detected using the West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and visualized using FUSION Solo 6S EDGE (Vilber Lourmat Sté, Collégien, France). The band intensity was quantified using ImageJ software (Bethesda, MD, USA).

The harvested hearts were perfused with PBS and 4% PFA. The brains were excised and fixed in PFA overnight at 4 °C. Subsequently, they were sliced into 30 μm thick sections using a vibratome (VT-1200S; Leica Biosystems, Germany). The slice tissues were washed with PBS three times for 5 min each and incubated with a blocking solution (1% bovine serum albumin, 3% normal goat serum, and 0.4% Triton X-100 in PBS) for 1 hr at RT followed by incubation with anti-ionized calcium-binding adapter molecule 1 (Iba-1; Wako, 019–19741, 1:500) and anti-glial fibrillary acidic protein (GFAP; Dako, Z0334, 1:500) in blocking solution overnight at 4 °C. Then the tissue sections were washed with PBS three times for 5 min each and incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies for 1 hr at RT in light-shielded conditions. The tissue slices were washed and mounted on glass slides. The images were captured using a K1-Fluo confocal microscope.

All data presented as mean ± standard error of the mean. The differences between the two groups were analyzed using the student's t test. One-way or Two-way analysis of variance followed by Dunnett’s multiple comparison post-hoc test was used for multiple group comparisons. For behavioral data obtained from the Morris water maze acquisition trials, mixed-effects model was used. Treatment group and training day were included as fixed effects, and mouse was specified as a random effect. The group × day interaction was incorporated into the model. Multiple comparisons were performed within each training day to compare treatment groups. All analyses were performed using GraphPad Prism (Version 8.0a; Graph Pad Software Inc, San Diego, CA, USA). A value of p < 0.05 was determined as statistically significant.

We treated SH-Tau cells with omipalisib to assess its cytotoxic effects. As omipalisib showed cytotoxicity at 10 nM but not at 1 nM, we analyzed the effects of omipalisib at concentrations below 1 nM (Fig 1B). To determine whether omipalisib reduced p-tau expression, SH-Tau cells were incubated with omipalisib (0.1, 0.3, or 1 nM) for 24 hr. At 1 nM, omipalisib significantly decreased the expression of Ser262 (p = 0.0139) and Ser404 (p = 0.0071), but not Tau5 in SH-Tau cells (Fig 1C-E). Additionally, the expression of p-tau was reduced by omipalisib treatment (1 nM) in a time-dependent manner (3 hr, p = 0.0074; 6 hr, p = 0.0057; 12 hr, p = 0.0003; 24 hr, p = 0.0006 for Ser262, 12 hr, p = 0.0063; 24 hr, p = 0.0007 for Ser404) (Fig 1F-H).

Autophagy is a main pathway that clears accumulated p-tau [15]. Considering that omipalisib can inhibit the activation of mTOR, a factor that inhibits autophagy [10], we analyzed mTOR pathway-related autophagy factors. The levels of p-mTOR were significantly reduced following omipalisib treatment in a dose- (0.3 nM, p = 0.0044; 1 nM, p < 0.0001) and time- (3 hr, p < 0.0001; 6 hr, p < 0.0001; 12 hr, p < 0.0001; 24 hr, p < 0.0001) dependent manner (Fig 2A and B). To investigate the activation of the autophagy pathway by omipalisib (1 nM) treatment, the p62 and LC3B expression levels were analyzed. We observed a significant decrease in p62 levels following omipalisib treatment (3 hr, p = 0.0021; 6 hr, p = 0.0004; 12 hr, p < 0.0001; 24 hr, p < 0.0001) (Fig 2C). Although LC3B-II/LC3B-I ratios showed a trend towards an increase, these changes were not significant (3 hr, p = 0.1237; 6 hr, p = 0.2776; 12 hr, p = 0.0857; 24 hr, p = 0.7543) (Fig 2D). Furthermore, omipalisib significantly reduced Ser262 levels (p = 0.0207), whereas co-treatment with the autophagy inhibitor 3-MA blocked this reduction (p = 0.0007) (S4 Fig). These data indicated that omipalisib decreased the levels of accumulated p-tau by modulating ALP signaling through mTOR inhibition.

To examine whether omipalisib reduced p-tau levels in vivo, we used PS19 mice, a well-known animal model of tauopathy. PS19 mice were administered omipalisib (1 mg/kg/day) for 2 months. We separated the brain proteins into soluble and insoluble fractions and measured the levels of p-tau in each fraction. Omipalisib treatment significantly reduced the levels of soluble p-tau at Ser396 (p = 0.0041) and AT8 (p = 0.0001), whereas soluble p-tau at Ser262 did not show a significant reduction (p = 0.1175) (Fig 3A-D). Moreover, levels of insoluble p-tau (Ser262, p = 0.0492; Ser396, p = 0.0055; AT8, p = 0.0383) were also markedly reduced in the brains of omipalisib-treated PS19 mice (Fig 3F-I). It appears that the number of Ser396-positive cells was reduced in omipalisib-treated PS19 mice similar to the WB data for Ser396 (S5 Fig). Expression of Tau5 did not change in both soluble and insoluble form (Fig 3E and J). We investigated whether the effect of omipalisib on p-tau degradation was mediated by mTOR signaling. As expected, p-mTOR expression of omipalisib-treated PS19 mice was significantly lower than those of vehicle-treated PS19 mice (p = 0.0017) (Fig 4).

Next, we stained the brains for Iba-1 (a microglial marker) and GFAP (an astrocyte marker) to determine changes in neuroinflammation, which is another hallmark of tauopathy. Notably, omipalisib treatment did not alleviate astrocyte and microglia activation in PS19 mice (). S6 Fig

Finally, to determine whether the reduction in p-tau expression by omipalisib influences learning and memory, the MWM test was performed. The escape time (s) of omipalisib-treated PS19 mice was shorter than that of the PS19 mice (Fig 5A). The representative navigation paths on day 8 of training showed that PS19 mice treated with omipalisib displayed a navigation pattern similar to that of WT mice (Fig 5B). Moreover, omipalisib-treated PS19 mice passed the platform more times than the PS19 mice (Fig 5C), and the time spent in the quadrant where the platform was located was longer (Fig 5D), without a decrease in motor function (Fig 5E and F). It should also be noted that the partial behavioral rescue observed in our study may reflect the complex molecular signatures and diverse pathological pathways involved in tauopathy, suggesting that multiple mechanisms likely contribute to functional outcomes in tauopathy models [16].