Ketamine induces delirium-like behavior and interferes with endosomal tau trafficking

Ketamine induces delirium-like behavior and interferes with endosomal tau trafficking | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ketamine induces delirium-like behavior and interferes with endosomal tau trafficking Xinghua Ren, Siyi Zhang, Yongyan Yang, Annie Song, Feng Liang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1362130/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ketamine is an anesthetic and antidepressant drug. However, whether ketamine can induce neurotoxicity and neurobehavioral deficits remains largely unknown. Delirium is a syndrome of acute brain dysfunction that is very similar to the presentation after ketamine administration. The onset of postoperative delirium in patients is often accompanied by elevated tau in cerebrospinal fluid. And ketamine may affect endosome, the key organelle for tau release from neurons. Therefore, we set out to determine the effects of ketamine on delirium-like behavior in mice and on tau trafficking in cultured cells. Methods We used the buried food test, open field test, and Y-maze test in adult mice to assess the occurrence of delirium-like behavior induced by ketamine. Quantified tau in the serum of mice with delirium-like behavior. And used cell fraction methods to determine the effects of ketamine on tau intracellular transfer, extracellular release and endosomes in cultured cells. Results Ketamine induced delirium-like behavior and increased tau in mouse serum. Ketamine treatment also led to increased accumulation of endosomes as evidenced by increased endosomal markers Rab5 and Rab7. Moreover, ketamine inhibited endosome maturation, demonstrated by decreased membrane-bound but increased cytoplasm amounts of Rab5 and Rab7. Consequently, ketamine increased tau in the endosomes of cultured cells and the cell culture medium. Conclusion These data suggest that ketamine may interfere with intracellular tau trafficking and induce delirium-like behavior, promoting future research regarding neurotoxicity of anesthetics. Ketamine tau endosome anesthetic delirium-like behavior neurotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Ketamine is a phencyclidine analogue and a non-competitive antagonist of the N-methyl-D-aspartic acid (NMDA) receptor (Sinner and Graf, 2008 ). It is a commonly used clinical anesthetic and can also be used to treat depression (Jansen, 2000 ; Sassano-Higgins et al., 2016 ). Ketamine also is a recreational drug owing to its ability to induce visual hallucinations in subanesthetic doses (Jansen, 2000 ; Morgan and Curran, 2012 ; Sassano-Higgins et al., 2016 ; Siegel, 1978 ). Ketamine induces cognitive impairment (Zhang et al., 2020 ), with acute effects including confusion, impaired judgment, and delirium in humans (Sassano-Higgins et al., 2016 ; Smith, 1999 ). However, whether ketamine can induce delirium-like behavior in rodents remains unknown. Delirium is a syndrome of acute brain dysfunction potentially associated with anesthesia, surgery, pain, disease, or medication, manifesting with impaired consciousness, disorganized thinking, lack of purpose, and inability to focus (Setters and Solberg, 2017 ). Previous studies (Witlox et al., 2011 ; Xie et al., 2014 ) have demonstrated an association between cerebrospinal fluid microtubule-associated protein τ (MAPT or tau) and amyloid β ratio with postoperative delirium in patients. Tau, a microtubule-associated protein, is expressed mainly in neurons and has a role in stabilizing the cytoskeleton, maintaining anchoring of membrane material, and participating in axonal transport (Martin et al., 2013 ). Tauopathy is a hallmark of Alzheimer’s disease neuropathogenesis (Querfurth and LaFerla, 2010 ) and contributes to cognitive impairment. However, it is unknown whether ketamine can induce tauopathy. A recent study showed that anesthetic sevoflurane can induce tau trafficking from neurons to microglia (Dong et al., 2021 ). Specifically, sevoflurane causes tau and phosphorylated tau to exit from neurons via a tau phosphorylation-associated mechanism. However, the pathways responsible for tau trafficking inside cells before its transition to the extracellular space remain largely unknown. Endosomes are vesicle organelles of the endomembrane system of eukaryotic cells. They originate from the trans-Golgi network (TGN) and have extensive bidirectional transport relationships with the TGN, cell membrane, and lysosome, which are responsible for sorting, transport, and degradation of intracellular cargo (Langemeyer et al., 2018 ; Stoorvogel et al., 1991 ; Yang, 2014 ). Endosomes are classified into three main categories—early endosomes (EEs), late endosomes (LEs) and recycling endosomes—based on the order of endocytic cargo transport to various endosomal vesicles (Futter et al., 1996 ; Stenmark, 2009 ). The three types of endosomes are defined mainly based on their electron microscopic morphology, the cargo transported within the lumen, and surface markers (Thery et al., 2018 ). Surface markers of EEs are EEA1 and Rab5. EEs can endo-emerge to form intralumenal vesicles and gradually transform into multivesicular bodies (i.e., LEs) (Neefjes et al., 2017 ). Maturation of EEs is demonstrated by increased binding of Rab5 on the endosome membrane (Delevoye and Goud, 2015 ; Nagano et al., 2019 ). LEs are mainly responsible for degradation in the endosomal system, and their surface markers are CD63 and Rab7. Similarly, LE maturation is demonstrated by increased binding of Rab7 on the endosome membrane (Langemeyer et al., 2018 ). In this study, we investigated the effects of ketamine on delirium-like behavior and changes in tau amounts in mouse serum. Mechanistically, we assessed the effects of ketamine on the number and maturation of EEs and LEs, intracellular transport, and extracellular release of tau in the SH-SY5Y cell line. The hypothesis of the present study was that ketamine causes delirium-like behavior and increases endosomal uptake and extracellular release of tau. Results Ketamine induces delirium-like behavior and increases serum tau in mice. We used our established animal model, consisting of the Y-maze test (YMT), buried food test (BFT), and open field test (OFT) (Liufu et al., 2020 ; Peng et al., 2016 ), to study the effects of ketamine on delirium-like behavior in mice ( Supplemental Figure S1 ). A single injection of ketamine in mice significantly increased the latency to eat food in the BFT and freezing time in the OFT compared to control mice at 1, but not 2, h after ketamine administration ( Supplemental Figure S2 ). Composite Z-scores were obtained by pooling data from six measurements. The composite Z-score increased in the ketamine-treated mice at 1, but not 2, h after ketamine treatment compared to control mice [KET: 7.899 (10.03–6.927) versus CON: 4.657 (6.371–3.365), P = 0.0005, Mann-Whitney test; Fig. 1 A,B]. These data suggest that ketamine may induce delirium-like behaviors in mice. Blood was collected from the mice 1, 2, and 6 h after administration of a single injection of ketamine, and serum was extracted. We found increased amounts of tau in the serum of mice at 1, but not 2 or 6, h after ketamine administration compared to control mice (KET: 90.36 pg/mL ± 47.41 pg/mL versus CON: 53.81 pg/mL ± 11.64 pg/mL, P = 0.0232, Student’s t-test; Fig. 1 C–E). Ketamine increases tau protein concentration in the endosome lumen and culture medium of SH-SY5Y cells. Ketamine decreased viability of cultured SH-SY5Y cells in a dose-dependent manner (F = 155.7, P < 0.0001, one-way ANOVA; Supplemental Figure S3 ). Hence, we chose 156.3 µg/mL ketamine (KET1), 312.5 µg/mL ketamine (KET2), 625 µg/mL ketamine (KET3), and a control condition (CON) to treat SH-SY5Y cells. We specifically assessed the effects of ketamine on the amount of tau inside the endosome lumen and outside of the SH-SY5Y cells. Ketamine increased the amounts of tau in whole cells (Fig. 2 A,B). ELISA showed that ketamine also increased the amounts of tau in the endosomal lumen (Fig. 2 C) and the cell culture medium (Fig. 2 D). However, a lactate dehydrogenase (LDH) assay showed that treatment with ketamine did not increase LDH amounts in the cell culture medium (F = 1.015, P = 0.4068, one-way ANOVA; Fig. 2 E), indicating that the ketamine-induced increase in tau in the cell culture medium was not due to rupture of cell membranes. Taken together, these data indicate that ketamine increases endosomal uptake and extracellular release of tau. Ketamine increases early and late endosome accumulation in SH-SY5Y cells. Given that ketamine increased tau concentration in both the endosome and extracellular space, next we assessed the effects of ketamine on endosome amounts and activation in SH-SY5Y cells. Quantitative western blot analysis demonstrated that after 24 h of incubation, whole cell protein amounts of EE markers EEA1 (F = 36.48, P < 0.0001, one-way ANOVA; Fig. 3 A,B) and Rab5 (F = 31.23, P < 0.0001, one-way ANOVA; Fig. 3 A,C) were significantly increased, suggesting that ketamine increased amounts of EEs in the cells. Moreover, ketamine increased whole cell protein amounts of LE marker Rab7 (F = 32.05, P < 0.0001, one-way ANOVA; Fig. 3 A,E) but not CD63 (F = 2.358, P = 0.1102, one-way ANOVA; Fig. 3 A,D). In addition, ketamine decreased whole cell protein amounts of lysosome marker LAMP2 (F = 4.892, P = 0.0323, one-way ANOVA; Fig. 3 A,F). These data demonstrate that ketamine increased accumulation of EEs and LEs and decreased lysosome amounts in SH-SY5Y cells. Ketamine decreases endosome membrane-bound Rab5 and Rab7 but increases Rab5 and Rab7 in the cytoplasm of SH-SY5Y cells. Given that ketamine increased the amounts of proteins associated with EEs in whole cells, we further assessed the effects of ketamine on the amounts of these proteins bound with the endosome membrane or in the cytoplasm. First, we demonstrated that ketamine did not significantly change the amounts of Na-K ATPase (F = 1.483, P = 0.2569, one-way ANOVA; Fig. 4 A,B), indicating that Na-K ATPase could be used as an internal loading control for the amounts of protein associated with endosome membranes. Quantitative western blot analysis demonstrated that ketamine significantly increased the amounts of cytoplasmic Rab5 (F = 11.15, P = 0.0003, KET3: 237.2% ± 84.99% versus CON: 100% ± 16.32%, one-way ANOVA; Fig. 4 C,D) and Rab7 (F = 9.200, P = 0.0009, KET3: 277.6% ± 113.5% versus CON: 100% ± 26.04%, one-way ANOVA; Fig. 4 C,E) 24 h after ketamine administration. Conversely, ketamine treatment significantly decreased the amounts of membrane-bound Rab5 (F = 7.410, P = 0.0025, KET1: 69.00% ± 26.86% and KET3: 55.86% ± 6.633% versus CON: 100% ± 7.792%, one-way ANOVA; Fig. 4 C,F) and Rab7 (F = 8.174, P = 0.0016, KET2: 55.23% ± 16.50% and KET3: 28.56% ± 11.05% versus CON: 100% ± 41.50%, one-way ANOVA; Fig. 4 C,G). These data demonstrate that ketamine could decrease endosome membrane-bound Rab5 and Rab7 but increase cytoplasmic amounts of Rab5 and Rab7, suggesting that ketamine may impair EE and LE maturation. Discussion Our results show that ketamine induced delirium-like behavior and increased serum tau amounts in mice. In mechanistic studies, ketamine impaired intracellular tau trafficking, evidenced by increasing amounts of tau inside the endosome lumen and extracellular space of SH-SY5Y cells. Moreover, ketamine might impair intracellular tau trafficking by increasing the number of endosomes but inhibiting endosome maturation. These data, using ketamine as a clinically relevant tool, demonstrate that tau could contribute to the neuropathogenesis of delirium, promoting future research to study delirium. These data also show that anesthetic ketamine could induce tauopathy and delirium-like behavior. We firstly found time-dependent effects of ketamine in inducing delirium-like behavior, evidenced by the findings that ketamine increased composite Z-score compared to controls. Consistently, previous studies show anesthesia/surgery induces time-dependent (Peng et al., 2016 ) and age-dependent (Liufu et al., 2020 ) delirium-like behavior in mice. However, previous studies did not assess the effects of anesthetic without surgery on the delirium-like behavior. The present study shows that anesthetic ketamine without surgery could still induce delirium-like behavior in mice. Ketamine induced delirium-like behavior and increased serum tau amounts 1 h after administration, showing an association between delirium-like behavior and increased serum tau. Consistently, a clinical observational study in 2021 concluded that postoperative plasma tau amounts could serve as a biomarker for postoperative delirium in patients (Ballweg et al., 2021 ). Endosomes are responsible for intracellular transport of cargo, including proteins, lipids, and nucleic acids, which are important for cell function (Elkin et al., 2016 ). Cell function is severely impaired when endosomes accumulate (Langemeyer et al., 2018 ). We found that ketamine increased endosome accumulation, which could explain the findings that ketamine increased endosomal tau amounts because more endosomes could uptake more tau. However, ketamine may also enhance the ability of single endosomes to uptake tau. We will test this hypothesis in future studies. Previous studies show that increased amounts of Rab5 or Rab7 in the cytoplasm and decreased amounts of membrane-bound Rab5 or Rab7 indicate inhibition of endosome maturation (Delevoye and Goud, 2015 ; Nagano et al., 2019 ). Endosomes becomes exosomes when they bind to the cell membrane (Bebelman et al., 2020 ). Moreover, it has been suggested that inhibition of the endosome maturation process will increase protein release via exosomes (Hessvik et al., 2016 ). In the present study, ketamine inhibited endosome maturation, evidenced by increased cytoplasm amounts of Rab5 and Rab7 and decreased membrane-bound Rab5 and Rab7. Meanwhile, ketamine also increased amounts of tau in the extracellular space. Together, these findings indicate that ketamine inhibits endosome maturation while increasing tau uptake by endosomes, causing migration of endosomes to the cell membrane to form exosomes and releasing more tau into the extracellular space. The mechanism of tau release into the extracellular space is unclear, but increasing evidence shows that tau relies mainly on the non-classical protein secretion pathway and extracellular vesicles for extracellular transport (Chen et al., 2019 ; Jiang et al., 2019 ). Our previous study also demonstrates that trafficking of tau to the extracellular space is dependent on tau phosphorylation and generation of extracellular vesicles (Dong et al., 2021 ). In the present study, we illustrated that ketamine increases endosome accumulation but inhibits endosome maturation, leading to more release of tau into the extracellular space. Moreover, we postulate that the effect of ketamine on EEs includes blocking conversion of EEs to LEs but does not affect conversion of other organelles, such as TGN, to EEs. In LEs, we found that ketamine did not significantly change CD63 protein amounts but increased Rab7 amounts. Further, ketamine decreased amounts of membrane Rab7 yet increased cytoplasmic amounts of Rab7. These data suggest that ketamine inhibits LE maturation, as demonstrated in previous studies (Takeda et al., 2019 ; Zerial and McBride, 2001 ). There are several limitations of this study. First, we did not study whether inhibition of the ketamine-induced increase in serum tau could mitigate ketamine-induced delirium-like behavior because we could not find a specific inhibitor of tau trafficking from the intracellular to extracellular space. We will use tau knockout mice to determine the role of tau in delirium-like behavior using the established system in future studies. Second, we did not determine the effects of ketamine on intracellular trafficking of phosphorylated tau in the present study because we wanted to focus on tau. We also will use the established system to study intracellular trafficking of both tau and phosphorylated tau in future studies. Finally, we only assessed the effects of ketamine on membrane Rab5 and Rab7 but not the specific endosome membrane Rab5 and Rab7. However, Rab5 and Rab7 are only known to bind to the endosome membrane. Conclusion In conclusion, this study shows that ketamine induces delirium-like behavior and increases serum tau in mice. In the in vitro studies, we found that ketamine increases tau uptake in endosomes by increasing endosome accumulation. Ketamine then increases release of tau to the extracellular space by inhibiting endosomal maturation. These findings will promote future studies of tau trafficking and delirium and the effects of anesthetic on tauopathy and delirium-like behavior. Materials And Methods Antibodies and reagents Full details of the primary antibodies used are in Table 1 . Table 1 Antibody information Antigen Host species Dilution Manufacturer Catalog No. CD63 Rabbit 1:1000 Abcam ab216230 EEA1 Mouse 1:1000 Cell Signaling Technology 48453S GAPDH Rabbit 1:2000 Cell Signaling Technology 5174S LAMP2 Rabbit 1:1000 Abcam ab199947 Na-K ATPase Rabbit 1:20000 Abcam ab76020 Rab5 Mouse 1:1000 Cell Signaling Technology 46449S Rab7 Rabbit 1:1000 Abcam ab126712 Total tau Rabbit 1:2000 Abcam ab32057 Mouse (secondary antibody) Goat 1:2500 Sigma-Aldrich SAB3701095 Rabbit (secondary antibody) Goat 1:10000 Invitrogen G-21234 Primary antibodies were diluted with western blot blocking buffer (see western blot subsection for details), and secondary antibodies were diluted with 1×TBST. Ketamine injection (Ketalar) was purchased from Hikma Pharmaceuticals PLC 2019. Animals A total of 48 8-week-old naïve adult male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) with an average weight of 23 g. No significant difference in mouse weight was found between groups at the beginning of the study ( Fig. S1 A1,A2 ). Mice were housed four per cage and maintained on a 12-h light/dark cycle (lights out at 18:00). Mice had unlimited access to water and food in their home cages. Sample size was decided by a previous pilot experiment and power analysis (β = 0.1). Mice were equally divided into two post-injection duration groups, a 1 h group and 2 h + group, with 24 mice in each group using the complete randomization method. Each post-injection duration group was divided equally into two treatment subgroups, a saline control group (CON) and a ketamine group (KET), using the complete randomization method, with 12 mice in each subgroup. Each mouse was injected with 2 µl/g i.p. of injection according to body weight. The dosage of KET group was 40 mg/kg. Within each subgroup of the 2 h + group, mice were divided into two additional subgroups using the complete randomization method, and tissue was harvested at 2 h and 6 h post-injection. Behavioral tests All mice had multiple behavioral tests starting with the YMT training trail followed by OFT, BFT, and finally the YMT retention trail at 24 h before (baseline) injection and 1 h or 2 h after injection (Fig. 5 ). We performed behavior tests in groups of four mice and finished tests within 50 min, mimicking certain features of clinical evaluation of delirium in patients. Y maze test . The YMT was performed as described previously (Chen et al., 2014 ; Li et al., 2017 ; Rayatnia et al., 2011 ) with modifications. Specifically, the Y maze, made of gray polyvinylene, was placed in a quiet and illuminated room. Each maze consisted of three arms (8 cm wide × 30 cm long × 15 cm tall), with an angle of 120º between each arm. The three arms included the start arm, in which the mouse starts to explore (always open); the novel arm, which is blocked at the first trial but opened at the second trial; and another familiar arm (always open). In the experiment, the start arm and the familiar arm were designed randomly to avoid spatial memory error. The YMT consisted of two trials separated by an inter-trial interval. The first trial (training) was 10 min in duration, which allowed the mouse to explore two arms (start arm and familiar arm) of the maze, with the novel arm blocked. After a 1 h inter-trial interval, the second trial (retention) was conducted. For the second trial, the mouse was placed back in the maze in the same start arm with free access to all three arms for 5 min. A video camera linked to AnyMaze (Stoelting Co.) animal tracking system software was installed 60 cm above the chamber to monitor and analyze the number of entries and the time spent in each arm. The time spent in and entries into the novel arms indicated spatial recognition memory (learned behavior). Each arm of the Y maze was cleaned with 70% ethanol solution between trials. Open field test . The OFT was performed as described previously (Li et al., 2014 ; Li et al., 2017 ) with modifications. Specifically, the mouse was gently placed in the center of an open field chamber (40 cm × 40 cm × 40 cm) under dim light and was allowed to move freely for 5 min. Movement parameters of the mouse were monitored and analyzed via a video camera connected to the AnyMaze animal tracking system software. The total distance moved (meters), time (seconds) spent in the center of the open field, freezing time (seconds), and latency (time in seconds for mouse to reach location of the first attempt) to the center of the open field were recorded and analyzed. The floor of the open field was cleaned with 70% ethanol solution between each test. Buried food test . The BFT was performed as described in previous studies (Lehmkuhl et al., 2014 ; Nathan et al., 2004 ) with modifications. Specifically, two days before the test, we gave each mouse two pieces of sweetened cereal. On all test days, we habituated the mice for one hour prior to the test by placing the home cage with mice in the testing room. The test cage was prepared with clean bedding (3-cm high). We buried one sunflower seed 0.5 cm below the surface of the bedding so that it was not visible. The location of the food pellet was changed every time in a random fashion. We placed the mouse in the center of the test cage and measured the latency of the mouse to eat the food. Latency was defined as the time from when the mouse was placed in the test cage until the mouse uncovered the food pellet and grasped it in their forepaws and/or teeth. Mice were allowed to consume the pellet they found and were then returned to their home cage. Observation time was 5 min. If the mouse could not find the pellet within 5 min, the testing session ended and the latency was defined as 300 s for that mouse. We emptied the bedding from the test cage and cleaned the cage with 70% ethanol solution after each test to prevent transmission of olfactory cues. We changed gloves after each test. Animal tissue harvesting and euthanasia Mice were anesthetized with 5% isoflurane at 1, 2, and 6 h after KET or CON injection. Retro-orbital sinus blood sampling was performed, and then mice were euthanized by guillotine. Blood was transferred to a 4℃ refrigerator for 15 min and centrifuged at 4℃ for 15 min at 1,200 × g. We then carefully extracted and transferred the supernatant as serum to a clean centrifuge tube. The serum was frozen in liquid nitrogen and stored in a -80℃ freezer. Cell culture SH-SY5Y cells were cultured at 37°C with 5% CO 2 in DMEM/F12 1:1 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich), 1% MEM non-essential amino acids solution (100X) (Gibco), and 1% sodium pyruvate (100 mM) (Gibco). When incubating with drugs, we replaced DMEM/F12 1:1 with DMEM/F12 1:1 with HEPES without phenol red and added 10% ketamine solution or saline and 0.1% DMSO. Cell viability assays MTT was purchased from Invitrogen, and all MTT assays were performed following the instructions provided with the kit to obtain cell viability data. LDH assay kit (Abcam) was used for quantitative analysis of LDH concentration in the cell culture medium. Standard curves were plotted for each assay using positive control LDH provided in the kit with gradient concentration (0 ng/mL, 125 ng/mL, 250 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 4000 ng/mL, and 8000 ng/mL). All operations were performed according to the kit instructions. Whole cell and cell fraction protein extraction Whole cell protein extraction. We obtained treated SH-SY5Y cells, discarded the medium in the culture dish, and rinsed gently with DPBS (Lonza) three times, shaking lightly each time. We then discarded as much residue as possible from the culture dish and added an appropriate amount of RIPA (Thermo Scientific) lysate with pre-dissolved Pierce protease and phosphatase inhibitor mini tablets (Thermo Scientific). After incubation, we scraped off all cells and lysate using a cell scraper and transferred them to a clean centrifuge tube. Tubes were shook in a vortex machine three times at 50% amplitude for 3 s with an interval of 3 s. The solution was frozen in liquid nitrogen and stored in a -80℃ freezer. Cytoplasmic protein and membrane protein extraction. We obtained treated SH-SY5Y cells, discarded the medium in the culture dish, and rinsed gently with DPBS three times, shaking lightly each time. We then discarded as much residue as possible from the culture dish and added an appropriate amount of M-PER (Thermo Scientific) lysate with pre-dissolved Pierce protease and phosphatase inhibitor mini tablets. We sealed the culture dish with plastic wrap and transferred it to a 4℃ refrigerated room for 30 min incubation on a shaker. After incubation, we scraped off all hanging cell debris and lysate using a cell scraper and transferred to a clean centrifuge tube. We centrifuged tubes at 16,000 × g for 30 min at 4℃ and carefully extracted the upper clarified solution into a clean centrifuge tube. This solution contained cytoplasmic and nuclear proteins, and the pellet contained membrane protein. The membrane protein pellet was dissolved into solution using RIPA. The solution was frozen in liquid nitrogen and stored in a -80 ℃ freezer. Endosome isolation. Minute endosome isolation and cell fractionation kit (Invent Biotechnologies) was used to isolate endosomes as described previously (Thapa et al., 2020 ; Xu et al., 2018 ) with modifications. Briefly, 4 × 10 7 cells were collected, and 1 ml buffer A was added. The cell suspension was incubated on ice for 15 min, loaded into a filter cartridge and centrifuged with 16,000 × g for 30 s. A filter cartridge re-pass-through was performed to enrich the field. The supernatant was transferred to a fresh tube, and 500 µl buffer B was added to the tube and incubated at 4°C overnight. After incubation, vortex briefly and centrifuge with 10,000 × g for 30 min. We removed the supernatant and washed the pellet with 750 µl 2:1 mixture of buffer A/buffer B. After centrifugation with 10,000 × g for 30 min, the pellet containing endosomes was frozen in liquid nitrogen and stored in a -80℃ freezer. All the centrifugations should be performed at 4°C. Endosome lumen proteins and endosome membrane proteins were extracted by a similar method as that used for cytoplasmic protein and membrane protein extraction. ELISA Concentrations of human total tau in the medium or protein lysate, and mouse total tau in serum were measured by ELISA kits (Invitrogen). Experiments were performed according to the manufacturer’s instructions. Protein concentration Protein concentration of lysates was obtained using the Pierce BCA protein assay kit (Thermo Scientific). Standard curves were plotted for each assay using Pierce bovine serum albumin standard (Thermo Scientific) with gradient concentration (0 mg/mL, 0.025 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, and 0.5 mg/mL). Western blot Proteins were separated by NuPAGE 4–12% Bis-Tris protein gel (Invitrogen) and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked in 50% SuperBlock (TBS) blocking buffer (Invitrogen), 5% bovine serum albumin (Sigma-Aldrich) in 1× TBST (8 g/L NaCl, 2.42 g/L Tris-base, 1 mL/L Tween 20, adjusted pH to 7.6 with HCl). Immunoreactive bands were detected using a SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific). The signal intensity was analyzed using ChemiDoc XRS + with Image Lab 5.0 software (Bio-Rad, Hercules, CA). The quantification of western blot was performed in two steps. At the first step, we used β-Actin levels to standardize protein amounts and to limit the disparities in the protein quantity loaded. At the second step, we expressed the protein levels obtained from the anesthesia/surgery mice as a percentage in relation to the control condition. Composite Z-score When pooling the battery of behavioral test data, we transformed outcome data according to the composite Z-score introduced in previous studies (Moller et al., 1998 ; Peng et al., 2016 ), with some modifications. We first calculated the difference between the measured value after injection and the baseline value \(\varDelta X\) for each mouse. The \(\varDelta X\) from the CON group was then used to calculate mean \({M}_{\varDelta {X}_{CON}}\) and SD \({S}_{\varDelta {X}_{CON}}\) . The composite Z-score of each mouse was obtained by the formula \({C}_{{Z}_{X}}=\sum \left|{Z}_{X}\right|=\sum \left|\frac{\varDelta X-{M}_{\varDelta {X}_{CON}}}{{S}_{\varDelta {X}_{CON}}}\right|\) . Finally, \({C}_{{Z}_{X}}\) was included in the statistical analysis. Statistical analysis Statistical analyses were performed using Prism (GraphPad Software) to generate curves or bar graphs. All error bars represent standard deviation (SD). Data were first tested for conformity to a normal distribution using the Shapiro-Wilk test, and then tested for equality of variances using the F test. Two-tailed unpaired t test was used for statistical analysis of two groups of samples. One-way ANOVA with a Turkey’s honestly significant difference test was used to evaluate statistical significance of multiple groups of samples. Normally distributed data were described as "mean ± SD". Data that did not conform to the normal distribution were combined with the experimental records to exclude outliers using the Grubbs’ test and then re-examined for normality and F tests. Data that still did not conform to the normal distribution were tested using the non-parametric independent samples Mann-Whitney exact probability test. Data were described using "median [interquartile spacing]". All tests were set at α = 0.05 and β = 0.1. Abbreviations BFT buried food test CON control group EE early endosome KET ketamine group LDH lactate dehydrogenase LE late endosome OFT open field test TGN trans-Golgi network Tau microtubule-associated protein τ YMT Y-maze test Declarations Ethical Approval and Consent to participate All experimental procedures involving mice were approved by the Standing Committee on Animals at Massachusetts General Hospital, Boston, MA (protocol number: 2006N000219) and conformed to National Institutes of Health (Bethesda, MD) guidelines. This article was written according to applicable ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Efforts were made to minimize the number of mice used in the studies. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Funding This study was supported by a grant from the National Institutes of Health (grant No. R01AG062509 to Zhongcong Xie). Authors’ contributions X inghua R en : conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing of original manuscript draft; Siyi Zhang : data curation, investigation, methodology, validation, writing of original manuscript draft; Yongyan Yang : formal analysis, investigation, methodology, resources; Annie Song : investigation; Feng Liang : conceptualization, investigation, methodology, reviewing and editing manuscript; Yiying Zhang : investigation, methodology, resources, reviewing and editing manuscript; Yuanlin Dong : conceptualization, investigation, methodology, project administration, resources, reviewing and editing manuscript; Xu Wu : conceptualization, methodology, project administration, resources, supervision, reviewing and editing manuscript; Zhongcong Xie : conceptualization, funding acquisition, methodology, project administration, resources, supervision, reviewing and editing manuscript. All authors reviewed and approved the final manuscript. References Ballweg, T., M. White, M. Parker, C. Casey, A. Bo, Z. Farahbakhsh, A. Kayser, A. Blair, H. Lindroth, R.A. Pearce, K. Blennow, H. Zetterberg, R. Lennertz, and R.D. Sanders. 2021. Association between plasma tau and postoperative delirium incidence and severity: a prospective observational study. Br J Anaesth. 126:458–466. Bebelman, M.P., C. Crudden, D.M. Pegtel, and M.J. Smit. 2020. The convergence of extracellular vesicle and gpcr biology. Trends Pharmacol Sci. 41:627–640. Chen, J.J., D.L. Nathaniel, P. Raghavan, M. Nelson, R. Tian, E. Tse, J.Y. Hong, S.K. See, S.A. Mok, M.Y. Hein, D.R. Southworth, L.T. Grinberg, J.E. Gestwicki, M.D. Leonetti, and M. Kampmann. 2019. Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation. J Biol Chem. 294:18952–18966. Chen, Y., X. Liu, X. Jia, W. Zong, Y. Ma, F. Xu, and J. Wang. 2014. Anxiety- and depressive-like behaviors in olfactory deficient Cnga2 knockout mice. Behav Brain Res. 275:219–224. Delevoye, C., and B. Goud. 2015. Rab GTPases and kinesin motors in endosomal trafficking. Methods Cell Biol. 130:235–246. Dong, Y., F. Liang, L. Huang, F. Fang, G. Yang, R.E. Tanzi, Y. Zhang, Q. Quan, and Z. Xie. 2021. The anesthetic sevoflurane induces tau trafficking from neurons to microglia. Commun Biol. 4:560. Elkin, S.R., A.M. Lakoduk, and S.L. Schmid. 2016. Endocytic pathways and endosomal trafficking: a primer. Wien Med Wochenschr. 166:196–204. Futter, C.E., A. Pearse, L.J. Hewlett, and C.R. Hopkins. 1996. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J Cell Biol. 132:1011–1023. Hessvik, N.P., A. Overbye, A. Brech, M.L. Torgersen, I.S. Jakobsen, K. Sandvig, and A. Llorente. 2016. PIKfyve inhibition increases exosome release and induces secretory autophagy. Cell Mol Life Sci. 73:4717–4737. Jansen, K.L. 2000. A review of the nonmedical use of ketamine: use, users and consequences. J Psychoactive Drugs. 32:419–433. Jiang, L., H. Dong, H. Cao, X. Ji, S. Luan, and J. Liu. 2019. Exosomes in pathogenesis, diagnosis, and treatment of Alzheimer's disease. Med Sci Monit. 25:3329–3335. Langemeyer, L., F. Fröhlich, and C. Ungermann. 2018. Rab GTPase function in endosome and lysosome biogenesis. Trends Cell Biol. 28:957–970. Lehmkuhl, A.M., E.R. Dirr, and S.M. Fleming. 2014. Olfactory assays for mouse models of neurodegenerative disease. J Vis Exp:e51804. Li, X.M., F. Su, M.H. Ji, G.F. Zhang, L.L. Qiu, M. Jia, J. Gao, Z. Xie, and J.J. Yang. 2014. Disruption of hippocampal neuregulin 1-ErbB4 signaling contributes to the hippocampus-dependent cognitive impairment induced by isoflurane in aged mice. Anesthesiology. 121:79–88. Li, Y., R. Shen, G. Wen, R. Ding, A. Du, J. Zhou, Z. Dong, X. Ren, H. Yao, R. Zhao, G. Zhang, Y. Lu, and X. Wu. 2017. Effects of ketamine on levels of inflammatory cytokines IL-6, IL-1beta, and TNF-alpha in the hippocampus of mice following acute or chronic administration. Front Pharmacol. 8:139. Liufu, N., L. Liu, S. Shen, Z. Jiang, Y. Dong, Y. Wang, D. Culley, G. Crosby, M. Cao, Y. Shen, E. Marcantonio, Z. Xie, and Y. Zhang. 2020. Anesthesia and surgery induce age-dependent changes in behaviors and microbiota. Aging (Albany NY). 12:1965–1986. Martin, L., X. Latypova, C.M. Wilson, A. Magnaudeix, M.L. Perrin, C. Yardin, and F. Terro. 2013. Tau protein kinases: involvement in Alzheimer's disease. Ageing Res Rev. 12:289–309. Moller, J.T., P. Cluitmans, L.S. Rasmussen, P. Houx, H. Rasmussen, J. Canet, P. Rabbitt, J. Jolles, K. Larsen, C.D. Hanning, O. Langeron, T. Johnson, P.M. Lauven, P.A. Kristensen, A. Biedler, H. van Beem, O. Fraidakis, J.H. Silverstein, J.E. Beneken, and J.S. Gravenstein. 1998. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet. 351:857–861. Morgan, C.J., and H.V. Curran. 2012. Ketamine use: a review. Addiction. 107:27–38. Nagano, M., J.Y. Toshima, D.E. Siekhaus, and J. Toshima. 2019. Rab5-mediated endosome formation is regulated at the trans-Golgi network. Commun Biol. 2:419. Nathan, B.P., J. Yost, M.T. Litherland, R.G. Struble, and P.V. Switzer. 2004. Olfactory function in apoE knockout mice. Behav Brain Res. 150:1–7. Neefjes, J., M.M.L. Jongsma, and I. Berlin. 2017. Stop or go? Endosome positioning in the establishment of compartment architecture, dynamics, and function. Trends Cell Biol. 27:580–594. Peng, M., C. Zhang, Y. Dong, Y. Zhang, H. Nakazawa, M. Kaneki, H. Zheng, Y. Shen, E.R. Marcantonio, and Z. Xie. 2016. Battery of behavioral tests in mice to study postoperative delirium. Sci Rep. 6:29874. Querfurth, H.W., and F.M. LaFerla. 2010. Alzheimer's disease. N Engl J Med. 362:329–344. Rayatnia, F., M. Javadi-Paydar, N. Allami, M. Zakeri, H. Rastegar, A. Norouzi, and A.R. Dehpour. 2011. Nitric oxide involvement in consolidation, but not retrieval phase of cognitive performance enhanced by atorvastatin in mice. Eur J Pharmacol. 666:122–130. Sassano-Higgins, S., D. Baron, G. Juarez, N. Esmaili, and M. Gold. 2016. A review of ketamine abuse and diversion. Depress Anxiety. 33:718–727. Setters, B., and L.M. Solberg. 2017. Delirium. Prim Care . 44:541–559. Siegel, R.K. 1978. Phencyclidine and ketamine intoxication: a study of four populations of recreational users. NIDA Res Monogr:119–147. Sinner, B., and B.M. Graf. 2008. Ketamine. Handb Exp Pharmacol .:313–333. Smith, K.M. 1999. Drugs used in acquaintance rape. J Am Pharm Assoc (Wash). 39:519–525; quiz 581 – 513. Stenmark, H. 2009. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol. 10:513–525. Stoorvogel, W., G.J. Strous, H.J. Geuze, V. Oorschot, and A.L. Schwartz. 1991. Late endosomes derive from early endosomes by maturation. Cell. 65:417–427. Takeda, M., J. Koseki, H. Takahashi, N. Miyoshi, N. Nishida, J. Nishimura, T. Hata, C. Matsuda, T. Mizushima, H. Yamamoto, H. Ishii, Y. Doki, M. Mori, and N. Haraguchi. 2019. Disruption of endolysosomal RAB5/7 efficiently eliminates colorectal cancer stem cells. Cancer Res. 79:1426–1437. Thapa, N., M. Chen, H.T. Horn, S. Choi, T. Wen, and R.A. Anderson. 2020. Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4. Nat Cell Biol. 22:1357–1370. Thery, C., K.W. Witwer, E. Aikawa, M.J. Alcaraz, J.D. Anderson, R. Andriantsitohaina, A. Antoniou, T. Arab, F. Archer, G.K. Atkin-Smith, D.C. Ayre, J.M. Bach, D. Bachurski, H. Baharvand, L. Balaj, S. Baldacchino, N.N. Bauer, A.A. Baxter, M. Bebawy, C. Beckham, A. Bedina Zavec, A. Benmoussa, A.C. Berardi, P. Bergese, E. Bielska, C. Blenkiron, S. Bobis-Wozowicz, E. Boilard, W. Boireau, A. Bongiovanni, F.E. Borras, S. Bosch, C.M. Boulanger, X. Breakefield, A.M. Breglio, M.A. Brennan, D.R. Brigstock, A. Brisson, M.L. Broekman, J.F. Bromberg, P. Bryl-Gorecka, S. Buch, A.H. Buck, D. Burger, S. Busatto, D. Buschmann, B. Bussolati, E.I. Buzas, J.B. Byrd, G. Camussi, D.R. Carter, S. Caruso, L.W. Chamley, Y.T. Chang, C. Chen, S. Chen, L. Cheng, A.R. Chin, A. Clayton, S.P. Clerici, A. Cocks, E. Cocucci, R.J. Coffey, A. Cordeiro-da-Silva, Y. Couch, F.A. Coumans, B. Coyle, R. Crescitelli, M.F. Criado, C. D'Souza-Schorey, S. Das, A. Datta Chaudhuri, P. de Candia, E.F. De Santana, O. De Wever, H.A. Del Portillo, T. Demaret, S. Deville, A. Devitt, B. Dhondt, D. Di Vizio, L.C. Dieterich, V. Dolo, A.P. Dominguez Rubio, M. Dominici, M.R. Dourado, T.A. Driedonks, F.V. Duarte, H.M. Duncan, R.M. Eichenberger, K. Ekstrom, S. El Andaloussi, C. Elie-Caille, U. Erdbrugger, J.M. Falcon-Perez, F. Fatima, J.E. Fish, M. Flores-Bellver, A. Forsonits, A. Frelet-Barrand, et al. 2018. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 7:1535750. Witlox, J., K.J. Kalisvaart, J.F. de Jonghe, N.A. Verwey, M.F. van Stijn, A.P. Houdijk, H.S. Traast, A.M. MacLullich, W.A. van Gool, and P. Eikelenboom. 2011. Cerebrospinal fluid beta-amyloid and tau are not associated with risk of delirium: a prospective cohort study in older adults with hip fracture. J Am Geriatr Soc. 59:1260–1267. Xie, Z., C.A. Swain, S.A. Ward, H. Zheng, Y. Dong, N. Sunder, D.W. Burke, D. Escobar, Y. Zhang, and E.R. Marcantonio. 2014. Preoperative cerebrospinal fluid beta-Amyloid/Tau ratio and postoperative delirium. Ann Clin Transl Neurol. 1:319–328. Xu, X., J. Xu, J. Wu, Y. Hu, Y. Han, Y. Gu, K. Zhao, Q. Zhang, X. Liu, J. Liu, B. Liu, and X. Cao. 2018. Phosphorylation-mediated IFN-gammaR2 membrane translocation is required to activate macrophage innate response. Cell. 175:1336–1351 e1317. Yang, T. 2014. [Medical Cell Biology]. Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2:107–117. Zhang, C., Y. Xu, B. Zhang, W. Hao, and W.K. Tang. 2020. Cognitive impairment in chronic ketamine abusers. Psychiatry Res. 291:113206. Additional Declarations No competing interests reported. Supplementary Files figS1.tif Fig. S1. Mouse weight and behavior test baseline data. figS2.tif Fig. S2. Results of behavioral tests 1 and 2 h after single ketamine injection. figS3KET.tif Fig. S3. MTT assay results for KET incubation statistical plots. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1362130","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":84424489,"identity":"622555a7-801f-4865-8901-eac1046c36a1","order_by":0,"name":"Xinghua Ren","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"Ren","suffix":""},{"id":84424490,"identity":"2c676a81-19e2-4084-8641-89105beb56a2","order_by":1,"name":"Siyi Zhang","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siyi","middleName":"","lastName":"Zhang","suffix":""},{"id":84424491,"identity":"66cb1116-3b39-455c-8483-37a1d2acc74b","order_by":2,"name":"Yongyan Yang","email":"","orcid":"","institution":"Tianjin Medical University General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongyan","middleName":"","lastName":"Yang","suffix":""},{"id":84424492,"identity":"f8b38bb8-fe67-4ab1-ba25-0a5b5d1107fe","order_by":3,"name":"Annie Song","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Annie","middleName":"","lastName":"Song","suffix":""},{"id":84424493,"identity":"8ef78fb1-d584-42c5-b077-9f320dbebe9f","order_by":4,"name":"Feng Liang","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Liang","suffix":""},{"id":84424494,"identity":"ae543ba5-2c62-4439-a162-3ef2629796f5","order_by":5,"name":"Yiying Zhang","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiying","middleName":"","lastName":"Zhang","suffix":""},{"id":84424495,"identity":"0e47e0b5-8420-4316-b83e-13888a31b499","order_by":6,"name":"Yuanlin Dong","email":"","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanlin","middleName":"","lastName":"Dong","suffix":""},{"id":84424496,"identity":"456733eb-cbc5-4e26-b2da-ab9b027bb3ff","order_by":7,"name":"Xu Wu","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Wu","suffix":""},{"id":84424497,"identity":"1f630bdf-0122-4aae-aea1-163b46f0b7d1","order_by":8,"name":"Zhongcong Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFACxgaGDwwMCQwScC6DAUEtjDNI1MLAwMxDkhb5GcmNj21zavMYpJuffeapuSfbwN68TQKvs3oONhvnbjtezCBzzHg2z7Fi4waeY2V4tTCzN7ZJ5247ltggkWDMOIMtAcjIMcOrhY2Zsf23JVhL+mfGGf+AWuTf4NfCA7SFmXFbDchwY4aPbSBbePBrkeA52CzZu+1AYptETjHDx74E4zaetGILfFrkZ6Q//PBzW11iv0T6ZoaEbwmy/eyHN97ApwUKDjOwwX1HhHIQqCNS3SgYBaNgFIxIAACrZ0fkP3eRkQAAAABJRU5ErkJggg==","orcid":"","institution":"Massachusetts General Hospital, Harvard Medical School","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhongcong","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2022-02-15 13:29:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1362130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1362130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18372936,"identity":"57160182-fad4-42df-85d2-999fc190f670","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70114,"visible":true,"origin":"","legend":"\u003cp\u003eKetamine induces delirium-like behavior and increases serum total tau protein concentration in mice. A: Composite Z-scores 1 h after ketamine administration, n = 12 in each group. B: Composite Z-scores 2 h after ketamine administration, n = 12 in each group. C: Serum total tau protein concentration 1 h after ketamine administration, n = 11 in CON group, n = 12 in KET group. D: Serum total tau protein concentration 2 h after ketamine administration, n = 6 in each group. E: Serum total tau protein concentration 6 h after ketamine administration, n = 6 in each group. Mann-Whitney test was applied to analyze data in panels A and B. Grubbs’ test was applied to identify an outlier owing to hemolysis in data in panel C. Student’s t-test was applied to analyze data in panels C, D, and E. Error bars represent 25%–75% interquartile range in panels A and B, and SD in panels C, D, and E; * indicates P \u0026lt; 0.05 by statistical analysis; ns indicates no significant difference between groups (P \u0026gt; 0.05) by statistical analysis.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/98f21f68b8609d70b7ca31d1.png"},{"id":18372937,"identity":"218da6b4-09c2-4dbe-bd30-64ffe89428f7","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66140,"visible":true,"origin":"","legend":"\u003cp\u003eKetamine induces total tau protein changes in endosomes and medium. A: Western blot analysis of tau in whole cells and the internal loading control GAPDH. One band is shown for each experimental condition. B: Quantitation of the western blot analysis of tau in whole cells, n = 3 biological variables in each experimental group. C: ELISA of tau concentrations in the endosomal lumen, n = 3 biological variables in each group. D: ELISA of total tau concentrations in the cell culture medium, n = 6 biological variables in the CON and KET1 groups, n = 5 biological variables in KET2 and KET3 groups. E: LDH concentration in the cell culture medium, n = 6 biological variables in each group. One-way ANOVA was applied to analyze data in panels B–E. Dunnett’s test was applied for ANOVA post-hoc. SH-SY5Y cells were treated with gradient concentrations of ketamine medium. Grubbs’ test was applied to identify outliers in data in KET2 and KET3 groups in panel C. Error bars represent SD; * indicates P \u0026lt; 0.05 by statistical analysis.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/7607fa80124eada7e39fa50e.png"},{"id":18372938,"identity":"dc130956-4838-4446-9253-387c42974541","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79553,"visible":true,"origin":"","legend":"\u003cp\u003eKetamine increases endosome accumulation in SH-SY5Y cells. A: Effects of ketamine on whole cell proteins (EEA1, Rab5, CD63, Rab7, LAMP2) and the internal loading control GAPDH. One band is shown for each condition. B–F: Quantitation of western blot analysis of whole cell EEA1 (B), Rab5 (C), CD63 (D), Rab7 (E), and LAMP2 (F) proteins. n = 5 biological variables for panels B – E, n = 3 biological variables for panel F in each group. One-way ANOVA and post-hoc analysis with Dunnett’s test were applied for statistical analysis. Error bars represent SD; * indicates P \u0026lt; 0.05 by statistical analysis; ns indicates no significant difference between groups (P \u0026gt; 0.05) by statistical analysis.\u003c/p\u003e","description":"","filename":"fig3n.png","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/89ff5c9601211ce8b9663554.png"},{"id":18372941,"identity":"ae87e1a9-4663-42fd-ab16-ba6847b5be19","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKetamine decreases endosome membrane-bound Rab5 and Rab7 but increases cytoplasmic amounts of Rab5 and Rab7. A\u003c/strong\u003e: Effects of ketamine on whole cell Na-K ATPase and the internal loading control GAPDH. One band is shown for each experimental condition. \u003cstrong\u003eB\u003c/strong\u003e: Quantitation of western blot analysis of whole cell Na-K ATPase, n = 5 biological variables. \u003cstrong\u003eC\u003c/strong\u003e: Western blot analysis of cytoplasmic and membrane proteins Rab5 and Rab7 and internal loading control proteins Na-K ATPase and GAPDH. One band is shown for each experimental condition. \u003cstrong\u003eD–G\u003c/strong\u003e: Quantitation of western blot analysis of cytoplasmic Rab5 (\u003cstrong\u003eD\u003c/strong\u003e), cytoplasmic Rab7 (\u003cstrong\u003eE\u003c/strong\u003e), membrane Rab5 (\u003cstrong\u003eF\u003c/strong\u003e), and membrane Rab7 (\u003cstrong\u003eG\u003c/strong\u003e), n = 5 biological variables in each group. One-way ANOVA and post-hoc analysis with Dunnett’s test were applied to analyze data in panels D–G. Error bars represent SD; * indicates P \u0026lt; 0.05 by statistical analysis; ns indicates no significant difference among groups (P \u0026gt; 0.05) by statistical analysis.\u003c/p\u003e","description":"","filename":"fig4n.png","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/03d490a9fea19fabe96b3301.png"},{"id":18373011,"identity":"4291d8f3-4727-4910-9d97-c7ee9fc6f11e","added_by":"auto","created_at":"2022-02-18 17:08:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27240,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral test timeline.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/40d3d86d0399481c51cd42ee.png"},{"id":18474864,"identity":"9075101c-3a8a-4129-87d2-54b26c75a85d","added_by":"auto","created_at":"2022-02-22 11:59:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":809784,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/0d6dad36-f1f9-418a-ac83-c836dcb548d1.pdf"},{"id":18373012,"identity":"84633db8-0034-434e-823f-f45887d549cd","added_by":"auto","created_at":"2022-02-18 17:08:55","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1051512,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1. Mouse weight and behavior test baseline data. \u003c/p\u003e","description":"","filename":"figS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/d7c7a627f267a3166dd2d499.tif"},{"id":18372945,"identity":"03337c6d-846e-4eec-ab34-178e69c484c4","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":43172456,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2. Results of behavioral tests 1 and 2 h after single ketamine injection.\u0026nbsp;\u003c/p\u003e","description":"","filename":"figS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/6d8a6abc4f45beefa61c4a31.tif"},{"id":18372942,"identity":"7de9ddc1-f5e3-4ea5-85f1-6b6491b078f6","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9147268,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3. MTT assay results for KET incubation statistical plots.\u003c/p\u003e","description":"","filename":"figS3KET.tif","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/77086276cc803e6811c219a9.tif"},{"id":18372943,"identity":"6a5ce1c0-21d7-4e5e-bb40-8f720e4126e0","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1288042,"visible":true,"origin":"","legend":"","description":"","filename":"SourceDataFig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/3de8295b6df364f711186a49.pdf"},{"id":18373013,"identity":"1e0707d3-ba70-42ed-bab5-bdc0f2d007c7","added_by":"auto","created_at":"2022-02-18 17:08:55","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5284217,"visible":true,"origin":"","legend":"","description":"","filename":"SourceDataFig3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/9572931237960844199ed3c8.pdf"},{"id":18372944,"identity":"6e279c52-379a-4852-8885-564e8991fa17","added_by":"auto","created_at":"2022-02-18 17:05:55","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4507923,"visible":true,"origin":"","legend":"","description":"","filename":"SourceDataFig4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1362130/v1/8909f3582b3f6855c3ff9512.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ketamine induces delirium-like behavior and interferes with endosomal tau trafficking","fulltext":[{"header":"Background","content":"\u003cp\u003eKetamine is a phencyclidine analogue and a non-competitive antagonist of the N-methyl-D-aspartic acid (NMDA) receptor (Sinner and Graf, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It is a commonly used clinical anesthetic and can also be used to treat depression (Jansen, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sassano-Higgins et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Ketamine also is a recreational drug owing to its ability to induce visual hallucinations in subanesthetic doses (Jansen, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Morgan and Curran, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sassano-Higgins et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Siegel, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1978\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eKetamine induces cognitive impairment (Zhang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with acute effects including confusion, impaired judgment, and delirium in humans (Sassano-Higgins et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Smith, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, whether ketamine can induce delirium-like behavior in rodents remains unknown.\u003c/p\u003e \u003cp\u003eDelirium is a syndrome of acute brain dysfunction potentially associated with anesthesia, surgery, pain, disease, or medication, manifesting with impaired consciousness, disorganized thinking, lack of purpose, and inability to focus (Setters and Solberg, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Previous studies (Witlox et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) have demonstrated an association between cerebrospinal fluid microtubule-associated protein τ (MAPT or tau) and amyloid β ratio with postoperative delirium in patients. Tau, a microtubule-associated protein, is expressed mainly in neurons and has a role in stabilizing the cytoskeleton, maintaining anchoring of membrane material, and participating in axonal transport (Martin et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Tauopathy is a hallmark of Alzheimer\u0026rsquo;s disease neuropathogenesis (Querfurth and LaFerla, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and contributes to cognitive impairment. However, it is unknown whether ketamine can induce tauopathy.\u003c/p\u003e \u003cp\u003eA recent study showed that anesthetic sevoflurane can induce tau trafficking from neurons to microglia (Dong et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Specifically, sevoflurane causes tau and phosphorylated tau to exit from neurons via a tau phosphorylation-associated mechanism. However, the pathways responsible for tau trafficking inside cells before its transition to the extracellular space remain largely unknown.\u003c/p\u003e \u003cp\u003eEndosomes are vesicle organelles of the endomembrane system of eukaryotic cells. They originate from the trans-Golgi network (TGN) and have extensive bidirectional transport relationships with the TGN, cell membrane, and lysosome, which are responsible for sorting, transport, and degradation of intracellular cargo (Langemeyer et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Stoorvogel et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Yang, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Endosomes are classified into three main categories\u0026mdash;early endosomes (EEs), late endosomes (LEs) and recycling endosomes\u0026mdash;based on the order of endocytic cargo transport to various endosomal vesicles (Futter et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Stenmark, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The three types of endosomes are defined mainly based on their electron microscopic morphology, the cargo transported within the lumen, and surface markers (Thery et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Surface markers of EEs are EEA1 and Rab5. EEs can endo-emerge to form intralumenal vesicles and gradually transform into multivesicular bodies (i.e., LEs) (Neefjes et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Maturation of EEs is demonstrated by increased binding of Rab5 on the endosome membrane (Delevoye and Goud, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nagano et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). LEs are mainly responsible for degradation in the endosomal system, and their surface markers are CD63 and Rab7. Similarly, LE maturation is demonstrated by increased binding of Rab7 on the endosome membrane (Langemeyer et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effects of ketamine on delirium-like behavior and changes in tau amounts in mouse serum. Mechanistically, we assessed the effects of ketamine on the number and maturation of EEs and LEs, intracellular transport, and extracellular release of tau in the SH-SY5Y cell line. The hypothesis of the present study was that ketamine causes delirium-like behavior and increases endosomal uptake and extracellular release of tau.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eKetamine induces delirium-like behavior and increases serum tau in mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe used our established animal model, consisting of the Y-maze test (YMT), buried food test (BFT), and open field test (OFT) (Liufu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), to study the effects of ketamine on delirium-like behavior in mice (\u003cb\u003eSupplemental Figure S1\u003c/b\u003e). A single injection of ketamine in mice significantly increased the latency to eat food in the BFT and freezing time in the OFT compared to control mice at 1, but not 2, h after ketamine administration (\u003cb\u003eSupplemental Figure S2\u003c/b\u003e). Composite Z-scores were obtained by pooling data from six measurements. The composite Z-score increased in the ketamine-treated mice at 1, but not 2, h after ketamine treatment compared to control mice [KET: 7.899 (10.03\u0026ndash;6.927) versus CON: 4.657 (6.371\u0026ndash;3.365), P\u0026thinsp;=\u0026thinsp;0.0005, Mann-Whitney test; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B]. These data suggest that ketamine may induce delirium-like behaviors in mice. Blood was collected from the mice 1, 2, and 6 h after administration of a single injection of ketamine, and serum was extracted. We found increased amounts of tau in the serum of mice at 1, but not 2 or 6, h after ketamine administration compared to control mice (KET: 90.36 pg/mL\u0026thinsp;\u0026plusmn;\u0026thinsp;47.41 pg/mL versus CON: 53.81 pg/mL\u0026thinsp;\u0026plusmn;\u0026thinsp;11.64 pg/mL, P\u0026thinsp;=\u0026thinsp;0.0232, Student\u0026rsquo;s t-test; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKetamine increases tau protein concentration in the endosome lumen and culture medium of SH-SY5Y cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eKetamine decreased viability of cultured SH-SY5Y cells in a dose-dependent manner (F\u0026thinsp;=\u0026thinsp;155.7, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, one-way ANOVA; \u003cb\u003eSupplemental Figure S3\u003c/b\u003e). Hence, we chose 156.3 \u0026micro;g/mL ketamine (KET1), 312.5 \u0026micro;g/mL ketamine (KET2), 625 \u0026micro;g/mL ketamine (KET3), and a control condition (CON) to treat SH-SY5Y cells. We specifically assessed the effects of ketamine on the amount of tau inside the endosome lumen and outside of the SH-SY5Y cells. Ketamine increased the amounts of tau in whole cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,B). ELISA showed that ketamine also increased the amounts of tau in the endosomal lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and the cell culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, a lactate dehydrogenase (LDH) assay showed that treatment with ketamine did not increase LDH amounts in the cell culture medium (F\u0026thinsp;=\u0026thinsp;1.015, P\u0026thinsp;=\u0026thinsp;0.4068, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), indicating that the ketamine-induced increase in tau in the cell culture medium was not due to rupture of cell membranes. Taken together, these data indicate that ketamine increases endosomal uptake and extracellular release of tau.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKetamine increases early and late endosome accumulation in SH-SY5Y cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that ketamine increased tau concentration in both the endosome and extracellular space, next we assessed the effects of ketamine on endosome amounts and activation in SH-SY5Y cells. Quantitative western blot analysis demonstrated that after 24 h of incubation, whole cell protein amounts of EE markers EEA1 (F\u0026thinsp;=\u0026thinsp;36.48, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,B) and Rab5 (F\u0026thinsp;=\u0026thinsp;31.23, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,C) were significantly increased, suggesting that ketamine increased amounts of EEs in the cells. Moreover, ketamine increased whole cell protein amounts of LE marker Rab7 (F\u0026thinsp;=\u0026thinsp;32.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,E) but not CD63 (F\u0026thinsp;=\u0026thinsp;2.358, P\u0026thinsp;=\u0026thinsp;0.1102, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,D). In addition, ketamine decreased whole cell protein amounts of lysosome marker LAMP2 (F\u0026thinsp;=\u0026thinsp;4.892, P\u0026thinsp;=\u0026thinsp;0.0323, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA,F). These data demonstrate that ketamine increased accumulation of EEs and LEs and decreased lysosome amounts in SH-SY5Y cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKetamine decreases endosome membrane-bound Rab5 and Rab7 but increases Rab5 and Rab7 in the cytoplasm of SH-SY5Y cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that ketamine increased the amounts of proteins associated with EEs in whole cells, we further assessed the effects of ketamine on the amounts of these proteins bound with the endosome membrane or in the cytoplasm. First, we demonstrated that ketamine did not significantly change the amounts of Na-K ATPase (F\u0026thinsp;=\u0026thinsp;1.483, P\u0026thinsp;=\u0026thinsp;0.2569, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B), indicating that Na-K ATPase could be used as an internal loading control for the amounts of protein associated with endosome membranes. Quantitative western blot analysis demonstrated that ketamine significantly increased the amounts of cytoplasmic Rab5 (F\u0026thinsp;=\u0026thinsp;11.15, P\u0026thinsp;=\u0026thinsp;0.0003, KET3: 237.2% \u0026plusmn; 84.99% versus CON: 100% \u0026plusmn; 16.32%, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D) and Rab7 (F\u0026thinsp;=\u0026thinsp;9.200, P\u0026thinsp;=\u0026thinsp;0.0009, KET3: 277.6% \u0026plusmn; 113.5% versus CON: 100% \u0026plusmn; 26.04%, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,E) 24 h after ketamine administration. Conversely, ketamine treatment significantly decreased the amounts of membrane-bound Rab5 (F\u0026thinsp;=\u0026thinsp;7.410, P\u0026thinsp;=\u0026thinsp;0.0025, KET1: 69.00% \u0026plusmn; 26.86% and KET3: 55.86% \u0026plusmn; 6.633% versus CON: 100% \u0026plusmn; 7.792%, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,F) and Rab7 (F\u0026thinsp;=\u0026thinsp;8.174, P\u0026thinsp;=\u0026thinsp;0.0016, KET2: 55.23% \u0026plusmn; 16.50% and KET3: 28.56% \u0026plusmn; 11.05% versus CON: 100% \u0026plusmn; 41.50%, one-way ANOVA; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,G). These data demonstrate that ketamine could decrease endosome membrane-bound Rab5 and Rab7 but increase cytoplasmic amounts of Rab5 and Rab7, suggesting that ketamine may impair EE and LE maturation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results show that ketamine induced delirium-like behavior and increased serum tau amounts in mice. In mechanistic studies, ketamine impaired intracellular tau trafficking, evidenced by increasing amounts of tau inside the endosome lumen and extracellular space of SH-SY5Y cells. Moreover, ketamine might impair intracellular tau trafficking by increasing the number of endosomes but inhibiting endosome maturation. These data, using ketamine as a clinically relevant tool, demonstrate that tau could contribute to the neuropathogenesis of delirium, promoting future research to study delirium. These data also show that anesthetic ketamine could induce tauopathy and delirium-like behavior.\u003c/p\u003e \u003cp\u003eWe firstly found time-dependent effects of ketamine in inducing delirium-like behavior, evidenced by the findings that ketamine increased composite Z-score compared to controls. Consistently, previous studies show anesthesia/surgery induces time-dependent (Peng et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and age-dependent (Liufu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) delirium-like behavior in mice. However, previous studies did not assess the effects of anesthetic without surgery on the delirium-like behavior.\u003c/p\u003e \u003cp\u003eThe present study shows that anesthetic ketamine without surgery could still induce delirium-like behavior in mice. Ketamine induced delirium-like behavior and increased serum tau amounts 1 h after administration, showing an association between delirium-like behavior and increased serum tau. Consistently, a clinical observational study in 2021 concluded that postoperative plasma tau amounts could serve as a biomarker for postoperative delirium in patients (Ballweg et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndosomes are responsible for intracellular transport of cargo, including proteins, lipids, and nucleic acids, which are important for cell function (Elkin et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Cell function is severely impaired when endosomes accumulate (Langemeyer et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We found that ketamine increased endosome accumulation, which could explain the findings that ketamine increased endosomal tau amounts because more endosomes could uptake more tau. However, ketamine may also enhance the ability of single endosomes to uptake tau. We will test this hypothesis in future studies.\u003c/p\u003e \u003cp\u003ePrevious studies show that increased amounts of Rab5 or Rab7 in the cytoplasm and decreased amounts of membrane-bound Rab5 or Rab7 indicate inhibition of endosome maturation (Delevoye and Goud, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nagano et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Endosomes becomes exosomes when they bind to the cell membrane (Bebelman et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, it has been suggested that inhibition of the endosome maturation process will increase protein release via exosomes (Hessvik et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the present study, ketamine inhibited endosome maturation, evidenced by increased cytoplasm amounts of Rab5 and Rab7 and decreased membrane-bound Rab5 and Rab7. Meanwhile, ketamine also increased amounts of tau in the extracellular space. Together, these findings indicate that ketamine inhibits endosome maturation while increasing tau uptake by endosomes, causing migration of endosomes to the cell membrane to form exosomes and releasing more tau into the extracellular space.\u003c/p\u003e \u003cp\u003eThe mechanism of tau release into the extracellular space is unclear, but increasing evidence shows that tau relies mainly on the non-classical protein secretion pathway and extracellular vesicles for extracellular transport (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jiang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our previous study also demonstrates that trafficking of tau to the extracellular space is dependent on tau phosphorylation and generation of extracellular vesicles (Dong et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, we illustrated that ketamine increases endosome accumulation but inhibits endosome maturation, leading to more release of tau into the extracellular space. Moreover, we postulate that the effect of ketamine on EEs includes blocking conversion of EEs to LEs but does not affect conversion of other organelles, such as TGN, to EEs.\u003c/p\u003e \u003cp\u003eIn LEs, we found that ketamine did not significantly change CD63 protein amounts but increased Rab7 amounts. Further, ketamine decreased amounts of membrane Rab7 yet increased cytoplasmic amounts of Rab7. These data suggest that ketamine inhibits LE maturation, as demonstrated in previous studies (Takeda et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zerial and McBride, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are several limitations of this study. First, we did not study whether inhibition of the ketamine-induced increase in serum tau could mitigate ketamine-induced delirium-like behavior because we could not find a specific inhibitor of tau trafficking from the intracellular to extracellular space. We will use tau knockout mice to determine the role of tau in delirium-like behavior using the established system in future studies. Second, we did not determine the effects of ketamine on intracellular trafficking of phosphorylated tau in the present study because we wanted to focus on tau. We also will use the established system to study intracellular trafficking of both tau and phosphorylated tau in future studies. Finally, we only assessed the effects of ketamine on membrane Rab5 and Rab7 but not the specific endosome membrane Rab5 and Rab7. However, Rab5 and Rab7 are only known to bind to the endosome membrane.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study shows that ketamine induces delirium-like behavior and increases serum tau in mice. In the in vitro studies, we found that ketamine increases tau uptake in endosomes by increasing endosome accumulation. Ketamine then increases release of tau to the extracellular space by inhibiting endosomal maturation. These findings will promote future studies of tau trafficking and delirium and the effects of anesthetic on tauopathy and delirium-like behavior.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and reagents\u003c/h2\u003e \u003cp\u003eFull details of the primary antibodies used are in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eAntibody information\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntigen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHost species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eManufacturer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCatalog No.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab216230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEEA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48453S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5174S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLAMP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab199947\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa-K ATPase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:20000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab76020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRab5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMouse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCell Signaling Technology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46449S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRab7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab126712\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal tau\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbcam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eab32057\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMouse (secondary antibody)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSigma-Aldrich\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSAB3701095\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRabbit\u003c/p\u003e \u003cp\u003e(secondary antibody)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGoat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:10000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInvitrogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG-21234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePrimary antibodies were diluted with western blot blocking buffer (see western blot subsection for details), and secondary antibodies were diluted with 1\u0026times;TBST.\u003c/p\u003e \u003cp\u003eKetamine injection (Ketalar) was purchased from Hikma Pharmaceuticals PLC 2019.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eA total of 48 8-week-old na\u0026iuml;ve adult male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) with an average weight of 23 g. No significant difference in mouse weight was found between groups at the beginning of the study (\u003cb\u003eFig. S1 A1,A2\u003c/b\u003e). Mice were housed four per cage and maintained on a 12-h light/dark cycle (lights out at 18:00). Mice had unlimited access to water and food in their home cages. Sample size was decided by a previous pilot experiment and power analysis (β\u0026thinsp;=\u0026thinsp;0.1). Mice were equally divided into two post-injection duration groups, a 1 h group and 2 h\u0026thinsp;+\u0026thinsp;group, with 24 mice in each group using the complete randomization method. Each post-injection duration group was divided equally into two treatment subgroups, a saline control group (CON) and a ketamine group (KET), using the complete randomization method, with 12 mice in each subgroup. Each mouse was injected with 2 \u0026micro;l/g i.p. of injection according to body weight. The dosage of KET group was 40 mg/kg. Within each subgroup of the 2 h\u0026thinsp;+\u0026thinsp;group, mice were divided into two additional subgroups using the complete randomization method, and tissue was harvested at 2 h and 6 h post-injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests\u003c/h2\u003e \u003cp\u003e All mice had multiple behavioral tests starting with the YMT training trail followed by OFT, BFT, and finally the YMT retention trail at 24 h before (baseline) injection and 1 h or 2 h after injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We performed behavior tests in groups of four mice and finished tests within 50 min, mimicking certain features of clinical evaluation of delirium in patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eY maze test\u003c/b\u003e. The YMT was performed as described previously (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rayatnia et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) with modifications. Specifically, the Y maze, made of gray polyvinylene, was placed in a quiet and illuminated room. Each maze consisted of three arms (8 cm wide \u0026times; 30 cm long \u0026times; 15 cm tall), with an angle of 120\u0026ordm; between each arm. The three arms included the start arm, in which the mouse starts to explore (always open); the novel arm, which is blocked at the first trial but opened at the second trial; and another familiar arm (always open). In the experiment, the start arm and the familiar arm were designed randomly to avoid spatial memory error. The YMT consisted of two trials separated by an inter-trial interval. The first trial (training) was 10 min in duration, which allowed the mouse to explore two arms (start arm and familiar arm) of the maze, with the novel arm blocked. After a 1 h inter-trial interval, the second trial (retention) was conducted. For the second trial, the mouse was placed back in the maze in the same start arm with free access to all three arms for 5 min. A video camera linked to AnyMaze (Stoelting Co.) animal tracking system software was installed 60 cm above the chamber to monitor and analyze the number of entries and the time spent in each arm. The time spent in and entries into the novel arms indicated spatial recognition memory (learned behavior). Each arm of the Y maze was cleaned with 70% ethanol solution between trials.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOpen field test\u003c/b\u003e. The OFT was performed as described previously (Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) with modifications. Specifically, the mouse was gently placed in the center of an open field chamber (40 cm \u0026times; 40 cm \u0026times; 40 cm) under dim light and was allowed to move freely for 5 min. Movement parameters of the mouse were monitored and analyzed via a video camera connected to the AnyMaze animal tracking system software. The total distance moved (meters), time (seconds) spent in the center of the open field, freezing time (seconds), and latency (time in seconds for mouse to reach location of the first attempt) to the center of the open field were recorded and analyzed. The floor of the open field was cleaned with 70% ethanol solution between each test.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBuried food test\u003c/b\u003e. The BFT was performed as described in previous studies (Lehmkuhl et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nathan et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) with modifications. Specifically, two days before the test, we gave each mouse two pieces of sweetened cereal. On all test days, we habituated the mice for one hour prior to the test by placing the home cage with mice in the testing room. The test cage was prepared with clean bedding (3-cm high). We buried one sunflower seed 0.5 cm below the surface of the bedding so that it was not visible. The location of the food pellet was changed every time in a random fashion. We placed the mouse in the center of the test cage and measured the latency of the mouse to eat the food. Latency was defined as the time from when the mouse was placed in the test cage until the mouse uncovered the food pellet and grasped it in their forepaws and/or teeth. Mice were allowed to consume the pellet they found and were then returned to their home cage. Observation time was 5 min. If the mouse could not find the pellet within 5 min, the testing session ended and the latency was defined as 300 s for that mouse. We emptied the bedding from the test cage and cleaned the cage with 70% ethanol solution after each test to prevent transmission of olfactory cues. We changed gloves after each test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimal tissue harvesting and euthanasia\u003c/h2\u003e \u003cp\u003eMice were anesthetized with 5% isoflurane at 1, 2, and 6 h after KET or CON injection. Retro-orbital sinus blood sampling was performed, and then mice were euthanized by guillotine. Blood was transferred to a 4℃ refrigerator for 15 min and centrifuged at 4℃ for 15 min at 1,200 \u0026times; g. We then carefully extracted and transferred the supernatant as serum to a clean centrifuge tube. The serum was frozen in liquid nitrogen and stored in a -80℃ freezer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eSH-SY5Y cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in DMEM/F12 1:1 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich), 1% MEM non-essential amino acids solution (100X) (Gibco), and 1% sodium pyruvate (100 mM) (Gibco). When incubating with drugs, we replaced DMEM/F12 1:1 with DMEM/F12 1:1 with HEPES without phenol red and added 10% ketamine solution or saline and 0.1% DMSO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assays\u003c/h2\u003e \u003cp\u003eMTT was purchased from Invitrogen, and all MTT assays were performed following the instructions provided with the kit to obtain cell viability data. LDH assay kit (Abcam) was used for quantitative analysis of LDH concentration in the cell culture medium. Standard curves were plotted for each assay using positive control LDH provided in the kit with gradient concentration (0 ng/mL, 125 ng/mL, 250 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 4000 ng/mL, and 8000 ng/mL). All operations were performed according to the kit instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWhole cell and cell fraction protein extraction\u003c/h2\u003e \u003cp\u003e \u003cb\u003eWhole cell protein extraction.\u003c/b\u003e We obtained treated SH-SY5Y cells, discarded the medium in the culture dish, and rinsed gently with DPBS (Lonza) three times, shaking lightly each time. We then discarded as much residue as possible from the culture dish and added an appropriate amount of RIPA (Thermo Scientific) lysate with pre-dissolved Pierce protease and phosphatase inhibitor mini tablets (Thermo Scientific). After incubation, we scraped off all cells and lysate using a cell scraper and transferred them to a clean centrifuge tube. Tubes were shook in a vortex machine three times at 50% amplitude for 3 s with an interval of 3 s. The solution was frozen in liquid nitrogen and stored in a -80℃ freezer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytoplasmic protein and membrane protein extraction.\u003c/b\u003e We obtained treated SH-SY5Y cells, discarded the medium in the culture dish, and rinsed gently with DPBS three times, shaking lightly each time. We then discarded as much residue as possible from the culture dish and added an appropriate amount of M-PER (Thermo Scientific) lysate with pre-dissolved Pierce protease and phosphatase inhibitor mini tablets. We sealed the culture dish with plastic wrap and transferred it to a 4℃ refrigerated room for 30 min incubation on a shaker. After incubation, we scraped off all hanging cell debris and lysate using a cell scraper and transferred to a clean centrifuge tube. We centrifuged tubes at 16,000 \u0026times; g for 30 min at 4℃ and carefully extracted the upper clarified solution into a clean centrifuge tube. This solution contained cytoplasmic and nuclear proteins, and the pellet contained membrane protein. The membrane protein pellet was dissolved into solution using RIPA. The solution was frozen in liquid nitrogen and stored in a -80 ℃ freezer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEndosome isolation.\u003c/b\u003e Minute endosome isolation and cell fractionation kit (Invent Biotechnologies) was used to isolate endosomes as described previously (Thapa et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with modifications. Briefly, 4 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells were collected, and 1 ml buffer A was added. The cell suspension was incubated on ice for 15 min, loaded into a filter cartridge and centrifuged with 16,000 \u0026times; g for 30 s. A filter cartridge re-pass-through was performed to enrich the field. The supernatant was transferred to a fresh tube, and 500 \u0026micro;l buffer B was added to the tube and incubated at 4\u0026deg;C overnight. After incubation, vortex briefly and centrifuge with 10,000 \u0026times; g for 30 min. We removed the supernatant and washed the pellet with 750 \u0026micro;l 2:1 mixture of buffer A/buffer B. After centrifugation with 10,000 \u0026times; g for 30 min, the pellet containing endosomes was frozen in liquid nitrogen and stored in a -80℃ freezer. All the centrifugations should be performed at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eEndosome lumen proteins and endosome membrane proteins were extracted by a similar method as that used for cytoplasmic protein and membrane protein extraction.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eConcentrations of human total tau in the medium or protein lysate, and mouse total tau in serum were measured by ELISA kits (Invitrogen). Experiments were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein concentration\u003c/h2\u003e \u003cp\u003eProtein concentration of lysates was obtained using the Pierce BCA protein assay kit (Thermo Scientific). Standard curves were plotted for each assay using Pierce bovine serum albumin standard (Thermo Scientific) with gradient concentration (0 mg/mL, 0.025 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, and 0.5 mg/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eProteins were separated by NuPAGE 4\u0026ndash;12% Bis-Tris protein gel (Invitrogen) and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked in 50% SuperBlock (TBS) blocking buffer (Invitrogen), 5% bovine serum albumin (Sigma-Aldrich) in 1\u0026times; TBST (8 g/L NaCl, 2.42 g/L Tris-base, 1 mL/L Tween 20, adjusted pH to 7.6 with HCl). Immunoreactive bands were detected using a SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific). The signal intensity was analyzed using ChemiDoc XRS\u0026thinsp;+\u0026thinsp;with Image Lab 5.0 software (Bio-Rad, Hercules, CA). The quantification of western blot was performed in two steps. At the first step, we used β-Actin levels to standardize protein amounts and to limit the disparities in the protein quantity loaded. At the second step, we expressed the protein levels obtained from the anesthesia/surgery mice as a percentage in relation to the control condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eComposite Z-score\u003c/h2\u003e \u003cp\u003eWhen pooling the battery of behavioral test data, we transformed outcome data according to the composite Z-score introduced in previous studies (Moller et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), with some modifications. We first calculated the difference between the measured value after injection and the baseline value \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\varDelta X\\)\u003c/span\u003e\u003c/span\u003e for each mouse. The \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\varDelta X\\)\u003c/span\u003e\u003c/span\u003e from the CON group was then used to calculate mean \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({M}_{\\varDelta {X}_{CON}}\\)\u003c/span\u003e\u003c/span\u003e and SD \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({S}_{\\varDelta {X}_{CON}}\\)\u003c/span\u003e\u003c/span\u003e. The composite Z-score of each mouse was obtained by the formula \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({C}_{{Z}_{X}}=\\sum \\left|{Z}_{X}\\right|=\\sum \\left|\\frac{\\varDelta X-{M}_{\\varDelta {X}_{CON}}}{{S}_{\\varDelta {X}_{CON}}}\\right|\\)\u003c/span\u003e\u003c/span\u003e. Finally, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({C}_{{Z}_{X}}\\)\u003c/span\u003e\u003c/span\u003e was included in the statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Prism (GraphPad Software) to generate curves or bar graphs. All error bars represent standard deviation (SD). Data were first tested for conformity to a normal distribution using the Shapiro-Wilk test, and then tested for equality of variances using the F test. Two-tailed unpaired t test was used for statistical analysis of two groups of samples. One-way ANOVA with a Turkey\u0026rsquo;s honestly significant difference test was used to evaluate statistical significance of multiple groups of samples. Normally distributed data were described as \"mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\". Data that did not conform to the normal distribution were combined with the experimental records to exclude outliers using the Grubbs\u0026rsquo; test and then re-examined for normality and F tests. Data that still did not conform to the normal distribution were tested using the non-parametric independent samples Mann-Whitney exact probability test. Data were described using \"median [interquartile spacing]\". All tests were set at α\u0026thinsp;=\u0026thinsp;0.05 and β\u0026thinsp;=\u0026thinsp;0.1.\u003c/p\u003e \u003c/div\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eBFT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;buried food test\u003c/p\u003e\n\u003cp\u003eCON\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;control group\u003c/p\u003e\n\u003cp\u003eEE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;early endosome\u003c/p\u003e\n\u003cp\u003eKET\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;ketamine group\u003c/p\u003e\n\u003cp\u003eLDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eLE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;late endosome\u003c/p\u003e\n\u003cp\u003eOFT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;open field test\u003c/p\u003e\n\u003cp\u003eTGN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;trans-Golgi network\u003c/p\u003e\n\u003cp\u003eTau\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;microtubule-associated protein\u0026nbsp;\u0026tau;\u003c/p\u003e\n\u003cp\u003eYMT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Y-maze test\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Approval and Consent to participate\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving mice were approved by the Standing Committee on Animals at Massachusetts General Hospital, Boston, MA (protocol number: 2006N000219) and conformed to National Institutes of Health (Bethesda, MD) guidelines. This article was written according to applicable ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Efforts were made to minimize the number of mice used in the studies.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the National Institutes of Health (grant No. R01AG062509 to Zhongcong Xie).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eX\u003c/strong\u003e\u003cstrong\u003einghua R\u003c/strong\u003e\u003cstrong\u003een\u003c/strong\u003e: conceptualization, data curation,\u0026nbsp;formal analysis, investigation, methodology, validation, visualization, writing of original manuscript draft; \u003cstrong\u003eSiyi Zhang\u003c/strong\u003e: data curation,\u0026nbsp;investigation, methodology, validation, writing of original manuscript draft; \u003cstrong\u003eYongyan Yang\u003c/strong\u003e: formal analysis, investigation, methodology, resources; \u003cstrong\u003eAnnie Song\u003c/strong\u003e: investigation; \u003cstrong\u003eFeng Liang\u003c/strong\u003e: conceptualization, investigation, methodology, reviewing and editing manuscript; \u003cstrong\u003eYiying Zhang\u003c/strong\u003e: investigation, methodology, resources, reviewing and editing manuscript; \u003cstrong\u003eYuanlin Dong\u003c/strong\u003e: conceptualization, investigation, methodology, project administration, resources, reviewing and editing manuscript; \u003cstrong\u003eXu Wu\u003c/strong\u003e: conceptualization, methodology, project administration, resources, supervision, reviewing and editing manuscript; \u003cstrong\u003eZhongcong Xie\u003c/strong\u003e: conceptualization, funding acquisition, methodology, project administration, resources, supervision, reviewing and editing manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBallweg, T., M. White, M. Parker, C. Casey, A. Bo, Z. Farahbakhsh, A. Kayser, A. Blair, H. Lindroth, R.A. Pearce, K. Blennow, H. Zetterberg, R. Lennertz, and R.D. Sanders. 2021. Association between plasma tau and postoperative delirium incidence and severity: a prospective observational study. Br J Anaesth. 126:458\u0026ndash;466.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBebelman, M.P., C. Crudden, D.M. Pegtel, and M.J. Smit. 2020. The convergence of extracellular vesicle and gpcr biology. Trends Pharmacol Sci. 41:627\u0026ndash;640.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J.J., D.L. Nathaniel, P. Raghavan, M. Nelson, R. Tian, E. Tse, J.Y. Hong, S.K. See, S.A. Mok, M.Y. Hein, D.R. Southworth, L.T. Grinberg, J.E. Gestwicki, M.D. Leonetti, and M. Kampmann. 2019. Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation. J Biol Chem. 294:18952\u0026ndash;18966.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Y., X. Liu, X. Jia, W. Zong, Y. Ma, F. Xu, and J. Wang. 2014. Anxiety- and depressive-like behaviors in olfactory deficient Cnga2 knockout mice. Behav Brain Res. 275:219\u0026ndash;224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelevoye, C., and B. Goud. 2015. Rab GTPases and kinesin motors in endosomal trafficking. Methods Cell Biol. 130:235\u0026ndash;246.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong, Y., F. Liang, L. Huang, F. Fang, G. Yang, R.E. Tanzi, Y. Zhang, Q. Quan, and Z. Xie. 2021. The anesthetic sevoflurane induces tau trafficking from neurons to microglia. Commun Biol. 4:560.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElkin, S.R., A.M. Lakoduk, and S.L. Schmid. 2016. Endocytic pathways and endosomal trafficking: a primer. Wien Med Wochenschr. 166:196\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFutter, C.E., A. Pearse, L.J. Hewlett, and C.R. Hopkins. 1996. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J Cell Biol. 132:1011\u0026ndash;1023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHessvik, N.P., A. Overbye, A. Brech, M.L. Torgersen, I.S. Jakobsen, K. Sandvig, and A. Llorente. 2016. PIKfyve inhibition increases exosome release and induces secretory autophagy. Cell Mol Life Sci. 73:4717\u0026ndash;4737.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJansen, K.L. 2000. A review of the nonmedical use of ketamine: use, users and consequences. J Psychoactive Drugs. 32:419\u0026ndash;433.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, L., H. Dong, H. Cao, X. Ji, S. Luan, and J. Liu. 2019. Exosomes in pathogenesis, diagnosis, and treatment of Alzheimer's disease. Med Sci Monit. 25:3329\u0026ndash;3335.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangemeyer, L., F. Fr\u0026ouml;hlich, and C. Ungermann. 2018. Rab GTPase function in endosome and lysosome biogenesis. Trends Cell Biol. 28:957\u0026ndash;970.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehmkuhl, A.M., E.R. Dirr, and S.M. Fleming. 2014. Olfactory assays for mouse models of neurodegenerative disease. J Vis Exp:e51804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, X.M., F. Su, M.H. Ji, G.F. Zhang, L.L. Qiu, M. Jia, J. Gao, Z. Xie, and J.J. Yang. 2014. Disruption of hippocampal neuregulin 1-ErbB4 signaling contributes to the hippocampus-dependent cognitive impairment induced by isoflurane in aged mice. Anesthesiology. 121:79\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y., R. Shen, G. Wen, R. Ding, A. Du, J. Zhou, Z. Dong, X. Ren, H. Yao, R. Zhao, G. Zhang, Y. Lu, and X. Wu. 2017. Effects of ketamine on levels of inflammatory cytokines IL-6, IL-1beta, and TNF-alpha in the hippocampus of mice following acute or chronic administration. Front Pharmacol. 8:139.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiufu, N., L. Liu, S. Shen, Z. Jiang, Y. Dong, Y. Wang, D. Culley, G. Crosby, M. Cao, Y. Shen, E. Marcantonio, Z. Xie, and Y. Zhang. 2020. Anesthesia and surgery induce age-dependent changes in behaviors and microbiota. Aging (Albany NY). 12:1965\u0026ndash;1986.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin, L., X. Latypova, C.M. Wilson, A. Magnaudeix, M.L. Perrin, C. Yardin, and F. Terro. 2013. Tau protein kinases: involvement in Alzheimer's disease. Ageing Res Rev. 12:289\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoller, J.T., P. Cluitmans, L.S. Rasmussen, P. Houx, H. Rasmussen, J. Canet, P. Rabbitt, J. Jolles, K. Larsen, C.D. Hanning, O. Langeron, T. Johnson, P.M. Lauven, P.A. Kristensen, A. Biedler, H. van Beem, O. Fraidakis, J.H. Silverstein, J.E. Beneken, and J.S. Gravenstein. 1998. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet. 351:857\u0026ndash;861.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorgan, C.J., and H.V. Curran. 2012. Ketamine use: a review. Addiction. 107:27\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagano, M., J.Y. Toshima, D.E. Siekhaus, and J. Toshima. 2019. Rab5-mediated endosome formation is regulated at the trans-Golgi network. Commun Biol. 2:419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNathan, B.P., J. Yost, M.T. Litherland, R.G. Struble, and P.V. Switzer. 2004. Olfactory function in apoE knockout mice. Behav Brain Res. 150:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeefjes, J., M.M.L. Jongsma, and I. Berlin. 2017. Stop or go? Endosome positioning in the establishment of compartment architecture, dynamics, and function. Trends Cell Biol. 27:580\u0026ndash;594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng, M., C. Zhang, Y. Dong, Y. Zhang, H. Nakazawa, M. Kaneki, H. Zheng, Y. Shen, E.R. Marcantonio, and Z. Xie. 2016. Battery of behavioral tests in mice to study postoperative delirium. Sci Rep. 6:29874.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuerfurth, H.W., and F.M. LaFerla. 2010. Alzheimer's disease. N Engl J Med. 362:329\u0026ndash;344.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRayatnia, F., M. Javadi-Paydar, N. Allami, M. Zakeri, H. Rastegar, A. Norouzi, and A.R. Dehpour. 2011. Nitric oxide involvement in consolidation, but not retrieval phase of cognitive performance enhanced by atorvastatin in mice. Eur J Pharmacol. 666:122\u0026ndash;130.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSassano-Higgins, S., D. Baron, G. Juarez, N. Esmaili, and M. Gold. 2016. A review of ketamine abuse and diversion. Depress Anxiety. 33:718\u0026ndash;727.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSetters, B., and L.M. Solberg. 2017. Delirium. \u003cem\u003ePrim Care\u003c/em\u003e. 44:541\u0026ndash;559.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel, R.K. 1978. Phencyclidine and ketamine intoxication: a study of four populations of recreational users. NIDA Res Monogr:119\u0026ndash;147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinner, B., and B.M. Graf. 2008. Ketamine. \u003cem\u003eHandb Exp Pharmacol\u003c/em\u003e.:313\u0026ndash;333.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, K.M. 1999. Drugs used in acquaintance rape. J Am Pharm Assoc (Wash). 39:519\u0026ndash;525; quiz 581 \u0026ndash; 513.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStenmark, H. 2009. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol. 10:513\u0026ndash;525.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStoorvogel, W., G.J. Strous, H.J. Geuze, V. Oorschot, and A.L. Schwartz. 1991. Late endosomes derive from early endosomes by maturation. Cell. 65:417\u0026ndash;427.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeda, M., J. Koseki, H. Takahashi, N. Miyoshi, N. Nishida, J. Nishimura, T. Hata, C. Matsuda, T. Mizushima, H. Yamamoto, H. Ishii, Y. Doki, M. Mori, and N. Haraguchi. 2019. Disruption of endolysosomal RAB5/7 efficiently eliminates colorectal cancer stem cells. Cancer Res. 79:1426\u0026ndash;1437.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThapa, N., M. Chen, H.T. Horn, S. Choi, T. Wen, and R.A. Anderson. 2020. Phosphatidylinositol-3-OH kinase signalling is spatially organized at endosomal compartments by microtubule-associated protein 4. Nat Cell Biol. 22:1357\u0026ndash;1370.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThery, C., K.W. Witwer, E. Aikawa, M.J. Alcaraz, J.D. Anderson, R. Andriantsitohaina, A. Antoniou, T. Arab, F. Archer, G.K. Atkin-Smith, D.C. Ayre, J.M. Bach, D. Bachurski, H. Baharvand, L. Balaj, S. Baldacchino, N.N. Bauer, A.A. Baxter, M. Bebawy, C. Beckham, A. Bedina Zavec, A. Benmoussa, A.C. Berardi, P. Bergese, E. Bielska, C. Blenkiron, S. Bobis-Wozowicz, E. Boilard, W. Boireau, A. Bongiovanni, F.E. Borras, S. Bosch, C.M. Boulanger, X. Breakefield, A.M. Breglio, M.A. Brennan, D.R. Brigstock, A. Brisson, M.L. Broekman, J.F. Bromberg, P. Bryl-Gorecka, S. Buch, A.H. Buck, D. Burger, S. Busatto, D. Buschmann, B. Bussolati, E.I. Buzas, J.B. Byrd, G. Camussi, D.R. Carter, S. Caruso, L.W. Chamley, Y.T. Chang, C. Chen, S. Chen, L. Cheng, A.R. Chin, A. Clayton, S.P. Clerici, A. Cocks, E. Cocucci, R.J. Coffey, A. Cordeiro-da-Silva, Y. Couch, F.A. Coumans, B. Coyle, R. Crescitelli, M.F. Criado, C. D'Souza-Schorey, S. Das, A. Datta Chaudhuri, P. de Candia, E.F. De Santana, O. De Wever, H.A. Del Portillo, T. Demaret, S. Deville, A. Devitt, B. Dhondt, D. Di Vizio, L.C. Dieterich, V. Dolo, A.P. Dominguez Rubio, M. Dominici, M.R. Dourado, T.A. Driedonks, F.V. Duarte, H.M. Duncan, R.M. Eichenberger, K. Ekstrom, S. El Andaloussi, C. Elie-Caille, U. Erdbrugger, J.M. Falcon-Perez, F. Fatima, J.E. Fish, M. Flores-Bellver, A. Forsonits, A. Frelet-Barrand, et al. 2018. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 7:1535750.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWitlox, J., K.J. Kalisvaart, J.F. de Jonghe, N.A. Verwey, M.F. van Stijn, A.P. Houdijk, H.S. Traast, A.M. MacLullich, W.A. van Gool, and P. Eikelenboom. 2011. Cerebrospinal fluid beta-amyloid and tau are not associated with risk of delirium: a prospective cohort study in older adults with hip fracture. J Am Geriatr Soc. 59:1260\u0026ndash;1267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie, Z., C.A. Swain, S.A. Ward, H. Zheng, Y. Dong, N. Sunder, D.W. Burke, D. Escobar, Y. Zhang, and E.R. Marcantonio. 2014. Preoperative cerebrospinal fluid beta-Amyloid/Tau ratio and postoperative delirium. Ann Clin Transl Neurol. 1:319\u0026ndash;328.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, X., J. Xu, J. Wu, Y. Hu, Y. Han, Y. Gu, K. Zhao, Q. Zhang, X. Liu, J. Liu, B. Liu, and X. Cao. 2018. Phosphorylation-mediated IFN-gammaR2 membrane translocation is required to activate macrophage innate response. Cell. 175:1336\u0026ndash;1351 e1317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, T. 2014. [Medical Cell Biology].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2:107\u0026ndash;117.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, C., Y. Xu, B. Zhang, W. Hao, and W.K. Tang. 2020. Cognitive impairment in chronic ketamine abusers. Psychiatry Res. 291:113206.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ketamine, tau, endosome, anesthetic, delirium-like behavior, neurotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-1362130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1362130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eKetamine is an anesthetic and antidepressant drug. However, whether ketamine can induce neurotoxicity and neurobehavioral deficits remains largely unknown. Delirium is a syndrome of acute brain dysfunction that is very similar to the presentation after ketamine administration. The onset of postoperative delirium in patients is often accompanied by elevated tau in cerebrospinal fluid. And ketamine may affect endosome, the key organelle for tau release from neurons. Therefore, we set out to determine the effects of ketamine on delirium-like behavior in mice and on tau trafficking in cultured cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe used the buried food test, open field test, and Y-maze test in adult mice to assess the occurrence of delirium-like behavior induced by ketamine. Quantified tau in the serum of mice with delirium-like behavior. And used cell fraction methods to determine the effects of ketamine on tau intracellular transfer, extracellular release and endosomes in cultured cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eKetamine induced delirium-like behavior and increased tau in mouse serum. Ketamine treatment also led to increased accumulation of endosomes as evidenced by increased endosomal markers Rab5 and Rab7. Moreover, ketamine inhibited endosome maturation, demonstrated by decreased membrane-bound but increased cytoplasm amounts of Rab5 and Rab7. Consequently, ketamine increased tau in the endosomes of cultured cells and the cell culture medium.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese data suggest that ketamine may interfere with intracellular tau trafficking and induce delirium-like behavior, promoting future research regarding neurotoxicity of anesthetics.\u003c/p\u003e","manuscriptTitle":"Ketamine induces delirium-like behavior and interferes with endosomal tau trafficking","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-18 17:05:53","doi":"10.21203/rs.3.rs-1362130/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"838c3a56-4cd6-4fbe-aa79-3e89a1af8063","owner":[],"postedDate":"February 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-02-22T11:59:15+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-18 17:05:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1362130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1362130","identity":"rs-1362130","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}