Abstract
The development, persistence and relapse of drug addiction require drug memory that generally develops with drug administration-paired contextual stimuli. Adult hippocampal neurogenesis (AHN) contributes to cocaine memory formation; however, the underlying mechanism remains unclear. Male mice hippocampal expression of Tau was significantly decreased during the cocaine-associated memory formation. Genetic overexpression of four microtubule-binding repeats Tau (4R Tau) in the mice hippocampus disrupted cocaine memory by suppressing AHN. Furthermore, 4R Tau directly interacted with phosphoinositide 3-kinase (PI3K)-p85 and impaired its nuclear translocation and PI3K-AKT signaling, processes required for hippocampal neuron proliferation. Collectively, 4R Tau modulates cocaine memory formation by disrupting AHN, suggesting a novel mechanism underlying cocaine memory formation and provide a new strategy for the treatment of cocaine addiction.
SIGNIFICANCE STATEMENT Drug memory that generally develops with drug-paired contextual stimuli and drug administration is critical for the development, persistence and relapse of drug addiction. Previous studies have suggested that adult hippocampal neurogenesis (AHN) plays a role in cocaine memory formation. Here, we showed that Tau was significantly downregulated in the hippocampus in the cocaine memory formation. Tau knock-out (KO) promoted AHN in the hippocampal dentate gyrus (DG), resulting in the enhanced memory formation evoked by cocaine-cue stimuli. In contrast, genetically overexpressed 4R Tau in the hippocampus disrupted cocaine-cue memory by suppressing AHN. In addition, 4R Tau interacted directly with phosphoinositide 3-kinase (PI3K)-p85 and hindered its nuclear translocation, eventually repressing PI3K-AKT signaling, which is essential for hippocampal neuronal proliferation.
Introduction
Drug addiction is characterized by compulsive drug seeking behaviors, repeated attempts at abstinence and high rates of relapse. Similar to the other memories, long-lasting drug-associated memory formation is closely associated with the rewarding effect of drugs and environmental cues related to drug exposure. Re-exposure to drug-associated contextual cues reactivates drug memory and promotes compulsive drug-seeking behaviors during the period of abstinence (Wolf, 2016). An understanding of the neurobiological mechanisms underlying drug memory formation is important for exploring effective strategies for drug addiction treatment and relapse prevention.
The hippocampus plays a critical role in the formation of drug-associated contextual memory (Deschaux et al., 2014; Castilla-Ortega et al., 2016a; Liddie and Itzhak, 2016; Liu et al., 2018). Accumulating evidence has suggested that hippocampus-dependent activities, such as memory encoding and mood regulation, are regulated by adult hippocampal neurogenesis (AHN), a unique phenomenon that confers neuroplasticity to the hippocampal circuitry and plays a significant role in neurodegenerative and psychiatric disorders (Takashima and Mandyam, 2018; Moreno-Jiménez et al., 2019; Tobin et al., 2019). In this process, new neurons undergo continual proliferation, migration, differentiation and integration into the existing neuronal circuitry of the dentate gyrus (DG), eventually modulating the formation and updating of hippocampus-dependent memory (Lazarov and Hollands, 2016; Toda and Gage, 2018). AHN has been recently considered an important factor in the establishment and maintenance of addiction memory (Castilla-Ortega et al., 2016b, 2017; Castilla-Ortega and Santín, 2020). When animals are subjected to pro-AHN manipulations, such as exercise, the subsequently acquired cocaine memory becomes stronger and more resistant to extinction (Mustroph et al., 2011). However, the precise mechanism underlying the role of AHN in cocaine memories formation has not been completely elucidated.
Tau is a microtubule-associated scaffolding protein whose major function is the stabilization of microtubules and promotion of microtubule polymerization (Guo et al., 2017). Tau isoforms containing either three or four microtubule-binding repeats (3R Tau or 4R Tau) exhibit approximately equal expression in the normal adult human brain; however, 4R Tau is the predominant isoform expressed in the adult murine brain. Tau was recently shown to be associated with the proliferation of newborn hippocampal granule neurons that require a high level of cytoskeletal plasticity to divide, proliferate and differentiate in response to external stimuli (Pallas-Bazarra et al., 2016; Dioli et al., 2017; Criado-Marrero et al., 2020). During early brain development, 3R Tau predominates in the neonatal brain and exhibits a lower affinity for microtubules than 4R Tau. Therefore, 3R Tau participates in the morphologic differentiation and migration of immature neurons. In contrast, 4R Tau binds with higher affinity to microtubules, thereby maintaining the cytoskeletal stability and neuronal integrity (Lu and Kosik, 2001; Tuerde et al., 2018). To date, the role of Tau isoforms in the formation of drug-associated memory is unknown. In the present study, our findings reveal a novel mechanism by which 4R Tau modulates cocaine memory formation by regulating AHN.
Materials and Methods
Animals
Male C57BL/6J wild-type (WT) mice (8–12 weeks old) were purchased from Vital River Laboratory Animal Technology Co Ltd. Tau−/− knock-out (Tau-KO) mice on the C57BL/6J background were obtained from The Jackson Laboratory (#007251). Male and female homozygous mice were bred to generate Tau−/− homozygous mice, and 8- to 12-week-old male homozygous Tau-KO mice and WT littermates (C57BL/6J) were used in this study. All mice were housed in the animal room on a standard 12/12 h light/dark cycle with a constant temperature and food and water available ad libitum. All experimental procedures and use of the animals were conducted in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee of Sichuan University. All efforts were made to minimize the suffering of the mice.
Behavioral paradigm
Mice were acclimated for one week before experiments and habituated to handling for 2 d before each behavioral test. Conditioned place preference (CPP) and self-administration were used to assess cocaine-cue memory formation. The home cage CPP, food CPP and locomotor activity were employed as control behavioral tests. All behavioral experiments were performed in a double-blind manner.
CPP
The CPP test was conducted using a standard three-chambered apparatus equipped with two large conditioning compartments (black and white) that differed in their flooring (bar and grid) and a small middle chamber (gray, smooth PVC floor) that connected the two large compartments. Before each session, animals were habituated to the chambers for at least 10 min for a continuous 2 d.
Pre-test session
Baseline preference was assessed by placing the mice in the middle chamber and allowing them to habituate the entire chambers freely for 15 min; an initial measurement of baseline preference was defined as the time spent in the black chamber subtracted from the time spent in the white chamber (Timepre-test). Animals were excluded from the test whether they showed a strong unconditioned preference for either side chamber (chamber bias >300 s).
CPP training
Animals were randomly assigned to two groups and trained for 6 d with alternating injections of cocaine (20 or 2.5 mg/kg, i.p) or saline injections in both compartments. They were confined to the conditioning or unconditioning chambers for 30 min after the injection and then returned to their home cages. On the test day, the animals were placed in the middle compartment and the time of spent in the two compartments was recorded for 15 min. We defined the time spent in the black chamber minus the time spent in the white chamber as Timetest. The CPP scores were calculated by Timetest minus Timepre-test and defined as the extent of the shift in preference after cocaine injection.
Home cage intraperitoneal injection
Mice were exposed to cocaine as described for consolidation training, and then they were not confined to the conditioning or unconditioning chambers, but were returned to their home cages without any context exposure to the test apparatus. On the test day, mice were placed in the middle connecting chamber, and the time spent in each chamber was calculated as described in previous experiments to evaluate the CPP score.
Food CPP
The food CPP test used a similar apparatus and methodology as described above. Mice were restricted from food for one week before the test, and their weight was maintained at 80% of their original body weight. During the food conditioning sessions, the food-induced CPP group was transferred to the food-paired chamber (2–3 g of food were placed in the chamber) for 30 min. In the non-food conditioning sessions, mice were assigned to the non-food-paired chamber for 30 min. After conditioning training for 3 h, animals were only fed once daily (1 h). The alternating sessions of conditioning were repeated 3 times (a total of 6 d). On the test day, mice were placed in the central chamber and allowed to freely explore both compartments for 15 min; the time spent in each chamber was recorded to calculate the CPP score.
CPP and extinction
Mice were exposed to the CPP protocol, and the test day course was repeated each day until the preference of the cocaine group for the conditioned compartment had returned to the habituation baseline.
Cocaine behavioral sensitization
Animals were acclimated to a chamber (48 × 48 cm) equipped with a camera for 15 min once a day for three consecutive days. The mice were given an intraperitoneal injection of cocaine (15 mg/kg; days 1–5) or an equal volume of saline, immediately placed in the chamber, and allowed to explore for 15 min. After 10 d of no injection (withdrawal session), the mice received a challenge dose of cocaine (15 mg/kg), and locomotor activity was tested (day 16). The distance traveled was measured daily, and automated tracking was performed with EthoVision 7.0 software (EthoVision 7.0; Noldus Information Technology).
Cocaine self-administration
Mice were anesthetized with sodium pentobarbital (60 mg/kg) and implanted with a single sterilized SILASTIC catheter (0.51 ID × 0.94 mm OD, BB518-20, Scientific Commodities) into the right jugular vein. The distal end of the catheter was threaded through the skin on the back of the mice and exited the skin via a stainless-steel guide cannula (RWD Life Science). After surgery, catheters were flushed with 0.1 ml of a saline solution containing penicillin (160,000 U/ml) and heparin (30 U/ml) daily. One week after recovering from surgery, animals were trained to self-administer intravenous injections of cocaine (0.75 mg/kg/infusion) or saline during 2-h sessions daily over 10 d on a fixed ratio 1 (FR1) schedule in an operant chamber. The response to the active poke produced a cocaine injection and was accompanied with a blue light stimulus for 20 s and an audible tone for a 5-s timeout period. At the same time, the inactive poke failed to inject the drug and produce conditioning stimuli.
Locomotor activity
The locomotor activity was measured as the distance traveled. Animals were acclimated to the chambers (48 × 48 cm) equipped with a camera for 15 min once a day for a consecutive 2 d. Before the test, the baseline locomotor activity was not statistically different between groups. In the following week, animals were injected cocaine (20 mg/kg, i.p) or an equal volume of saline, immediately placed in the locomotor activity chamber, and allowed to explore for 15 min. The distance traveled was measured daily for one week, and automated tracking was performed with EthoVision 7.0 software (EthoVision 7.0; Noldus Information Technology).
Drugs
Cocaine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products and dissolved in saline. LY294002 (S1105, Selleck) were dissolved in saline containing 4% DMSO, 30% PEG300, and 5% Tween 80.
Stereotactic surgery and inhibitor administration
Animals were anesthetized with sodium pentobarbital (60 mg/kg) and mounted in a standard stereotaxic instrument (RWD Life Science). After shaving the hair and cleaning the incision site with medical-grade alcohol, the scalp was incised to expose the skull and permanent bilateral guide cannulas (RWD Life Science) were bilaterally implanted into the dorsal DG (dDG; AP, −2.0 mm; ML, ±1.4 mm; DV, −2.2 mm) with stereotaxic instruments. Dental cement was used to anchor the guide cannula and the stainless-steel stylet blocker was inserted into each cannula to prevent blockage and infection. All mice were used for subsequent training after one week of recovery from surgery.
LY294002, a specific inhibitor of the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, was also dissolved in saline containing 4% DMSO, 30% PEG300 and 5% Tween 80 at a final concentration of 4.6 µg/µl. LY294002 were administered bilaterally (1 µl/side, 0.5 µl/min) with a micro-injector, 15 min before cocaine or saline administration during the cocaine-associated memory formation.
Adeno-associated virus (AAV) injection
The AAVs AAV2/8-CMV-2N4R-3Flag (1.83 × 1013 vg/ml), AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag (8.53 × 1012 vg/ml) and AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag (2.83 × 1012 vg/ml) were purchased from Obio Technology Co Ltd. Point mutations of arginine 205, proline 208 and proline 210 to alanine were synthesized in the murine full-length Tau amino acid sequence, as these three residues are normally important for the interaction of PI3K-p85 SH3 domains, and their mutation has previously been shown to attenuate PI3K-p85 binding to human 2N4R Tau (Reynolds et al., 2008). The AAV2/8-CMV-2N4R-3Flag, AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag and AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag were constructed for use in animal studies.
The coding regions of 2N4R Tau were amplified from cDNAs obtained from C57BL/6J mice by PCR and cloned into pAAV-CMV-MCS-3Flag and pAAV-CMV-MCS-2A-mNeongreen-3Flag to produce AAV-2N4R Tau or mutant 2N4R Tau, respectively. The vector was confirmed by sequencing. The recombinant plasmids were then packaged into AAV2/8 particles together with the AAV helper plasmid and purified using an iodixanol step-gradient ultracentrifugation method. Recombinant AAVs were produced by transient transfection in HEK293T cells and the expression of 4R Tau was analyzed using Western blotting.
Mice (8–12 weeks old) were anesthetized with sodium pentobarbital (60 mg/kg) and placed on a stereotaxic apparatus (RWD Life Science) to inject the virus into the dDG (AP, −2.0 mm; ML, ±1.4 mm; DV, −2.2 mm). After shaving the hair and cleaning the incision site with medical-grade alcohol, the scalp was incised to expose the skull and the connective tissue was gently removed from the skull surface with cotton swabs. Small craniotomy holes were drilled with a skull rotor (RWD Life Science) for virus injection. The micro syringe needles were used to bilaterally infuse the tissue with AAV2/8-CMV-2N4R-3Flag (0.2 µl, 0.05 µl/min), AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag (0.4 µl, 0.05 µl/min), or AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag (1.2 µl, 0.05 µl/min). After each injection, the syringe was left in place for an additional 5 min and then slowly withdrawn to allow the virus to diffuse. Mice recovered for at least three weeks before behavioral tests.
Lentiviral vector (LV) construction
The LVs pLenti-EF1a-CMV-2N4R-3Flag (3.39 × 108 TU/ml), pLenti-EF1a-CMV-mutant 2N4R-3Flag (3.41 × 108 TU/ml), pLenti-CMV-p85α-MYC-2A-mCherry-PGK-blasticidin (3.94 × 108 TU/ml), and pLKD-CMV-EGFP-2A-Puro-U6-shRNA Tau (5.44 × 108 TU/ml) were also purchased from Obio Technology Co Ltd. The pLenti-EF1a-CMV-2N4R-3Flag, pLenti-EF1a-CMV-mutant 2N4R-3Flag, pLenti-CMV-p85α-MYC-2A-mCherry-PGK-blasticidin and pLKD-CMV-EGFP-2A-Puro-U6-shRNA Tau were constructed for cell-based studies.
The coding regions of 2N4R Tau and PI3K-P85α were amplified from murine cDNAs using PCR and cloned into pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS-3Flag and pLenti-CMV-MCS-MYC-2A-mCherry-PGK-blasticidin vectors to produce LV-2N4R Tau, LV-mutant 2N4R Tau and PI3K-P85α, respectively. The Tau shRNA was cloned into pLKD-CMV-EGFP-2A-Puro-U6-shRNA and confirmed by sequencing. The sequences of the scrambled control shRNA and Tau shRNA were 5′-TTCTCCGAACGTGTCACGT-3′ and 5′-GGAGTTTGACACAATGGAA-3′, respectively. Recombinant lentiviruses were also produced by transient transfection in the HEK293T cells and then the levels of 4R Tau and PI3K-p85 were analyzed using Western blotting.
Tissue isolation
Mice were killed by rapid decapitation at the end of the behavioral tests. The hippocampus or DG were removed from the brain, snap frozen in dry ice, and stored at −80°C until the assay.
Western blotting
Brain tissues and cells were lysed and proteins were extracted using a mammalian cell and tissue extraction kit (K269-500, Biovision) containing phosphatase inhibitors (4906845001, Roche) according to the manufacturer's protocols. The total protein concentration was analyzed with a Bradford assay kit (P0006, Beyotime). Twenty micrograms protein were loaded and separated on 10 or 12.5% sodium dodecyl sulfate-polyacrylamide gels. After separation, the gels were then transferred to a polyvinylidene difluoride membrane (Millipore) in a mixed solution of Tris-glycine buffer and 20% (v/v) methanol. The membrane was blocked in TBST buffer containing 5% non-fat dry milk (Cell Signaling Technology) for 1 h at room temperature, and then incubated and gently shaken overnight with the primary antibody at 4°C. On the next day, after three washes with TBST for 15 min, the blots were incubated with the secondary antibody at room temperature for 2 h. Immunoreactivity was visualized using a chemiluminescence substrate (WBKLS0500, Millipore) with a chemiluminescence imagine system (CLINX). The optical density of each band was quantified using Chemi Analysis software (CLINX). The following antibodies were used for Western blotting: mouse anti-Tau (1:1000, Thermo Scientific), mouse anti-3R Tau (1:1000, Millipore), mouse anti-4R Tau (1:1000, Millipore), rabbit anti-cAMP response element-binding protein (CREB; 1:1000, Cell Signaling Technology), rabbit anti-p-CREB (S133; 1:1000, Cell Signaling Technology), rabbit anti-extracellular signal-regulated kinase (ERK)1/2 (1:1000, Cell Signaling Technology), rabbit anti-p-ERK1/2 (T 202/T 204; 1:1000, Cell Signaling Technology), rabbit anti-cofilin (1:1000, Abcam), rabbit anti-p-cofilin (S3; 1:1000, Cell Signaling Technology), rabbit anti-c-fos (1:1000, Cell Signaling Technology), mouse anti-Flag (1:5000, Sigma), mouse anti-2A (1:1000, Millipore), rabbit anti-PI3K-p85 (1:1000, Cell Signaling Technology), rabbit anti-PI3K-p110α (1:1000, Abcam), rabbit anti-AKT (1:1000, Abcam), rabbit anti-p-AKT (S473; 1:1000, Cell Signaling Technology), rabbit anti-GSK-3β (1:1000, Abcam), rabbit anti-p-GSK-3β (S9; 1:1000, Cell Signaling Technology), rabbit anti-β-catenin (1:1000, Abcam), rabbit anti-p-β-catenin (S33/37/T41; 1:1000, Cell Signaling Technology), rabbit anti-β-actin (1:1000, Cell Signaling Technology), mouse anti-GAPDH (1:2000, ZSGB-BIO), rabbit anti-lamin B1 (1:1000, Abcam), goat anti-mouse (HRP; SAB), goat anti-rabbit (HRP; SAB), goat anti-mouse light chain specific (HRP), and mouse anti-rabbit light chain specific (HRP; Jackson ImmunoResearch).
Immunohistochemistry
Animals that had undergone behavioral training were deeply anesthetized with sodium pentobarbital (60 mg/kg) and perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde in 0.1 m PBS, pH 7.4. Brains were carefully extracted from the skull, postfixed with 4% PFA overnight, and then dehydrated with 30% sucrose at 4°C. Brains were sectioned into 45-µm coronal slices using a freezing microtome (Leica) and stored in 12-well plates filled with a cryoprotectant solution (glycerol, ethylene glycol and 0.1 m phosphate buffer, pH 7.4, 1:1:2 by volume) at −20°C until processing for immunohistochemical staining.
The sections were washed with TBS 3 times (10 min each) and incubated with a blocking solution containing 0.3% Triton X-100 and 5% normal donkey serum (017-000-121, Jackson ImmunoResearch) in TBS for 2 h at room temperature. Sections were then incubated with primary antibodies in the blocking solution at 4°C overnight. On the next day, the sections were also washed with TBS-0.3% Triton X-100 (TBST) three times, transferred to the blocking solution containing fluorescent dye-conjugated secondary antibodies, and then incubated for 1.5 h at room temperature. After another three washes with TBST, sections were coverslipped with anti-fade mounting medium containing DAPI (H-1200, Vector), and confocal images were acquired with a laser confocal microscope (Nikon). The corresponding DG areas and cell numbers were determined using Image J software (National Institutes of Health) to assess the cell densities. The following antibodies were used for immunostaining: rabbit anti-Tau (1:100, Protech), mouse anti-3R Tau (1:200, Millipore), mouse anti-4R Tau (1:50, Biolegend), rat anti-bromodeoxyuridine (BrdU; 1:100, Abcam), mouse anti-Ki67 (1:100, BD), goat anti-sex determining region Y-box 2 (SOX2; 1:100, RD), rabbit anti-glial fibrillary acidic protein (GFAP; 1:100, Abcam), mouse anti-c-fos (1:100, Abcam), rabbit anti-doublecortin (DCX; 1:100, Abcam), donkey anti-mouse (Alexa Fluor 488; 1:500, Invitrogen), donkey anti-rabbit (Alexa Fluor 488; 1:500, Invitrogen), donkey anti-goat (Alexa Fluor 488; 1:500, Invitrogen), donkey anti-rabbit (Alexa Fluor 568; 1:500, Invitrogen), and donkey anti-rat (Alexa Fluor 647; 1:500, Jackson ImmunoResearch).
For BrdU staining, DNA was denatured by incubating the section in 2 m hydrochloric acid for 30 min at 37°C and then renatured with 0.05 m boric acid (28341, Thermo Scientific) at room temperature for 30 min, followed by TBS rinses for 10 min. Immunohistochemistry was then performed.
The cannula placements were confirmed in 5-µm-thick coronal sections using hematoxylin eosin (HE) staining, and images were acquired using a light microscope. Mice with misplaced cannulas were excluded from the statistical analysis.
Immunocytochemistry
Cells cultured on glass coverslips coated with poly-D-lysine (P6407, Sigma) were washed three times with PBS and then fixed with 4% PFA for 10 min. After three washes with PBS, cells were blocked with blocking buffer (5% normal donkey serum and 0.3% Triton X-100 in PBS) for 1 h and then incubated with primary antibodies overnight. On the next day, coverslips were washed three times with PBS and incubated with the fluorescent dye-conjugated secondary antibodies for 1 h at room temperature. Coverslips were then washed three times with PBS, stained with Hoechst (H1399, Life Technologies) for 5 min, and mounted with anti-fade mounting medium (H-1000, Vector). The following antibodies were used for immunostaining: rabbit anti-PI3K-p85α (1:400, Abcam), mouse anti-4R Tau (1:100, Biolegend), donkey anti-mouse (Alexa Fluor 488; 1:500, Invitrogen), and donkey anti-rabbit (Alexa Fluor 568; 1:500, Invitrogen).
Golgi staining
Golgi staining was performed using the FD Rapid Golgi Stain kit (PK401, FD Neurotechnologies) to label neurons in the dDG. Briefly, animals were deeply anesthetized with sodium pentobarbital (60 mg/kg), and the brains were immediately removed and rinsed with MilliQ water. Afterwards, the retrieved brains were immersed in equal parts of Solutions A and B (containing potassium dichromate, mercuric chloride, and potassium chromate) and stored at room temperature for two weeks in the dark. Tissues were subsequently rinsed, placed in the cryoprotectant solution, and stored at room temperature for at least 72 h in the dark before cutting. The brain slices were cut in the coronal plane at approximately a 100-mm thickness with the freezing microtome (Leica). Tissue sections were placed on poly-D-lysine-coated slides and dried in the dark. After drying, sections were transferred to distilled water, subsequently stained with a developing solution, and then successively dehydrated with 50%, 75%, 95%, and 100% ethanol. Finally, the sections were defatted with a xylene substitute and coverslipped with DPX mounting medium (06522, Sigma).
Images were acquired from prepared slides using a confocal microscope (Olympus). Each neuron was scanned at high magnification (100×, oil immersion) to ensure that all parts of the dendrites were intact. At least 20 neurons per mouse were selected for dendritic spine analysis in each group (three mice per group), and the average of spine density of each mouse was collected for statistical analysis. Neuronal reconstruction was performed using a confocal microscope, and dendrite spine density was measured using ImageJ software (National Institutes of Health).
Co-immunoprecipitation (co-IP) analysis
Cells and tissues were harvested for the co-IP analysis using the simplified and reliable Pierce Crosslink Magnetic IP/co-IP kit (88 805, Thermo Scientific). Briefly, the Tau or PI3K-p85 primary antibody was bound to 50 µl of Protein A/G magnetic beads (B23201, Bimake) for 15 min and washed three times. After the incubation, the protein supernatants extracted using the mammalian cell and tissue extraction kit were collected, and the protein concentration was determined using a Bradford assay kit. The supernatant from each sample was incubated with the antibody-crosslinked beads overnight at 4°C. On the next day, beads were washed two times with IP lysis/wash buffer and one time with ultrapure water. Elution buffer was used to elute the bound antigen, and neutralization buffer was used to neutralize the low pH. The supernatants were collected for Western blot analysis.
Cell culture and transfection
Mouse neuroblastoma 2a (N2a) cells were cultured in DMEM (C1195500BT, Invitrogen) containing 10% fetal bovine serum (FBS; 10099-141, Invitrogen) and grown in a 5% CO2 atmosphere at 37°C.
N2a cells were grown in six-well plates to generate 2N4R Tau overexpression and Tau knock-down-resistant cells. DMEM was replaced with serum-free DMEM and incubated for 2 h before transfection, follow by incubations with the lentiviruses contain the mouse 2N4R Tau, mutant 2N4R Tau, PI3K-p85, Tau-shRNA (5′-GGAGTTTGACACAATGGAA-3′) and the non-specific target oligonucleotides (5′-TTCTCCGAACGTGTCACGT-3′) and selection with 2.5 µg/ml puromycin or 10 µg/ml blasticidin. Antibiotic-resistant clones were picked and cultured in DMEM containing puromycin or blasticidin.
Cytoplasmic and nuclear fractionation
Harvested cells and tissues were extracted using the ProteoExtract Subcellular Proteome Extraction kit (539791, Millipore). The cytoplasmic and nuclear fractions were separated according to the manufacturer's instructions.
Preparation of the hippocampal single-cell suspension
The brain tissue was dissected on ice and enzymatically digested using the Adult Brain Dissociation kit (130-107-677, Miltenyi). The single-cell suspension was prepared for the flow cytometry assay according to the manufacturer's instructions.
Flow cytometry
Single-cell suspensions from each group were centrifuged and the pellet was resuspended with a viability dye Fixable Viability Stain 780 (1:1000; BD) for cell live/dead discrimination, followed by an incubation with blocking buffer containing CD16/CD32 (1:100; BD) for 10 min. Cells were then incubated with Foxp3/transcription factor staining buffer set (00-5523, Invitrogen) at 4°C for 30 min in the dark. Finally, samples were incubated with the antibody mixture at 4°C for 30 min, washed, centrifuged, resuspended in staining buffer containing 2% FBS, and then sorted using a FACSAria SORP (BD). Gating parameters and data analysis were performed using FlowJo 10 software (Tree Star). The following antibodies were used for flow cytometry: mouse anti-SOX2 (Alexa Fluor 647; 1:100, Biolegend), mouse anti-DCX (PE; 1:100, BD), mouse anti-GFAP (Alexa Fluor 405; 1:100, Novus), rat anti-Ki67 (PerCP/Cy5.5; 1:100, Biolegend), mouse anti-AKT (Alexa Fluor 350; 1:100, R&D Systems), and mouse anti-pAKT (S473, BV421; 1:100, BD).
5-Ethynyl-2'-deoxyuridine (EdU) or BrdU incorporation assays
For the analysis of newborn neuron proliferation in the hippocampus, the mice were injected with 50 mg/kg (intraperitoneal) BrdU (00-0103, Invitrogen) or Edu (900584, Sigma) three times every 4 h on the last training day, and analyzed after the tests were completed (Wang et al., 2019). BrdU and EdU immunostaining were performed to assess the proliferation of newborn hippocampal cells during cocaine memory formation.
For the flow cytometry assay of proliferation, single-cell suspensions were prepared from mice injected intraperitoneally with EdU as described above. Cultured N2a cells were serum-starved overnight, exposed to the external stimuli, and then 10 µm EdU was added to the culture medium for 2 h before cells were collected.
For the flow cytometry detection of EdU expression, collected cells were transferred to 5 ml FACS tubes, fixed, washed with staining buffer and stained with the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay kit (C10634, Thermo Scientific) at 4°C for 30 min in the dark. Finally, samples were incubated with the antibody mixture at 4°C for 30 min in the dark and then evaluated using a FACSAria SORP (BD). Data were analyzed as described above.
Quantification of the tau-interacting proteome
The dDG was removed from the hippocampus for co-IP, and the co-IP method was employed to detect the Tau-interacting proteome. The proteins were extracted and the co-IP procedures were conducted as described above. The collected protein supernatants were loaded and separated on 10% sodium dodecyl sulfate-polyacrylamide gels, and then subjected to gel digestion followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis.
In-gel digestion
For in-gel tryptic digestion, gel pieces were destained in 50 mm NH4HCO3 in 50% acetonitrile (v/v) until they were clear. Gel pieces were dehydrated with 100 µl of 100% acetonitrile for 5 min, rehydrated in 10 mm dithiothreitol and incubated at 56°C for 60 min. Gel pieces were again dehydrated in 100% acetonitrile and rehydrated with 55 mm iodoacetamide. Samples were incubated at room temperature for 45 min in the dark. Gel pieces were washed with 50 mm NH4HCO3 and dehydrated with 100% acetonitrile, followed by rehydration with 10 ng/µl trypsin and resuspension in 50 mm NH4HCO3 on ice for 1 h. The excess liquid was removed and gel pieces were digested with trypsin at 37°C overnight. Peptides were extracted with 50% acetonitrile in 5% formic acid, followed by 100% acetonitrile. Finally, peptides were dried completely and resuspended in 2% acetonitrile in 0.1% formic acid.
LC-MS/MS analysis
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reverse-phase analytical column (15 cm length, 75-µm i.d.). The gradient was comprised of 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) in 16 min, 23% to 35% in 8 min, increasing to 80% in 3 min, and then maintained at 80% for the last 3 min with a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to an NSI source followed by MS/MS in Q Exactive Plus coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350–1800 for the full scan, and the intact peptides were detected in the Orbitrap at a 70,000 resolution. Peptides were then selected for MS/MS using NCE at a setting of 28%, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0-s dynamic exclusion was performed. Automatic gain control (AGC) was set to 5E4.
Data processing
The MS/MS data were processed using Proteome Discoverer 1.3. Tandem mass spectra were searched in the SwissProt Mouse database. Trypsin/P was specified as the cleavage enzyme, allowing up to 2 missing cleavages. The mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as the fixed modification and oxidation on Met was specified as the variable modification. Peptide confidence was set at high, and the peptide ion score was set to >20.
Protein interaction networks
All interesting gene name identifiers (mainly proteins involved in regulating cell proliferation) were searched against the STRING database version 10.5 for protein-protein interactions. Only interactions between the proteins belonging to the searched dataset were selected, thereby excluding external candidates. STRING defines a metric called a confidence score to define the interaction confidence, and we retrieved all interactions with a confidence score ≥0.7 (high confidence). The interaction network identified using STRING was visualized with Cytoscape.
Statistical analysis
All data were analyzed with GraphPad Prism 7 software, presented as means ± SEM, and subjected to the Kolmogorov–Smirnov test to assess the normality of the distribution. For simple comparisons, an unpaired two-tailed Student's t test was used. For multiple comparisons, one-way or repeated-measures two-way ANOVA test was used for each experiment. In all cases, n refers to the number of animals. For all results, statistical significance was defined as p < 0.05.
Results
Tau downregulation is involved in cocaine-cue memory formation
CPP test, an associative memory model linking drug reward with environment cues, is widely used to assess the formation of drug-associated memory (Zhou et al., 2019; Bardo and Bevins, 2000; Wang et al., 2020). In the present study, we first investigated the effect of cocaine on the expression of Tau in the hippocampus during the cocaine memory formation. Compared with the control group, the mice trained with CPP showed significantly reduced level of Tau in the hippocampus (CPP score: t(28) = 9.138, p < 0.0001, n = 15 per group; Tau: t(14) = 2.579, p < 0.05, n = 8 per group; Fig. 1A–C). We next performed two other reward tests, the home cage intraperitoneal injection test and food CPP tests, to investigate whether the changes in Tau level was specific to the formation of cocaine CPP memory. In the former test, mice were only injected with cocaine, but without cocaine-associated cue training (n = 8 per group; Fig. 2A,B). In the latter test, mice were only exposed to a palatable food reward to train the food-associated cue memory, but were not administered cocaine (n = 11 per group; Fig. 2D,E). Importantly, change in the level of Tau was not observed in the hippocampus of the mice subjected to these two tests (n = 7 per group; Fig. 2C,F). We further measured Tau level after cocaine CPP extinction. Interestingly, the level of Tau clearly returned to baseline values (n = 8 per group; Fig. 2G–I). In addition, in the cocaine behavioral sensitization model, the level of hippocampal Tau was not altered (F(6,84) = 20.28, p < 0.0001, n = 8 per group; Fig. 2J–L), and the level of hippocampal Tau was not significantly increased at day 1 after CPP test (n = 4 per group; Fig. 2M,N).
Cocaine-cue memory downregulates the expression of Tau in the hippocampus. A, Experimental schedule for cocaine CPP. B, Cocaine CPP score was significantly increased in cocaine group when compared with control group. C, Expression of Tau in the hippocampus with cocaine CPP conditioning. D, Timeline of cocaine self-administration experiment. E–G, Number of active pokes, inactive pokes and infusions in the cocaine self-administration. H, Expression of Tau in the hippocampus of cocaine self-administered mice. I, Schematic of the locomotor-activity procedure. J, Distance during the 15 min after daily cocaine administration. K, Expression of Tau in the hippocampus in cocaine-induced hyperlocomotion. Data are the means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Hab, habituation.
Tau downregulation may play an important role in cocaine-associated memory formation. A, Experimental schedule for home cage intraperitoneal injections. B, CPP score of mice after home cage intraperitoneal injections test. C, Western blotting of Tau in the hippocampus of mice with home cage intraperitoneal injections training. D, Experimental schedule for food CPP. E, CPP score of mice after food CPP training. F, Western blotting of Tau in the hippocampus with food CPP training. G, Timeline of cocaine CPP extinction experiment. H, CPP score of mice after cocaine CPP extinction training. I, Immunoblotting of Tau in the hippocampus after cocaine CPP extinction. J, Experimental schedule for cocaine behavioral sensitization. K, Distance during the 15 min after daily cocaine administration. L, Western blotting of Tau in the hippocampus of mice with cocaine behavioral sensitization. M, Schematic of tissue collection after cocaine CPP. N, Immunoblotting of Tau in the hippocampus after cocaine CPP test. Data are the means ± SEM; *p < 0.05 and ****p < 0.0001.
We continued to investigate the role of Tau in cocaine self-administration, a paradigm that incorporates memories of drug experience and drug-associated environmental cues. Cocaine-associated memory was formed when an instrumental action (active pokes) resulted in cocaine delivery (unconditioned stimulus) paired with an audiovisual cue (conditioned stimulus). Mice were trained on a FR1 schedule, where a single active poke produced an infusion of cocaine or saline. Compared with saline-treated mice, cocaine-self-administered mice showed cocaine reinforcing (active pokes: F(12,130) = 1.787, p < 0.01, p < 0.001, and p < 0.0001; infusions: F(12,130) = 1.556, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, n = 6 per group; Fig. 1D–G). Consistent with these findings, the level of Tau in the hippocampus was obviously reduced (t(10) = 2.466, p < 0.05, n = 6 per group; Fig. 1H). We further detected Tau level in the hippocampus of cocaine-treated mice subjected to a locomotor activity test that lacks cocaine-associated contextual stimuli. Interestingly, Tau expression was not altered after cocaine injection for 7 d (distance: F(7,176) = 9.619, p < 0.0001, n = 12 per group; Fig. 1I–K).
To further explore the role of Tau in cocaine memory formation, Tau-KO mice and non-transgenic WT mice were injected a low dose of cocaine (2.5 mg/kg; Salery et al., 2017). Notably, cocaine significantly induced a clear preference for the cocaine-paired chamber in Tau-KO mice that was not observed in WT mice (CPP score: F(3,28) = 6.165, p < 0.05, n = 8 per group; Fig. 3A–C). Collectively, Tau downregulation may play an important role in cocaine-associated memory formation.
Tau-KO mice show increased proliferating rates in hippocampal neuronal subpopulations with a low dose of cocaine CPP paradigm. A, Experiment timeline for WT and Tau-KO mice CPP test at a low dose of cocaine (2.5 mg/kg). B, Tau-KO mice more easily for cocaine memory formation compared with WT mice with a low dose of cocaine. C, Immunoblotting of Tau after cocaine CPP training. D–G, Proliferating rates of hippocampal neural subpopulations were elevated in Tau-KO mice after cocaine CPP training. Data are the means ± SEM; *p < 0.05. The upper right quadrants show the corresponding positive subpopulations.
Tau downregulation promotes AHN during cocaine memory formation
It has been known that AHN contributes to the formation of memory, including cocaine-associated memory (Castilla-Ortega et al., 2017; Castilla-Ortega and Santín, 2020). To explore the role of Tau in AHN and cocaine memory formation, a flow cytometry assay was performed to investigate whether Tau ablation would affect the proliferation of newborn hippocampal neurons during cocaine memory formation. Notably, compared with WT mice, Tau-KO mice showed a qualitative increase in the proliferation of the neuronal subpopulations positive for SOX2 (a marker of neural stem cells and progenitor cells), SOX2/GFAP double-labeled cells (a marker of radial glia-like cells), and DCX [a marker of neuroblasts and immature neurons; F(3,9) = 6.717, p < 0.05 (Fig. 3D); F(3,9) = 6.673, p < 0.05 (Fig. 3F); F(3,9) = 5.862, p < 0.05, n = 3 per group (Fig. 3G)]. Thus, Tau deficiency may enhance AHN and facilitate cocaine memory formation.
4R Tau overexpression reduces AHN and attenuates cocaine memory formation
To investigate whether Tau overexpression affects AHN and weakens cocaine memory formation, we first compared the expression of Tau isoforms in the DG, where AHN generally occurs (Altman and Das, 1965). Because the neuroanatomy of the hippocampus supports the segregation of neuronal outputs along a dorsal-ventral axis (Fanselow and Dong, 2010), the dDG and ventral DG (vDG) were separated from the hippocampus for immunoblotting analysis after CPP training, respectively. Both Tau and 4R Tau levels were significantly decreased in the dDG after cocaine CPP conditioning, whereas 3R Tau levels were not altered (Tau: t(13) = 2.715, p < 0.05; 4R Tau: t(11) = 2.287, p < 0.05, n = 6 per group; Fig. 4A). In addition, the levels of the memory formation-related proteins phosphorylated CREB and phosphorylated cofilin were significantly increased; however, the levels of phosphorylated ERK remained unchanged (p-CREB: t(12) = 2.306, p < 0.05; p-cofilin: t(12) = 2.209, p < 0.05, n = 6 per group; Fig. 4B). No significant differences in the levels of Tau, Tau isoforms, phosphorylated CREB or phosphorylated cofilin were observed in the vDG after cocaine CPP conditioning (n = 6 per group; Fig. 4C,D). Immunostaining further verified the decreased expression of both Tau and 4R Tau in the dDG of cocaine-conditioned mice (n = 3 per group; Fig. 4E–G). Collectively, 4R Tau, but not 3R Tau, may specifically modulate cocaine-induced memory formation.
Cocaine CPP represses Tau and 4R Tau expression in the dDG. A, B, Immunoblotting analysis for Tau, Tau isoforms, CREB, ERK, and cofilin in the dDG. C, D, Immunoblotting analysis for Tau, Tau isoforms, CREB, ERK, and cofilin in the vDG. D–G, The expression of Tau and Tau isoforms in the dDG was detected by immunostaining. Data are the means ± SEM; *p < 0.05. Scale bar: 25 μm.
We next explored the function of genetically overexpressed 4R Tau in AHN during cocaine memory formation. To this end, we constructed AAVs specifically expressing 2N4R Tau (AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag), the longest brain 4R Tau isoform containing an intact Tau amino acid sequence (Avila et al., 2016). AAV-2N4R Tau was stereotactically microinfused into the dDG before cocaine CPP training, and 2N4R Tau expression was visualized directly based on mNeonGreen expression (Fig. 5A). As expected, overexpression of AAV-2N4R Tau (AAV2/8-CMV-2N4R-3Flag) in the dDG significantly decreased the cocaine CPP score (F(3,44) = 6.687, p < 0.05 and p < 0.01, n = 12 per group; Fig. 5B,C). Furthermore, the proliferation rates of the neuronal subpopulations positive for SOX2, both SOX2 and GFAP, and DCX were reduced [F(3,9) = 9.380, p < 0.05 and p < 0.01 (Fig. 5D); F(3,9) = 8.868, p < 0.05 (Fig. 5F); F(3,9) = 13.08, p < 0.05 and p < 0.01, n = 4 AAV-saline group, n = 3 AAV-cocaine group, n = 3 AAV-2N4R-cocaine group, n = 3 AAV-2N4R-saline group (Fig. 5G)]. We further determined the effect of 4R Tau overexpression on the proliferation of newborn hippocampal neurons in the dDG during cocaine memory formation. Mice were administered three consecutive injections of BrdU (50 mg/kg, i.p.), a marker of the exogenous proliferation of newborn cells, in 1 day before the cocaine CPP test (Fig. 6A). Importantly, AAV-mediated 4R Tau overexpression markedly reduced the density of BrdU-labeled cells in the dDG of the hippocampus (F(3,8) = 13.85, p < 0.05 and p < 0.01, n = 3 per group; Fig. 6B,C). In addition, the density of Ki67/BrdU double-labeled cells was clearly reduced following the overexpression of 4R Tau (F(3,8) = 14.01, p < 0.05 and p < 0.01, n = 3 per group; Fig. 6B,D). By quantifying the densities of SOX2/BrdU double-labeled cells (F(3,8) = 14.98, p < 0.01, n = 3 per group; Fig. 6B,E), SOX2/GFAP/BrdU triple-labeled cells (F(3,8) = 14.52, p < 0.01, n = 3 per group; Fig. 6B,F) and DCX/BrdU double-labeled cells (F(3,8) = 13.79, p < 0.01, n = 3 per group; Fig. 6B,G), we found that 4R Tau exerted a deleterious effect on the proliferation of the aforementioned neuronal subpopulations. Furthermore, immunostaining and immunoblotting analyses revealed an increased expression of c-Fos in the dDG during cocaine memory formation; nevertheless, such increase was attenuated by AAV-mediated 4R Tau overexpression [F(3,8) = 9.080, p < 0.05, n = 3 per group (Fig. 6H,I); F(3,19) = 4.359, p < 0.05, n = 6 per group (Fig. 6J)]. These results support the hypothesis that 4R Tau overexpression inhibits the proliferation and activity of adult hippocampal neurons in the dDG during cocaine memory formation.
AAV-mediated 4R Tau overexpression attenuates the proliferation of adult hippocampal neurons during cocaine memory formation. A, Representative images of AAV2/8-mNeonGreen-2N4R Tau expression in the dDG (green); DAPI, nucleus (blue). B, Experiment timeline for AAV-mediated 4R Tau overexpression in the dDG three weeks before cocaine CPP training. C, AAV-mediated 4R Tau overexpression in the dDG weakened cocaine-induced CPP. D–G, Representative flow cytometry plots depict the effect of AAV-mediated 4R Tau overexpression on the proliferation rates of hippocampal neuron. The upper right quadrants show the corresponding positive subpopulations. Data are the means ± SEM; *p < 0.05 and **p < 0.01.
AAV-mediated 4R Tau overexpression suppresses cocaine CPP-promoted proliferating rates and activity of hippocampal neuron. A, Schematic representation of experiment timeline for mice receiving BrdU injection. B–D, The density of BrdU and BrdU/Ki67 double-labeled cells in the dDG was reduced by AAV-mediated 4R Tau overexpression. B, E, The rate of SOX2/BrdU double-labeled cells was reduced by AAV-mediated 4R Tau overexpression. B, F, Confocal images showed a decreased density of SOX2/GFAP/BrdU triple-labeled cells in the dDG after AAV-mediated 4R Tau overexpression. B, G, The density of DCX/BrdU double-labeled cells were reduced by AAV-mediated 4R Tau overexpression. H, I, Confocal images of c-fos immunostaining and quantification showed a decreased density of c-fos labeled cells in dDG by AAV-mediated 4R Tau overexpression. J, Immunoblotting for c-fos in the dDG. Data are the means ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001. Long scale bars: 100 μm. Short scale bars: 10 μm.
We continued to investigate the effect of 4R Tau overexpression on the levels of memory formation-related proteins and the synaptic structure of granule neuron in dDG. The phosphorylation levels of CREB and cofilin were increased in WT mice after cocaine CPP paradigm; however, these increases were attenuated by AAV-mediated 4R Tau overexpression. In contrast, the levels of total CREB and cofilin did not change (4R Tau: F(3,16) = 16.40, p < 0.05 and p < 0.01; Tau: F(3,16) = 18.88, p < 0.05 and p < 0.01; p-CREB: F(3,16) = 8.137, p < 0.01; p-cofilin: F(3,16) = 3.964, p < 0.05, n = 5 per group; Fig. 7A,B). Golgi staining revealed a significant increase in the dendritic spine density of granule neurons after the cocaine CPP paradigm, whereas this increase was inhibited by 4R Tau overexpression (F(3,8) = 29.26, p < 0.05, p < 0.01 and p < 0.001, n = 3 per group; Fig. 7C). In the cocaine self-administration model, the active pokes and numbers of cocaine infusions were significantly increased during cocaine memory formation; however, this increase was attenuated by 4R Tau overexpression (active pokes: F(27,280) = 4.185, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001; infusions: F(27,280) = 4.309, p < 0.01, p < 0.001, and p < 0.0001, n = 8 per group; Fig. 7D–G). Similarly, cocaine-induced increases in the levels of phosphorylated CREB and cofilin were decreased by 4R Tau overexpression (4R Tau: F(3,16) = 16.65, p < 0.05 and p < 0.01; Tau: F(3,16) = 16.97, p < 0.05 and p < 0.01; p-CREB: F(3,16) = 5.457, p < 0.01; p-cofilin: F(3,16) = 5.323, p < 0.05 and p < 0.01, n = 5 per group; Fig. 7H,I). However, 4R Tau overexpression did not attenuate cocaine-induced hyperlocomotion in the absence of cocaine contextual stimuli (4R Tau: t(10) = 2.299, p < 0.05; Tau: t(10) = 2.368, p < 0.05, n = 8 per group; Fig. 7J–L). Thus, 4R Tau upregulation disrupts the formation of cocaine-associated memory and dendritic structural plasticity of granule neuron.
AAV-mediated 4R Tau overexpression modulates the expression of memory formation-related proteins and granule neurons morphology during the cocaine memory formation. A, B, Expression of Flag, 4R Tau, Tau, CREB, phosphorylated CREB, cofilin, and phosphorylated cofilin after cocaine CPP paradigm. C, Golgi staining showed the dendritic spines density of granule neurons in the dDG. D, J, Schematics of AAV-mediated 4R Tau overexpression in cocaine self-administration and hyperlocomotor activity tests. E–G, Number of active pokes, inactive pokes and infusions after AAV-mediated 4R Tau overexpression in the dDG in cocaine self-administration (****p < 0.001, AAV-cocaine group vs AAV-saline group; ###p < 0.001 and ####p < 0.0001, AAV-2N4R-cocaine group vs AAV-cocaine group; Δp < 0.05 and ΔΔp < 0.01, AAV-2N4R-cocaine group vs AAV-2N4R-saline group). H, I, Immunoblotting of Flag, 4R Tau, Tau as well as CREB, phosphorylated CREB, cofilin, and phosphorylated cofilin in the dDG with cocaine self-administration paradigm. K, Total distance within 15 min after daily cocaine administration. L, Flag, 4R Tau and Tau expression in the dDG after hyperlocomotion paradigm. Data are the means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.001. Scale bars: 10 μm.
Previous studies showed that overexpression of human Tau in GABAergic interneurons induces AHN deficits and cognition impairment (Zheng et al., 2020); indicating that intracellular accumulation of Tau in GABAergic interneurons may impair AHN. To further confirm whether the AAV-mediated expression of exogenous Tau in GABAergic interneurons would block AHN and cocaine memory, the 2N4R Tau-expressing AAV (AAV2/8-GAD67-2N4R-3Flag) was infused into the dDG of Tau-KO mice before the cocaine CPP paradigm. We observed that overexpression of 2N4R Tau in GABAergic interneurons of dDG significantly decreased the cocaine CPP score (F(3,28) = 8.840, p < 0.05 and p < 0.01, n = 12 per group; Fig. 8A,B) and reduced the proliferation rates of the neuronal subpopulations positive for SOX2, SOX2/GFAP and DCX [F(3,8) = 6.398, p < 0.05 (Fig. 8C); F(3,8) = 6.918, p < 0.05 (Fig. 8E); F(3,8) = 11.88, p < 0.01, n = 3 AAV-saline group, n = 3 AAV-cocaine group, n = 3 AAV-2N4R-cocaine group, n = 3 AAV-2N4R-saline group (Fig. 8F)]. Therefore, we speculated that the 4R Tau in GABAergic interneurons may be a main factor that inhibits AHN and impairs cocaine memory formation.
AAV-mediated 4R Tau overexpression in dDG GABAergic interneurons inhibits the proliferation of adult hippocampal neurons during cocaine memory formation. A, Experiment timeline for AAV-mediated 4R Tau overexpression in the dDG three weeks before cocaine CPP training. B, AAV-mediated 4R Tau overexpression in the dDG weakened cocaine-induced CPP. C–F, Representative flow cytometry plots depict the effect of AAV-mediated 4R Tau overexpression on the proliferation rates of hippocampal neuron. The upper right quadrants show the corresponding positive subpopulations. Data are the means ± SEM; *p < 0.05 and **p < 0.01.
4R Tau modulates AHN by altering PI3K-AKT signaling during the cocaine memory formation
Tau is able to bind to a variety of other proteins, including a number of proteins functioning in cell signaling transduction (Brandt and Leschik, 2004; Reynolds et al., 2008). Tau-interacting proteome was analyzed to investigate the underlying mechanisms by which Tau regulates AHN. The dDG tissue was harvested, subjected to co-IP, and analyzed using LC-MS/MS. Interestingly, 20 specific Tau-interacting proteins, which are intimately involved in regulating cell proliferation, were identified (Extended Data Fig. 9-1). Eight of these proteins, such as PI3K-p85, a critical PI3K regulatory subunit, were enriched in the PI3K-AKT and GSK-3β-β-catenin pathways (Fig. 9A). Using an immunoblotting analysis, we confirmed that PI3K-AKT and GSK-3β-β-catenin signaling in the dDG were altered by cocaine CPP conditioning; however, AAV-mediated 4R Tau overexpression inhibited PI3K-AKT signaling, but not GSK-3β-catenin signaling (PI3K-p110α: F(3,20) = 7.115, p < 0.05 and p < 0.01; p-AKT: F(3,20) = 7.056, p < 0.01; p-GSK-3β: F(3,20) = 2.739, p < 0.05; p-β-catenin: F(3,20) = 3.984, p < 0.05, n = 6 per group; Fig. 9B,C). We employed a co-IP analysis to further determine whether the interaction of Tau with PI3K-p85 would be altered by the cocaine CPP paradigm and found that both 3R Tau and 4R Tau bound endogenous PI3K-p85 in the dDG. Notably, the interaction of 4R Tau with PI3K-p85 was markedly reduced by cocaine CPP conditioning and reversed by the genetic overexpression of 4R Tau. However, the interaction of 3R Tau with PI3K-p85 was unchanged by cocaine CPP conditioning (F(3,8) = 19.16, p < 0.01 and p < 0.001, n = 3 per group; Fig. 9D). Based on these results, the interaction of 4R Tau with PI3K-p85 may specifically modulate cocaine memory.
Analysis of Tau-interacting proteins involved in regulating cell proliferation, and AAV-mediated 4R Tau overexpression weakens PI3K-AKT singling in cocaine CPP test. A, The genes involved in regulating cell proliferation are mainly distributed the PI3K-AKT (light blue) and GSK-3β-β-catenin (light red) signaling pathway. Prediction gene refer to the typical genes involved in PI3K-AKT and GSK-3β-β-catenin signaling, which were identified by bioinformatics analysis. B, C, Western blot analysis for the expression of PI3K-p85, PI3K-p110α, AKT, GSK-3β, and β-catenin. D, Endogenous PI3K-p85 was immunoprecipitated with antibodies against PI3K-p85 from tissue lysates. Rabbit immunoglobulin G (IgG) was used as control. Data are the means ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001. Extended Data Figure 9-1 is supporting thus figure.
Extended Data Figure 9-1
Tau-interacting proteome analysis of dDG Download Figure 9-1, XLS file.
We subsequently performed immunofluorescence staining to track the cellular localization of these two proteins in the cultured N2a cells in vitro and to further clarify the interaction of 4R Tau with PI3K-p85. 4R Tau was primarily located in the cytosol of N2a cells and was clearly co-localized with PI3K-p85 (Fig. 10A). Co-IP assays further revealed the interaction of endogenous Tau with PI3K-p85 (n = 3 per group; Fig. 10B). An association of human Tau with PI3K-p85 has been shown in COS-7 cells, and the replacement of prolines 216 and 219 and arginine 221 of human Tau with alanine drastically decreased its binding to PI3K-p85 (Reynolds et al., 2008). By comparing the amino acid sequences of human and mouse Tau, prolines 216 and 219 and arginine 221 in human Tau correspond to prolines 205 and 208 and arginine 210 in mouse Tau, respectively. We then replaced these three amino acid residues of murine Tau with alanine and performed a co-IP assay. Notably, the interaction of mutant murine Tau with PI3K-p85 was markedly reduced (n = 3 per group; Fig. 10C), confirming the direct interaction of 4R Tau with PI3K-p85.
LV-mediated 4R Tau overexpression decreases the proliferating number of N2a cells. A, N2a cells were stained with Hochest and antibodies against 4R Tau and PI3K-p85. B, Endogenous PI3K-p85 was immunoprecipitated with antibodies against PI3K-p85 from cell lysates. Rabbit immunoglobulin G (IgG) was used as control. C, N2a cells were transfected with LV-EGFP, LV-2N4R-EGFP, and LV-mutant-2N4R-EGFP, and immunoprecipitates were analyzed by immunoblotting with antibody against PI3K-p85 and Flag. D–F, The number of the cell populations positive for EdU, AKT, and phosphorylated AKT was determined by flow cytometry. Data are the means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Scale bar: 10 μm. The upper right quadrants show the corresponding positive subpopulations. EGFP visualizes directly the 2N4R Tau expression in N2a cells.
We further explored the function of the 4R Tau interaction with PI3K-p85 in the cultured N2a cells. N2a cells were serum-starved overnight, and then cultured with EdU, an exogenous proliferation marker, for 2 h before the cells were collected for flow cytometry detection. LV-mediated 4R Tau overexpression significantly inhibited the proliferation of N2a cells, whereas mutant 4R Tau exerted no effect (F(2,6) = 103.7, p < 0.001 and p < 0.0001, n = 3 per group; Fig. 10D). Moreover, the level of phosphorylated AKT at serine 473, a marker of PI3K-AKT activation, was markedly decreased by the overexpression of 4R Tau, but not mutant 4R Tau (F(2,6) = 138.9, p < 0.05 and p < 0.01, n = 3 per group; Fig. 10E,F).
We further used a Tau shRNA to silence Tau expression in N2a cells and determined whether the downregulation of Tau expression promotes cell proliferation by reducing its association with PI3K-p85. The siRNA oligo pairs targeting Tau were transfected into N2a cells and efficiently knocked down Tau expression (n = 3 per group; Fig. 11A). The co-IP analysis revealed a decrease in the association of 4R Tau with PI3K-p85 (Fig. 11A). Not surprisingly, the Tau protein deficiency significantly increased N2a cell proliferation and the percentage of phosphorylated AKT-positive cells (EdU: t(5) = 3.121, p < 0.05; p-AKT: t(5) = 2.602, p < 0.05, n = 3 per group; Fig. 11B–D).
Silencing of Tau attenuates the interaction between Tau and PI3K-p85 and promotes PI3K-p85 nuclear translocation. A, N2a cells were transfected with LV-sh-EGFP and LV-sh-Tau-EGFP, and immunoprecipitates were analyzed by immunoblotting with antibody against Tau and PI3K-p85. B–D, After exposure of external stimuli, the rates of the cell population positive for EdU, AKT, and phosphorylated AKT were analyzed by flow cytometry. E, N2a cells were transfected with LV-sh-EGFP and LV-sh-Tau-EGFP, and the location of endogenous PI3K-p85 was analyzed by immunofluorescence. F, Cells were transfected with LV-sh-EGFP and LV-sh-Tau-EGFP, and then transfected with exogenous LV-PI3K-p85-mCherry. G, Proteins in the cytosolic and nuclear fraction of N2a cells were analyzed by immunoblotting. Data are the means ± SEM; *p < 0.05. Scale bar: 10 μm. EGFP visualizes directly the silencing of Tau expression of N2a cells; mCherry visualizes directly the exogenous PI3K-p85 expression.
4R Tau restricts PI3K-p85 to the cytoplasm
Because the nuclear accumulation of PI3K-p85 is required for PI3K-AKT signaling activity (Chiu et al., 2014), we examined whether 4R Tau could trap endogenous PI3K-p85 to reduce its nuclear translocation in cells. Immunofluorescence staining revealed the even distribution of endogenous PI3K-p85 (red) in the cytosol and nucleus of N2a cells. Strikingly, endogenous PI3K-p85 was predominantly expressed in the nucleus when the Tau protein was deleted by a Tau shRNA (t(4) = 2.822, p < 0.05, n = 3 per group; Fig. 11E). An exogenous PI3K-p85-expressing LV (red) was transfected into the N2a cells to further explore the effect of 4R Tau on the cellular distribution of PI3K-p85. Similarly, exogenously expressed PI3K-p85 was also mainly expressed in the cytoplasm. Importantly, the Tau-deficient cells exhibited a clear increase in the nuclear translocation of exogenous PI3K-p85 (t(4) = 17.59, p < 0.0001, n = 3 per group; Fig. 11F). We further separated cytosolic and nuclear fractions for Western blot analysis. Tau deletion significantly increased the level of nuclear PI3K-p85 and the cytosolic level of phosphorylated AKT (cytosol PI3K-p85: t(4) = 3.6, p < 0.05; cytosol p-AKT: t(4) = 3.247, p < 0.05; nucleus PI3K-p85: t(4) = 4.438, p < 0.05, n = 3 per group; Fig. 11G), suggesting that Tau, a cytoskeletal protein, may restrain PI3K-p85 in the cytoplasm, eventually blocking PI3K-AKT signaling.
To illustrate whether Tau deficiency enhancing neuronal proliferation depend on PI3K-AKT signaling. Tau-silenced N2a cells were pretreated with LY294002, a PI3K-AKT inhibitor, at a final concentration of 10 µm to determine the role of PI3K-AKT signaling in Tau-silenced N2a cells proliferation. The levels of PI3K-p110α and phosphorylated AKT were decreased by LY294002 in Tau-silenced N2a cells (n = 3 per group; Fig. 12A). The flow cytometry analysis showed a clear decrease in the subpopulations of Tau-silenced N2a cells expressing both EdU and phosphorylated AKT [t(6) = 3.28, p < 0.05 (Fig. 12B,E); t(6) = 2.699, p < 0.05, n = 3 vehicle-LV-sh-Tau-EGFP group; n = 4 LY294002-LV-sh-Tau-EGFP group (Fig. 12D,G)]. Based on these results, 4R Tau regulates PI3K-AKT signaling by binding to the PI3K-p85 subunit, subsequently modulating neuronal proliferation.
Pharmacological suppression of PI3K-AKT signaling inhibits the proliferation of LV-sh-Tau-EGFP cells. A, The expression of PI3K-p85, PI3K-p110α, AKT, and phosphorylated AKT were analyzed by Western blotting. B–G, The number of the cell populations positive for EdU, AKT, and phosphorylated AKT was analyzed by flow cytometry. Data are the means ± SEM; *p < 0.05. The upper right quadrants show the corresponding positive subpopulations.
The interaction of 4R tau with PI3K-p85 modulates cocaine memory formation
The 2N4R Tau-expressing AAV (AAV2/8-CMV-2N4R-2A-mNeonGreen-3Flag) was infused into the dDG of Tau-KO mice before the cocaine CPP paradigm to investigate the role of the 4R Tau and PI3K-p85 interaction in cocaine memory formation. Not surprisingly, 2N4R Tau overexpression significantly reduced the cocaine CPP score of Tau-KO mice; moreover, the co-IP assay showed an enhanced interaction of 4R Tau with PI3K-p85 (F(3,36) = 8.282, p < 0.05 and p < 0.01, n = 9 per group; Fig. 13A–C). In Tau-KO mice, flow cytometry assays revealed a significant increase in the hippocampal cell populations labeled with EdU or antibodies against phosphorylated AKT; however, this effect was obviously reversed by AAV-mediated 2N4R Tau re-expression in the dDG [F(3,8) = 6.041, p < 0.05 (Fig. 13D); F(3,8) = 6.684, p < 0.05, n = 3 per group (Fig. 13F)]. Similarly, the levels of nuclear PI3K-p85 and cytosolic phosphorylated AKT were also reduced by 2N4R Tau re-expression (cytosol PI3K-p85: F(3,8) = 10.41, p < 0.05; cytosol p-AKT: F(3,8) = 8.475, p < 0.05; nucleus PI3K-p85: F(3,8) = 18.06, p < 0.01, n = 3 per group; Fig. 13G). However, re-expression of 2N4R Tau in the dDG did not reverse cocaine-induced hyperlocomotion in Tau-KO mice (n = 6 per group; Fig. 13H–J).
Tau regulates AHN by interacting with PI3K-p85 in the dDG during cocaine-associated memory formation. A, Schematic diagram of re-expression 2N4R Tau and EdU injection in Tau-KO mice with cocaine CPP conditioning. B, Re-expression 2N4R Tau in the dDG significantly attenuated cocaine CPP. C, Endogenous PI3K-p85 was immunoprecipitated with antibodies against PI3K-p85 from tissue lysates. Rabbit immunoglobulin G (IgG) was used as control. D–F, The rates of the cell populations positive for EdU, AKT, and phosphorylated AKT in the hippocampus were analyzed by flow cytometry. G, Re-expression of 2N4R Tau disrupts the nuclear translocation of PI3K-p85 in the dDG with cocaine CPP paradigm. H, Schematics of re-expression of 2N4R Tau in Tau-KO mice in cocaine-induced hyperlocomotion. I, Immunoblotting of 2A expression. J, Total distance within 15 min after daily cocaine administration. Data are the means ± SEM; *p < 0.05 and **p < 0.01. mNeonGreen visualizes directly the expression of 2N4R Tau; 2A refers to a 2A peptide, a tag protein representing 2N4R Tau expression.
The mutant 2N4R Tau-expressing AAV (AAV2/8-CMV-mutant 2N4R-2A-mNeonGreen-3Flag) was stereotactically infused into the dDG of Tau-KO mice before cocaine CPP training. As expected, mutant 2N4R Tau exerted little effect on cocaine memory formation, and only a small amount of 2N4R Tau bound to PI3K-p85 (F(3,24) = 4.202, p < 0.05, n = 7 per group; Fig. 14A–C). Besides, mutant 4R Tau also failed to reduce the number of the cells labeled with EdU or antibodies against phosphorylated AKT, or the nuclear PI3K-p85 levels and cytosolic phosphorylated AKT levels [F(3,8) = 4.987, p < 0.05 (Fig. 14D); F(3,8) = 6.436, p < 0.05 (Fig. 14F); cytosol p-AKT: F(3,8) = 9.029, p < 0.05, n = 3 per group (Fig. 14G)].
Cocaine CPP promotes PI3K-p85 nuclear translocation in adult hippocampal neurons. A, Schematic diagram of cocaine CPP experimental design and EdU injection. B, Re-expression mutant 2N4R Tau in the dDG did not significantly attenuate cocaine CPP score. C, Endogenous PI3K-p85 from dDG tissue of Tau-KO mice was immunoprecipitated with the antibody against PI3K-p85. Rabbit immunoglobulin G (IgG) was used as control. D–F, Hippocampal neuronal subpopulations positive for EdU, AKT, and phosphorylated AKT were analyzed by flow cytometry. G, Re-expression mutant 2N4R Tau showed no effect on PI3K-p85 nuclear translocation in the dDG with cocaine CPP training. Data are the means ± SEM; *p < 0.05. The upper right quadrants show the corresponding positive subpopulations. mNeonGreen visualizes directly the mutant 2N4R Tau expression; 2A refers to 2A peptide, a tag protein representing mutant 2N4R Tau expression.
Finally, to explore whether Tau-mediated the hippocampus neurons proliferation relay on PI3K-AKT signaling during cocaine memory formation, we applied LY294002 to pharmacologically suppress PI3K-AKT signaling in the dDG of Tau-KO mice. LY294002 was micro-infused into the dDG of mice through an implanted cannula before CPP training (Fig. 15A). As expected, the mice pretreated with LY294002 displayed a significant decrease in the cocaine CPP score (F(3,32) = 5.225, p < 0.05, n = 9 per group; Fig. 15B,C). In addition, cocaine-induced changes in the levels of PI3K-p110α and phosphorylated AKT were attenuated by LY294002 (PI3K-p110α: F(3,19) = 3.715, p < 0.05; p-AKT: F(3,19) = 4.243, p < 0.05, n = 6 per group; Fig. 15D). The proliferation rates of the neuronal subpopulations labeled with Ki67/SOX2 and Ki67/DCX were also reduced by the LY294002 treatment [F(3,9) = 15.89, p < 0.05 and p < 0.01 (Fig. 15E); F(3,9) = 17.13, p < 0.01, n = 3 per group (Fig. 15F)].
Pharmacological inhibition of dDG PI3K-AKT signaling suppresses adult hippocampal proliferating cells of Tau-KO mice with cocaine CPP paradigm. A, Schematic representation of the injection sites and photomicrographs of representative cannula placements in the dDG. B, Experimental timeline for cocaine CPP procedure. C, Inhibition of PI3K-AKT signaling in the dDG by LY294002 significantly attenuated cocaine CPP. D, Immunoblotting for PI3K-p85, PI3K-p110α, AKT, and phosphorylated AKT expression in the dDG. E, F, LY294002 inhibited hippocampal neuronal proliferation. Data are the means ± SEM; *p < 0.05 and **p < 0.01. The upper right quadrants show the corresponding positive subpopulations.
In summary, downregulation of 4R Tau reduces its association with PI3K-p85 and thus promotes PI3K-p85 nuclear translocation to activate PI3K-AKT signaling, which eventually promotes AHN and evokes cocaine-cue memory formation.
Discussion
Tau accumulation plays an important role in memory impairment (Yin et al., 2016; Li et al., 2019), and a decrease in Tau expression prevents neuronal loss, amyloid β-induced axonal transport deficits as well as memory impairment in tauopathy models (Vossel et al., 2010, 2015; DeVos et al., 2017). In the present study, we found a decreased expression of hippocampal Tau during cocaine memory formation; however, Tau expression remained unchanged in response to non-cocaine-conditioned stimuli. Furthermore, 4R Tau overexpression in the dDG disrupted cocaine memory formation, hippocampal neuronal proliferation and activity, and cocaine-seeking behaviors. In addition, cocaine-induced downregulation of 4R Tau clearly increased the expression of phosphorylated CREB, which is preferentially recruited or allocated to cocaine engram cells to encode memory consolidation (Hsiang et al., 2014). These findings suggest a novel mechanism that 4R Tau modulates cocaine-associated memory formation.
Distinct functions of tau isoforms in the DG during cocaine memory formation
The segregation of neuronal outputs along the dorsal-ventral axis and their connectivity affect neurogenesis in the DG and hippocampus-associated memory (Pierard et al., 2017). During the cocaine contextual memory formation, 4R Tau expression was reduced in the dDG, but not in the vDG. The difference observed in these two brain regions may be attributed to the functional dissociation that exists along the dorsal-ventral gradient in the hippocampus (Fanselow and Dong, 2010). Indeed, the dorsal hippocampus is more important for spatial learning and contextual discrimination than the ventral region (Wells et al., 2016; Pierard et al., 2017). In contrast, the ventral hippocampus is strongly associated with negative affective symptoms, such as emotional behavior and stress responses (Anacker et al., 2018).
The functional neuronal expression of Tau isoforms has been a subject of controversy and debate. During brain development, the expression of 3R Tau and 4R Tau isoforms shifts during AHN (Fuster-Matanzo et al., 2012), and 3R Tau provides a dynamic microtubule network in DCX-positive cells, allowing proper axonal growth during AHN (Llorens-Martin et al., 2012). Interestingly, cocaine decreased hippocampal 4R Tau expression, but had little effect on 3R Tau expression, suggesting different functions of Tau isoforms in specific neuron populations. Indeed, 3R Tau binds with a lower affinity to microtubules, potentially contributing to the cytoskeletal plasticity. In contrast, 4R Tau is mainly expressed in the adult brain, exhibits higher affinity for microtubules, and functions in the establishment and maintenance of the synaptic structure of newly integrated neurons (Lu and Kosik, 2001). Therefore, based on our findings, 4R Tau may contribute to the structural remodeling and integration of newborn neurons into the hippocampal circuit in response to drug conditioning.
4R Tau regulates cocaine-associated memory by modulating AHN
AHN is also a process in which adult-born hippocampal neurons are functionally integrated into the addiction network by interacting with addiction-related brain regions to contribute to cocaine memory formation (Castilla-Ortega et al., 2016b). In our study, AHN in the dDG was tightly regulated by 4R Tau during cocaine memory formation, and a reduction in cell proliferation inhibited memory formation in both the cocaine-paired CPP and self-administration paradigms. Previous studies reported that cocaine is a potent suppressor of AHN and may frequently reduce cell proliferation in rodents (García-Fuster et al., 2010; Sudai et al., 2011). However, these results were obtained from experiments in which rodents were forcibly administered cocaine in their home cage; moreover, the measurement of cell proliferation was usually performed immediately or soon after cocaine administration in an open-field environment (Xie et al., 2009). More importantly, these experiments lacked the cocaine-associated contextual stimuli that is critical for cocaine-cue memory formation.
Besides, AHN induced by a genetic, pharmacological or environmental approach is able to exacerbate cocaine-seeking behavior, showing a role of AHN in increasing the vulnerability to the action of drugs (Castilla-Ortega et al., 2017; Deroche-Gamonet et al., 2019; Castilla-Ortega and Santín, 2020). For example, through reducing AHN, mice exhibited higher motivation to cocaine self-administration and drug-seeking behaviors in the phase of reinstatement (Deroche-Gamonet et al., 2019). On the contrary, exercise before CPP training, which increases AHN, promotes cocaine memory formation (Mustroph et al., 2011). Therefore, we infer that that if cocaine CPP conditioning is conducted in the animals with increased numbers of adult-born hippocampal neurons, more young neurons might be recruited for CPP memory formation. It would explain why the cocaine-associated memory is more easily formed after increasing AHN (Mustroph et al., 2011). Accordingly, reducing AHN in the acquisition phase of cocaine memory formation may suppress the survival, maturation, and/or function integration of the new neurons involved in the memory circuits and eventually impair cocaine memory formation. However, once cocaine memory has been formed, such as in cocaine withdrawal phase, reducing AHN may fail to weaken the previous cocaine memory, which further trigger animal drug seeking and relapse during the period of abstinence (Deroche-Gamonet et al., 2019). Our findings highlight the existence of a dynamic population of proliferating neurons in the DG, and AHN clearly emerges as a robust phenomenon during the cocaine memory formation.
The interaction of 4R Tau with PI3K-p85 modulates PI3K-AKT signaling
By analyzing the Tau-interacting proteome, we revealed that PI3K-p85 is the key regulator of Tau-mediated AHN. Although accumulating studies have shown that PI3K-AKT signaling regulates hippocampal neuron survival and proliferation in the DG on exposure to external chronic stress, the role of PI3K-AKT signaling in cocaine memory formation has not been completely elucidated (Tiwari et al., 2015; Dioli et al., 2017). Only one study published to date reported an association of human Tau with PI3K-p85 in COS-7 cells, and the replacement of a few amino acid residues of human Tau drastically decreased its binding with PI3K-p85 (Reynolds et al., 2008). Here, we provide strong evidence that 4R Tau directly binds to PI3K-p85 and may restrict its nuclear translocation, thus modulating PI3K-AKT signaling. In turn, the cocaine-induced downregulation of 4R Tau may diminish the cytoplasmic localization of PI3K-p85 and subsequently increase its nuclear transportation, which is essential for cell proliferation and AHN.
In fact, as a critical regulatory subunit, PI3K-p85 interacts with several proteins to regulate the activation of the PI3K-AKT signaling pathway. For instance, the interaction of PI3K-p85 with bromodomain-containing protein 7 (BRD7; Chiu et al., 2014), C-type lectin-like receptor 2 or leucine zipper tumor suppressor 2 suppresses PI3K-AKT signaling (Wang et al., 2016; Xu et al., 2018). In contrast, the interaction of PI3K-p85 with CD133 activates PI3K-AKT signaling (Wei et al., 2013). Therefore, through its interacting proteins, PI3K-p85 appears to exert distinct functions in the regulation of PI3K-AKT signaling. In the present study, the cellular distribution of PI3K-p85 was tightly regulated by 4R Tau, and the 4R Tau deficiency induced PI3K-p85 nuclear translocation, thus promoting neuronal proliferation. Further studies are needed to investigate the nuclear transport dynamics of PI3K-p85 and its _precise function in neuronal proliferation during cocaine memory formation.
Collectively, 4R Tau regulates cocaine-associated memory through an AHN-dependent mechanism. This finding suggests the presence of unexplored mechanisms of cocaine-induced circuit plasticity in the hippocampus, and may have implications for the development of novel therapeutic strategies aimed at restoring a normal level of AHN or the disruption of the Tau-mediated synaptic plasticity that occurs in response to cocaine.
Footnotes
This work was partially supported by the National Natural Science Foundation of China Grants 82071494, 81871043, 81571301, and 81401105 and the 1·3·5 Project for Disciplines of Excellence, West China Hospital, Sichuan University Grant ZYGD18024. We thank professor Peng Lei, State Key Laboratory of Biotherapy in West China hospital of Sichuan University, for providing Tau-KO mice.
The authors declare no competing financial interests.
- Correspondence should be addressed to Xiaobo Cen at xbcen{at}scu.edu.cn