All animal experiments were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee at Waseda University (Approval Nos. 2021-A022, 2022-A032, and A23-051). Mice were housed under standard laboratory conditions with a 12-h light/dark cycle and had ad libitum access to food and water. Heterozygote P301S tau transgenic mice (PS19 line), originally generated in a mixed-genetic background [25], were backcrossed to the C57BL/6 J strain and maintained on this background.

To generate P301S tau Tg; CRMP2KI/KI mice (referred to as tau Tg; CRMP2KI), male P301S tau Tg mice were crossed with female CRMP2KI/KI mice, producing F1 offspring heterozygous for CRMP2KI (P301S tau Tg; CRMP2KI/ +). These F1 P301S tau Tg; CRMP2KI/ + mice were bred with CRMP2 KI/KI mice to generate P301S tau Tg; CRMP2KI/KI mice. In this mating scheme, the P301S tau Tg was consistently maintained in the hemizygous state to ensure uniform transgene copy number. Mice with the same genetic background, including P301S tau Tg and P301S tau Tg; CRMP2KI/KI, were used for subsequent analyses to minimize genetic variability.

Aβ25–35 peptide monomers were obtained from Peptide Institute Inc. (Japan). The peptides were dissolved in sterile water at a concentration of 1 µg/µL, aliquoted, and stored at −20℃ until use. To induce oligomerization, the dissolved Aβ peptides were incubated at 37℃ for 4 days, then aliquoted and stored at −80℃ [26]. Sterile phosphate-buffered saline (1xPBS) was prepared and used as a control in the experiments.

Wild-type (WT), CRMP2KI, tau Tg, and tau Tg; CRMP2KI male mice (6–7 months old) were anesthetized using isoflurane (2–3% for induction and 1–2% for maintenance) and placed in a stereotaxic apparatus (Narishige SR-5 M-HT). Under aseptic surgical conditions, a midline incision was made above the designated injection site, and a segment of the cranial bone overlying the target area was carefully removed using a surgical drill.

Aβ25–35 or sterile PBS (control) was unilaterally injected into the dentate gyrus (DG) of the hippocampus. The injection coordinates were as follows: anterior/posterior: −1.94 mm, medial/lateral: + 1.4 mm, dorsal/ventral: −2.2 mm from the brain surface. Two minutes after the needle insertion to the targeted injection site, the solution was infused at a controlled flow at a rate of 1 µL/min at a volume of 3.0 µL per brain. A 5-µL Hamilton syringe fitted with a 10-gauge beveled needle and connected to an injector pump was used for the infusion. The syringe was secured to the stereotactic frame to ensure the stability during the injection process. To enhance the diffusion, the needle was kept in place for an additional 2 min after the infusion was complete. Subsequently, the incision site was sutured, and the mice were allowed to recover from anesthesia on a 37℃ heating pad before being returned to a sanitized housing environment.

At 30 days post-injection, the mice were perfused with 4% paraformaldehyde (PFA) in PBS and their brains were harvested for further analysis.

All mice used for immunohistochemistry were perfused with 4% PFA in PBS following intraperitoneal injection of a triple anesthesia cocktail (medetomidine hydrochloride 0.3 mg/kg, midazolam 4 mg/kg, and butorphanol tartrate 5 mg/kg). The brains were dissected and post-fixed overnight (O/N) in 4% PFA/PBS. Subsequently, the brains were rinsed in 1xPBS at 4 °C O/N, followed by sequential incubation in 10% sucrose/PBS and 20% sucrose/PBS for 4–8 h.

Brain samples were embedded in a mixture of Tissue Tek optimal cutting temperature (OCT) compound (Sakura) and 20% sucrose (2:1 ratio) for cryostat sectioning. Coronal Sects. (15 µm thick) were prepared using a Cryocut 1800 cryostat (Leica), mounted on MAS-coated glass slides (Matsunami), and stored at −20℃.

Tissue sections were washed in PBS for 30 min and blocked in 3% bovine serum albumin (BSA, SIGMA) diluted in 0.1% Triton X-100 (Wako) in PBS (PBSTr) for 10 min. Antigen retrieval was performed to unmask antigen epitopes by heating sections in citrate buffer (1.47 g citrate in 500 mL water) using a microwave or autoclave. Sections were then blocked in 0.01% PBSTr for 1 h at room temperature (RT), followed by incubation with the primary antibody at 4℃ O/N. After three 10-min washes in 0.01% PBSTr, the sections were incubated with secondary antibodies (Alexa Fluor 594 goat polyclonal antibody (pAb) anti-mouse IgG, Abcam, and Alexa Fluor 647 donkey pAb anti-rabbit IgG, Abcam) for 1 h at RT. The slides were then washed three times with 0.01% PBSTr, dried, and mounted using Fluormount™ Aqueous Mounting Medium (Sigma-Aldrich).

Fluorescent images were acquired using an inverted confocal laser microscope (FV3000, Olympus) equipped with UPlanSApo 40 × oil immersion objective lens (NA 0.95). Images were acquired as z-stacks with eight slices at 1.4-µm intervals. Immunostained signals were analyzed using NIH ImageJ software. The mean gray value of immunolabeling was measured by subtracting the background signal.

Mouse monoclonal anti-phospho-tau (S202, T205) (AT8; RRID: AB_223647) and anti-total tau (Tau-5; RRID: AB_2536235) antibodies were purchased from Invitrogen. Rabbit polyclonal anti-Iba1 antibody (RRID: AB_839504) was purchased from FUJIFILM. Rabbit polyclonal anti-CRMP2 phospho-T509 antibody (Rabbit/IgG1, 1:500) recognizes the pT509 epitope of CRMP2 [24].

Statistical analyses were conducted using GraphPad Prism 6 software. An unpaired two-tailed Student’s t-tests with Welch’s correction was applied for pairwise comparisons, while one-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used for multiple comparisons. Data are presented as the mean ± standard error of the mean (SEM).