Donepezil hydrochloride

Anti‐pT205 (SAB,11108‐2), anti‐pT231 (SAB,11110), anti‐pS396 (SAB,11102), anti‐pS199 (Invitrogen, 44734G) and anti‐human PHF‐tau (AT8) (Thermo, MN1020), anti‐HT7 (Thermo, MN1000), anti‐Tau5 (Abcam, ab80579) and β‐actin (Abcam, ab6276), NeuN (CST,12943), ChAT (Chemicon, AB144P), Nissl staining solution (Beyotime, C0117), DAB‐staining kit (ZLI‐9031, ZSGB‐BIO), and donepezil hydrochloride (MedChemExpress, E2020) were obtained and used in the present study.
The virus AAV‐CAG‐hTau‐mCherry‐3flag and AAV‐CAG‐vector‐mCherry‐3flag were constructed and packaged based on the plasmid mCherry‐tau‐2N4R and mCherry‐vector‐2N4R, encoding hTau as described in previous study.¹⁵ The target gene was human microtubule‐associated protein tau (hTau) with gene ID: 4137. The serotype of the virus was AAV8, which has been generally employed in the nervous system.

Donepezil hydrochloride (MedChemExpress, E2020) was dissolved in normal saline (NS, 0.9% NaCl) as 10 mg/ml stock solution and was stored at −20°C. The Mice were divided into four experimental groups as follows: vector group treated with vehicle (Vec:veh), hTau mice treated with vehicle (hTau:veh), hTau mice treated with donepezil at low dose (1.0 mg/kg) (hTau:DZ[L]) and high‐dose (2.0 mg/kg) (hTau:DZ[H]), respectively. The NS and donepezil were administrated daily through intraperitoneal injection for 4 weeks, started at 5 months after the brain infusion of the AAV virus. All solutions were freshly prepared before daily injection.

5xFAD (n = 6) and WT littermate (n = 4) male mice at 6 months of age were used. Mice were treated with donepezil at 1 mg*kg⁻¹*day⁻¹. Donepezil hydrochloride (MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in water with 1.8% 2-hydroxypropyl-β-cyclodextrin and administered through drinking water. During treatment, donepezil was freshly dissolved in water each week. Prior to treatment and after 4 weeks of treatment, ABR and DPOAE tests were performed to evaluate auditory function, following the same procedure described in Section 2.2.