Mouse Models
3xTg mice were sourced from Jackson Laboratory (strain: 034830). The mice carry a transgene containing mutated human APPK670N/M671L and MAPTP301L, as well as a knock–in mutation Psen1M146V, on a heterozygous C57BL/6;129 background44. The mice exhibit AD–like neuropathology and a decline in long–term memory around 3 to 4 months of age26. Fshr mutants were bred and maintained at the Icahn School of Medicine at Mount Sinai (ISMMS), with heterozygotes on 129T2svEmsJ27. The two strains were crossed to produce viable F1 hybrid 3xTg+/–;Fshr+/– littermates. The latter were subsequently crossed with 3xTg+/+ mice to generate compound 3xTg+/+;Fshr+/– mice, which were then crossed to create 3xTg+/+;Fshr+/+; 3xTg+/+; Fshr+/– and 3xTg+/+;Fshr–/– mice (hitherto termed 3xTg;Fshr+/+, 3xTg;Fshr+/–, and 3xTg;Fshr–/– mice). Half of the animals in each group underwent either ovariectomy or sham operation. 90–day, slow–release pellets containing 0.36 mg 17β-estradiol were inserted in the unoperated and sham–operated 3xTg;Fshr–/– to normalize their estrogen level, as before29. APP/PS1 mice were obtained from Jackson Laboratory (strain: 34829) and maintained at ISMMS. The mice carry a human transgene containing APPK670N/M671Land PSEN1ΔE9 mutations36. All experimental mice were grouped–housed to reduce single–house stress, under a 12–hour light/dark cycle with food and water ad libitum. Behavioral tests were performed at ages 5 and 10 months for the Fshr;3xTg mutants, and at 15 months for APP/PS1 mice. All tests were conducted in the light phase, and the order of behavioral tests was the same for each mouse. The protocols were reviewed and approved by the ISMMS Institutional Animal Care and Use Committee.
Surgery
For ovariectomy and sham operation, mice were anesthetized using ketamine/xylazine and received a prophylactic dose of meloxicam to alleviate potential post-surgical pain or distress. After anesthesia, the lower back was shaved, cleaned with 70% ethanol, and washed with povidone prior to the surgery. A ~1 cm skin incision was made, followed by an incision through the muscle layer to access the peritoneal cavity. The ovaries were identified, extracted one at a time through the incision, tied off, and removed. The muscle layer was sutured, and the external incision was closed using wound clips. For pellet implantation, a 0.5 cm incision was created in the skin at the nape of the neck of the 3xTg;Fshr–/– mice. A small pocket was carefully dissected towards the caudolateral area behind the ear, where the 90–day, slow–release pellet containing 0.36 mg 17β-estradiol was placed using tweezers. The incision was closed with a wound clip. Each mouse was placed in a clean cage, allowed to recover from anesthesia, and returned to the home cage after exhibiting normal behavior and ambulation. Meloxicam was continued 24, 48, and 72 hours after surgery.
Reagents
ELISA kits for human Aβ40 (Cat. #KHB3481) and Aβ42 (Cat. #KHB3544) were purchased from Invitrogen and FSH (Cat. #MPTMAG-49K) were purchased from Millipore. The 90–day, slow–release pellets containing 0.36 mg 17β-estradiol were purchased from Innovative Research of America (Cat. #NE121).
Digital PCR
Fshr mRNA levels were quantified using droplet digital PCR (ddPCR). In brief, RNA was isolated by TRIzol (Life Technologies). Reverse transcription was performed using SuperScript III reverse transcriptase (Life Technologies). Isolated RNA was used to perform ddPCR to determine Fshr mRNA levels using FSHR Probe (Life Technologies, Cat. #4331182). Droplets containing the cDNA were generated using a Biorad Droplet generator (QX200) by mixing with droplet generator oil (Cat. #D9161172A), and the formed droplets were amplified using a thermocycler (Applied Biosystems) and analyzed using droplet reader (Biorad).
Behavioral Tests
Behavioral testing, described by us previously45, consisted of two memory tests conducted in the order of increasing invasiveness in the following order: Novel Object Recognition and Morris Water Maze. Mice received 3 days of resting time between tests to decrease carryover effects from prior tests. Mice were habituated to the testing room for 30 minutes at the beginning of each test day. The order of tests in which mice were tested was the same across all mice. Each mouse was tested once per test. The behavioral room wall cues remained the same for all tasks and the same experimenter conducted all of the tests. All test trials were video–recorded, tracked, and analyzed with ANY-maze tracking software (v 7.2; Stoelting, Wood Dale, IL). Locomotor activity data for each test are summarized in Table S1.
Novel Object Recognition Test
The Novel Object Recognition test was performed in square test boxes (40x40x35 cm) with even lighting conditions (30 ± 5 lux). Each test box consisted of grey steel bottom plate, white Perspexunder a camera mounted above all boxes. A tower of Lego bricks and Falcon tissue culture flask filled with sand were used as objects27. Prior to the experiments, both objects were tested with a separate cohort of mice to exclude that mice showed a preference for either object or side preference due to the behavioral room conditions. Sample object and the novel object placement followed a counterbalanced design between trials to control for order and location effects.
The test consisted of two trials––training and testing––separated by 6 hours. In the training trial, mice were placed into the test box containing two equal sample objects (e.g., flasks), in front of the south wall facing away from the objects. Each mouse was allowed to explore the objects for 10 minutes before it was returned to its home cage. After 6 hours, the testing was conducted by placing the mouse into the same test box again, but containing one sample (familiar) object and one unfamiliar object (a flask and a Lego bricks tower) and object interaction was recorded for 10 minutes. After each trial, the objects and boxes were cleaned with a Quatricidedilution to eliminate odor cues. The maze was cleaned using 70% ethanol between each trial. Object interaction was defined as an event where a mouse’s head was within 2 cm of the object and directed towards the object, excluding sitting on the objects28,29. For the training trial, object Interaction [%] was calculated as [sample object interaction time]/[total test time] x 100%. For the testing trial, object interaction [%] was calculated as [novel object interaction time]/[total object interaction time] x 100%30. Mice with less than 5% of total object interaction in either trial were excluded from the analysis31.
Morris Water Maze
To test spatial memory accusation and retrieval, we used the Morris Water Maze test (adapted from Vorhees et al.)32. This utilized a circular pool (150 cm diameter) filled with water (26 ± 1°C; 10 cm distance from water surface to wall rim) made opaque with non–toxic tempera paint. A circular rescue platform (diameter: 11 cm; distance between platform center point and pool wall: 27 cm) was submerged 1–1.5 cm below the water surface and the testing area was illuminated with indirect lighting (150 ± 10 lux) to avoid reflections. To monitor animals during trials, a camera was mounted to the ceiling centrally above the pool. The water maze was surrounded by black–and–white extra–maze cues on the walls of the room. Repeated episodes of excessive floating (>10 seconds and/or ≥25% of trial across five days of training) was rare and found in only 6 mice during the entire study. These mice were excluded from the analysis a priori32.
For spatial acquisition trials, a submerged rescue platform, invisible to the mice was used. To locate the escape platform, mice used the extra–maze cues. The platform location remained the same for all trials, whereas the starting location was varied between trials. Mice had 60 seconds to find the rescue platform, after which they were guided there. Each mouse performed four trials per day over 5 days with an inter–trial interval of 15 to 20 minutes. Mice that failed to locate the platform during the 60 second trial, were placed on the platform for 15 seconds immediately after the end of the trial. At the end spatial acquisition day 5, mice were housed back in home cage for 24 hours (day 6). The retention trial was conducted on day 7 without additional training. For the retrieval trials, the rescue platform was removed from the pool and the mouse was allowed to swim for 60 seconds. Mice with extensive floating of >25% of the trial time were removed from the entire analysis a priori. For the spatial acquisition trials, the mean latency to reach the platform was calculated for each test. For the retention trials, the percent of the time spent in the platform zone (40 cm diameter surrounding the platform center point) was analyzed32 (See Supplementary Videos 1 and 2 for examples of unimpaired and impaired 3xTg mice in the Morris Water Maze testing).
Statistical Analysis
Statistical analyses were performed using GraphPad Prism v.10. For molecular analyses, the tests were either unpaired two-tailed Student’s t-test (two-group comparison) or one–way ANOVA followed by Fisher’s least significant difference post hoc test (more than two groups). Differences with P≤0.05 were considered significant. P values are annotated in the figures and are provided in the Source Data Files. For behavioral analyses, repeated measures two–way ANOVA followed by Fisher’s Least Significant Difference post–hoc test (more than two groups), one–way ANOVA or two–tailed Student’s t-test (two–group comparison) were utilized. Differences with P ≤0.05 were considered significant.