Generation and characterization of Prosapip1(flx/flx) mice

(A) Identification of guide RNA binding sites in intron 2 and the 3’ UTR of exon 5 of Prosapip1. (B) PAGE purified Ultramer single stranded DNA oligos that were homologous to the target loci in intron 2 and exon 5 were used as repair templates. (C) Genetic crossing scheme of the Prosapip1(flx/flx);Syn1-Cre mice. Male Prosapip1(flx/flx);Syn1-Cre(−/−) mice were mated with female Prosapip1(flx/flx);Syn1-Cre(+/−) mice, leading to litters of Prosapip1(flx/flx);Syn1-Cre(−/−) or Prosapip1(flx/flx);Syn1-Cre(+/−) mice. (D) The dHP of C57BL/6, Prosapip1(flx/flx);Syn1-Cre(-) and Prosapip1(flx/flx);Syn1-Cre(+) mice was dissected. Prosapip1 was detected using anti-Prosapip1 antibodies. GAPDH was used as a loading control. (E) Histogram of litter size from Prosapip1 mating pairs. X axis depicts number of pups per litter, while Y axis depicts numbers of litters at that size. (F) Proportion of male and female offspring from Prosapip1 matings. (G) Proportion of Cre(-) and Cre(+) offspring from Prosapip1 matings. (H) Body weight of male and female Prosapip1(flx/flx);Syn1-Cre(-) and Prosapip1(flx/flx);Syn1-Cre(+) mice was measured biweekly from birth to assess overt developmental deficits. Data are represented as mean±SEM and analyzed using three-way ANOVA (Table 1). n = 10 (Prosapip1(flx/flx);Syn1-Cre(-) male), 5 (Prosapip1(flx/flx);Syn1-Cre(-) female), 11 (Prosapip1(flx/flx);Syn1-Cre(+) male), 11 (Prosapip1(flx/flx);Syn1-Cre(+) female). (I-J) Mice were placed in an open field and locomotion was recorded for 20 minutes. Total distance traveled during the open field test (I) and time spent in the center of the open field (J). Data represented as mean±SEM and analyzed using an unpaired t-test (Table 1). ns, non-significant. n = 22 (Prosapip1(flx/flx);Syn1-Cre(-)), 21 (Prosapip1(flx/flx);Syn1-Cre(+)).

Statistics

Prosapip1 is required for synaptic localization of PSD proteins

(A) The levels of Prosapip1 (top), Synapsin (middle), and CREB (bottom) in the dorsal hippocampus of Prosapip1(flx/flx);Syn1-Cre(-) mice were measured in the total and crude synaptosomal fraction. (B-I) Total levels of SPAR (B), Shank3 (D, F), and PSD-95 (D, H) alongside the synaptic levels of SPAR (C), Shank3 (E, G), and PSD-95 (E, I) were measured in the dHP of Prosapip1(flx/flx);Syn1-Cre(-) and Prosapip1(flx/flx);Syn1-Cre(+) mice using western blot analysis. Protein levels were normalized to GAPDH and presented as a percentage of the average of the Prosapip1(flx/flx);Syn1-Cre(-) values. Data are represented as mean±SEM and analyzed using unpaired two-tailed t-test with Welch’s correction (Table 1). **p<0.01, ****p<0.0001; ns, non-significant. n = 5 per group (B-G), 9 (Prosapip1(flx/flx);Syn1-Cre(-)) and 10 (Prosapip1(flx/flx);Syn1-Cre(+)) (H-I). (J-Q) The total levels of GluN2A (J, L), GluN2B (J, N), and GluA1 (J, P) and the synaptic levels of GluN2A (K, M), GluN2B (K, O), and GluA1 (K, Q) were measured dHP of Prosapip1(flx/flx);Syn1-Cre(-) and Prosapip1(flx/flx);Syn1-Cre(+) mice via western blot analysis. Protein levels were normalized to GAPDH and presented as a percentage of the average of the Prosapip1(flx/flx);Syn1-Cre(-) values. Data are represented as mean±SEM and analyzed using unpaired two-tailed t-test with Welch’s correction (Table 1). *p<0.05, **p<0.01, ***p<0.001; ns, non-significant. n = 4 (Prosapip1(flx/flx);Syn1-Cre(-)), 5 (Prosapip1(flx/flx);Syn1-Cre(+)).

Prosapip1 in the dorsal hippocampus plays a role in NMDA receptor-mediated transmission and long-term potentiation

(A) Location of stimulating and recording electrodes within the hippocampal CA1 region. (B) Sample field excitatory postsynaptic potential (fEPSP) traces recorded before (pre) and after (post) administering high-frequency stimulation (HFS) (100Hz, 100 pulses every 20 seconds) in both Prosapip1(flx/flx);Syn1-Cre(-) (Cre(-)) and Prosapip1(flx/flx);Syn1-Cre(+) (Cre(+)) groups. (C) A stable baseline of fEPSPs was established for 10 minutes before application of HFS and fEPSPs were recorded for 30 minutes after HFS. Time course of fEPSPs before and after HFS. (D) Quantification of average of fEPSP amplitudes measured between 30-40 minutes. Data are represented as mean±SEM and analyzed using unpaired two-tailed t-test (Table 1). **p<0.01. n = 10 slices from 6 mice (10/6) (Prosapip1(flx/flx);Syn1-Cre(-)) and (9/5) (Prosapip1(flx/flx);Syn1-Cre(+)). (E) Cells were clamped at −65 mV and the bath contained both DNQX and PTX to block AMPA and GABA mediated responses. Voltage clamp whole cell recordings and representative electrically evoked NMDA currents in Prosapip1(flx/flx);Syn1-Cre(-) (Cre(-)) and Prosapip1(flx/flx);Syn1-Cre(+) (Cre(+)) mice at four stimulation intensities (left). Summarized responses for Prosapip1(flx/flx);Syn1-Cre(-) (Cre(-)) and Prosapip1(flx/flx);Syn1-Cre(+) (Cre(+)) CA1 neurons quantified by average at each stimulating intensity; #p < 0.05 by two-way repeated measures ANOVA followed by post-hoc Tukey test (Prosapip1(flx/flx);Syn1-Cre(-) vs. Prosapip1(flx/flx);Syn1-Cre(+)) at the same stimulating intensities, *p<0.05. n = 14/4 (Prosapip1(flx/flx);Syn1-Cre(-)) and 11/4 (Prosapip1(flx/flx);Syn1-Cre(+)). (F) Representative currents evoked in the CA1 neurons after NMDA bath application (20 uM, 30s) in Prosapip1(flx/flx);Syn1-Cre(-) (Cre(-)) and Prosapip1(flx/flx);Syn1-Cre(+) (Cre(+)) mice (left). Average of the peak current elicited by each mouse (right). Data are represented as mean±SEM and analyzed using unpaired two-tailed t-test (Table 1). **p<0.01. n = 13/4 (Prosapip1(flx/flx);Syn1-Cre(-)) and 11/4 (Prosapip1(flx/flx);Syn1-Cre(+)). (G) In voltage clamp recordings, 2 electrical stimulations (100 ms separation) were provided to elicit 2 responses in the CA1 neurons. PPR was calculated as the amplitude of peak 2/amplitude of peak 1. Representative paired-pulse ratio in Prosapip1(flx/flx);Syn1-Cre(-) (Cre(-)) and Prosapip1(flx/flx);Syn1-Cre(+) (Cre(+)) mice (left). Average PPR for Prosapip1(flx/flx); Syn1-Cre(-) (Cre(-)) and Prosapip1(flx/flx);Syn1-Cre(+) (Cre(+)) (right). Data are represented as mean±SEM and analyzed using unpaired two-tailed t-test (Table 1). *p<0.05. n = 11/3 (Prosapip1(flx/flx);Syn1-Cre(-)) and 13/4 (Prosapip1(flx/flx);Syn1-Cre(+)).

Prosapip1 contributes to recognition, social, and spatial memory

(A) Mice underwent the novel object recognition test, where they were first allowed to explore two similar objects. After 24 hours, one of the familiar objects was replaced by a novel object, and mice were again allowed to explore and interact with the objects. Time spent interacting with the familiar and novel object. n = 22 (Prosapip1(flx/flx);Syn1-Cre(-)), 20 (Prosapip1(flx/flx);Syn1-Cre(+)). (B-C) In the novelty T-maze test, mice were allowed to explore two arms of a three-armed, T-shaped maze. There were 5 training trials separated by a 1-minute inter-trial interval. During testing, the third “novel” arm was unblocked and allowed to be explored. (B) Difference score (time exploring novel arm – time exploring familiar arm) performance during the novelty T-maze test. A positive difference score indicates preference for the novel arm of the maze. n = 22 (Prosapip1(flx/flx);Syn1-Cre(-)), 19 (Prosapip1(flx/flx);Syn1-Cre(+)). (C) Heat map of group average time spent in each arm of the T-maze during the test trial. (D-E) Mice performed the 3-chamber social interaction test. Specifically, they were placed in the center chamber of a 3-chamber apparatus and allowed to freely explore for 15 minutes for two trials. During the first trial, one chamber was paired with a juvenile interaction partner (social), while the other chamber contained only the empty interaction cage (empty). During the second trial, one chamber was paired with the familiar mouse from the first trial (familiar), and the other chamber contained a novel juvenile interaction partner (novel). (D) Time spent in the empty and social-paired chamber respectively in the first portion of the 3-chamber social interaction test. n = 14 (Prosapip1(flx/flx);Syn1-Cre(-)), 17 (Prosapip1(flx/flx);Syn1-Cre(+)). (E) Time spent in the familiar and novel chamber during the second portion of the 3-chamber social interaction test. n = 14 (Prosapip1(flx/flx);Syn1-Cre(-)), 17 (Prosapip1(flx/flx);Syn1-Cre(+)). (F-K) Mice performed the Barnes maze test, where they were placed in the center of a white plastic platform with 40 uniformly distributed holes around the perimeter, one of which had an exit compartment placed underneath. The goal of the trial was to escape into the exit compartment. There were 4 training trials a day over the course of 4 days, separated by an inter-trial interval of 30 minutes. 24 hours after the last training trial mice were placed back onto the platform but with the exit compartment removed (probe trial) and allowed to explore for 5 minutes. (F) Average distance traveled from start point to exit during the Barnes maze training trials. n = 9 (Prosapip1(flx/flx);Syn1-Cre(-)), 12 (Prosapip1(flx/flx);Syn1-Cre(+)). (G) Primary (filled circles) and secondary (hollow circles) errors committed during Barnes maze training. Primary errors are an incorrect hole visit and secondary errors are an incorrect hole revisit. n = 9 (Prosapip1(flx/flx);Syn1-Cre(-)), 12 (Prosapip1(flx/flx);Syn1-Cre(+)). (H) The method of searching utilized by each mouse for each training trial was qualified. Example path to exit from mice exhibiting random, serial, and spatial search strategies. (I-J) Ratio of search strategy utilization by Prosapip1(flx/flx);Syn1-Cre(-) (I) and Prosapip1(flx/flx);Syn1-Cre(+) (J) mice during Barnes maze training. (K-L) Time spent in exit-associated quartile during the probe trial and associated heatmaps (L). n = 9 (Prosapip1(flx/flx);Syn1-Cre(-)), 12 (Prosapip1(flx/flx);Syn1-Cre(+)). Data are represented as mean±SEM and analyzed using two-way ANOVA (A,C,D,E), Welch’s t-test (B), three-way ANOVA (F), or Mann-Whitney test (J) (Table 1). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Prosapip1 in the dorsal hippocampus contributes to recognition, social, and spatial memory

(A) Images of AAV-GFP and AAV-Cre overexpression in the dHP of Prosapip1(flx/flx) mice. (B) Western blot analysis of Prosapip1 protein level in the dHP in non-infected mice compared to mice infected with AAV-Cre. (C-D) Mice infected with AAV-GFP or AAV-Cre in the dHP were placed in an open field, and their behavior was recorded for 20 minutes. The total distance traveled (C) and the time spent in the center of the field (D) were measured during the test. n = 19 (AAV-GFP), 15 (AAV-Cre). (E) Mice infected with AAV-GFP or AAV-Cre in the dHP were placed on the light side of a light/dark box apparatus and allowed to explore for 10 minutes. Time spent in the dark and light chamber during the light/dark box test. n = 19 (AAV-GFP), 15 (AAV-Cre). (F) Mice infected with AAV-GFP or AAV-Cre in the dHP underwent the novel object recognition test. Briefly, they were familiarized with two similar objects before one was switched for a novel object after 24 hours. Cumulative time spent exploring the familiar and novel object during the novel object recognition test. n = 16 (AAV-GFP), 17 (AAV-Cre). (G) Mice infected with AAV-GFP or AAV-Cre in the dHP underwent the novelty T-maze test, where they were first allowed to explore two arms of a three-armed, T-shaped maze. During the testing phase, the third “novel” arm was unblocked and made available for exploration. Difference score (time exploring novel arm – time exploring familiar arm) of time spent exploring the novel arm of the T-maze. A heatmap of each average group performance during the test is also presented. n = 18 (AAV-GFP), 15 (AAV-Cre). (H-I) Mice infected with AAV-GFP or AAV-Cre in the dHP performed the 3-chamber social interaction test. Briefly, they were placed in the center chamber and allowed to freely explore for 15 minutes during two trials. In the first trial, one chamber had a juvenile interaction partner, and the other was empty. In the second trial, one chamber had the familiar mouse from the first trial, and the other had a new juvenile interaction partner. (H) Cumulative time spent in the empty and social chamber of the 3-chamber social interaction test. n = 18 (AAV-GFP), 15 (AAV-Cre). (I) Cumulative time spent in the familiar and novel chambers in the 3-chamber social interaction test. n = 18 (AAV-GFP), 15 (AAV-Cre). (J-K) Mice infected with AAV-GFP or AAV-Cre in the dHP underwent the Barnes maze experiment. They were placed in the center of a platform with 40 evenly spaced holes around the perimeter, one of which led to an exit compartment. Mice underwent 4 training trials per day for 4 days, with 30-minute intervals between trials. Twenty-four hours after the last training trial, they were placed back on the platform without the exit compartment (probe trial) and allowed to explore for 5 minutes. (J) Average distance traveled from start point to exit during the Barnes maze training trials. n = 8 (AAV-GFP), 6 (AAV-Cre). (K) Time spent in the exit quartile during the probe trial. n = 8 (AAV-GFP), 6 (AAV-Cre). Data are represented as mean±SEM and analyzed using two-way ANOVA (E, F, H, I, J), Welch’s t-test (C, D, G), or Mann-Whitney test (K) (Table 1). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Prosapip1 knockout did not affect Barnes maze exploratory behavior or baseline anxiety

(A) Before the Barnes maze training trials began, mice were placed on the platform to assess baseline exploratory behavior and locomotion in the environment. The exit compartment was in a different location than training trials. Distance traveled during the Barnes maze habituation trial. Data represented as mean±SEM and analyzed using unpaired t-test (Table 1). ns, non-significant. n = 6 (Prosapip1(flx/flx);Syn1-Cre(-)), 12 Prosapip1(flx/flx);Syn1-Cre(+). (B) To assess anxiety, mice were placed on the light side of a light/dark box apparatus and allowed to explore for 10 minutes. Time spent in the light and dark chamber during the test. Data represented as mean±SEM and analyzed using a two-way ANOVA (Table 1). ****p<0.0001. n = 15 (Prosapip1(flx/flx);Syn1-Cre(-)), 16 (Prosapip1(flx/flx);Syn1-Cre(+)). (C) Mice were placed in the center of a plus-shaped maze and allowed to explore two closed and two open arms for 5 minutes to assess anxiety and exploratory behavior. Time spent in the open arm of the elevated plus maze. Open-arm time was measured and analyzed using unpaired two-tailed t-test (Table 1). ns, non-significant. n = 7 (Prosapip1(flx/flx);Syn1-Cre(-)), 17 (Prosapip1(flx/flx);Syn1-Cre(+)).