1. Neuroscience

Prosapip1 (encoded by the Lzts3 gene) in the dorsal hippocampus mediates synaptic protein composition, long-term potentiation, and spatial memory

  1. Zachary W Hoisington
  2. Himanshu Gangal
  3. Khanhky Phamluong
  4. Chhavi Shukla
  5. Jeffrey J Moffat
  6. Alexandra Salvi
  7. Gregg Homanics
  8. Jun Wang
  9. Yann Ehinger  Is a corresponding author
  10. Dorit Ron  Is a corresponding author
  1. Alcohol and Addiction Research Group, Department of Neurology, University of California, San Francisco, United States
  2. Department of Neuroscience and Experimental Therapeutics, School of Medicine, Texas A&M University Health Science Center, United States
  3. Department of Anesthesiology and Perioperative Medicine, University of Pittsburgh, United States
https://doi.org/10.7554/eLife.100653.3
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Cite this article
  1. Zachary W Hoisington
  2. Himanshu Gangal
  3. Khanhky Phamluong
  4. Chhavi Shukla
  5. Jeffrey J Moffat
  6. Alexandra Salvi
  7. Gregg Homanics
  8. Jun Wang
  9. Yann Ehinger
  10. Dorit Ron
(2025)
Prosapip1 (encoded by the Lzts3 gene) in the dorsal hippocampus mediates synaptic protein composition, long-term potentiation, and spatial memory
eLife 13:RP100653.
https://doi.org/10.7554/eLife.100653.3
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7 figures, 2 tables and 1 additional file

Figures

Figure 1
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Generation and characterization of Prosapip1 cKO mice.

(A) Identification of guide RNA binding sites in intron 2 and the 3’ UTR of exon 5 of Lzts3. (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 Lzts3fl/fl;Syn1-Cre mice. Male Lzts3fl/fl;Syn1-Cre(-) mice were mated with female Lzts3fl/fl;Syn1-Cre(+) (Prosapip1 cKO) mice, leading to litters of control (Lzts3fl/fl;Syn1-Cre(-)) or Prosapip1 cKO (Lzts3fl/fl;Syn1-Cre(+)) mice. (D) The dHP of C57BL/6, control and Prosapip1 cKO mice was dissected. Prosapip1 was detected using anti-Prosapip1 antibodies. GAPDH was used as a loading control. (E) Histogram of litter size from Lzts3fl/fl × Syn1-Cre 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 Lzts3fl/fl × Syn1-Cre matings. (G) Proportion of Cre(-) and Cre(+) offspring from Lzts3fl/fl × Syn1-Cre matings. (H) Body weight of male and female Prosapip1 cKO and control 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 (control male), 5 (control female), 11 (Prosapip1 cKO male), 11 (Prosapip1 cKO female). (I–J) Mice were placed in an open field and locomotion was recorded for 20 min. 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 (control), 21 (Prosapip1 cKO).

Figure 1—source data 1

File containing raw data for Figure 1.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig1-data1-v1.zip
Figure 1—source data 2

Original files for western blot analysis displayed in Figure 1D.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig1-data2-v1.zip
Figure 2 with 1 supplement
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Prosapip1 is required for synaptic localization of PSD proteins.

(A) The levels of Prosapip1 (top), Synapsin (middle), and cAMP response element-binding protein (CREB) (bottom) in the dorsal hippocampus of control (Lzts3fl/fl;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 cKO and control mice using western blot analysis. Protein levels were normalized to GAPDH and presented as a percentage of the average of the control mice values. Data are represented as mean ± SEM and analyzed using an 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 (control) and 10 (Prosapip1 cKO) (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 in the dHP of Prosapip1 cKO and control mice using western blot analysis. Protein levels were normalized to GAPDH and presented as a percentage of the average of the control mice values. Data are represented as mean ± SEM and analyzed using an unpaired two-tailed t-test with Welch’s correction (Table 1). **p<0.01; ns, non-significant. n=4 (control), 5 (Prosapip1 cKO).

Figure 2—source data 1

File containing raw data for Figure 2.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig2-data1-v1.xlsx
Figure 2—source data 2

Original files for western blot analysis displayed in Figure 2A–E, J and K.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig2-data2-v1.zip
Figure 2—figure supplement 1
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Verification of the crude synaptic preparation.

(A) Tissue was first homogenized in Krebs buffer. A portion of the homogenate was saved as total homogenate, and the remaining homogenate was diluted and centrifuged. The supernatant (S1) was collected, and the pellet (P1) was saved. The process was repeated, and the S2 supernatant was saved, while the pellet (P2) was kept on ice. The resulting pellet contained the synaptosomal fraction. (B) The levels of PSD-95, Synapsin, CREB, GAPDH, Actin, GluN2B, GluN2A, and GluA1 were measured via western blot analysis in the total homogenate (T), the nuclei and large debris fraction (P1), the non-synaptic fraction (S2), and the crude synaptosomal fraction (P2).

Figure 2—figure supplement 1—source data 1

File containing raw data for Figure 2—figure supplement 1.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 1—source data 2

Original files for western blot analysis displayed in Figure 2—figure supplement 1B.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig2-figsupp1-data2-v1.zip
Figure 3
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Prosapip1 in the dHP 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 of field excitatory postsynaptic potential (fEPSP) traces recorded before (pre) and after (post) administering high-frequency stimulation (HFS) (100 Hz, 100 pulses every 20 s) in Prosapip1 cKO (Cre(+)) and control (Cre(-)) mice. (C) A stable baseline of fEPSPs was established for 10 min before application of HFS and fEPSPs were recorded for 30 min after HFS. Time course of fEPSPs before and after HFS. (D) Quantification of average fEPSP amplitudes measured between 30–40 min. 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) (Cre(-)) and (9/5) (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 control (Cre(-)) and Prosapip1 cKO (Cre(+)) mice at four stimulation intensities (left). Summarized responses of control (Cre(-)) and Prosapip1 cKO (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 (Cre(-) vs. Cre(+)) at the same stimulating intensities, *p<0.05. n=14/4 (Cre(-)) and 11/4 (Cre(+)). (F) Representative currents evoked in the CA1 neurons after NMDA bath application (20 μM, 30 s) in control (Cre(-)) and Prosapip1 cKO (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 (Cre(-)) and 11/4 (Cre(+)). (G) In voltage clamp recordings, two electrical stimulations (100 ms separation) were provided to elicit two responses in the CA1 neurons. Paired-pulse ratio (PPR) was calculated as the amplitude of peak 2/amplitude of peak 1. Representative paired-pulse ratio in control (Cre(-)) and Prosapip1 cKO (Cre(+)) mice (left). Average PPR for control (Cre(-)) and Prosapip1 cKO (Cre(+)) (right). Data are represented as mean ± SEM and analyzed using unpaired two-tailed t-test (Table 1). *p<0.05. n=11/3 (Cre(-)) and 13/4 (Cre(+)).

Figure 3—source data 1

File containing raw data for Figure 3.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig3-data1-v1.zip
Figure 4 with 1 supplement
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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 hr, 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 objects. n=22 (control), 20 (Prosapip1 cKO). (B–C) In the novelty T-maze test, mice were allowed to explore two arms of a three-armed, T-shaped maze. There were five training trials separated by a 1 min 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 (control), 19 (Prosapip1 cKO). (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 min 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 (control), 17 (Prosapip1 cKO). (E) Time spent in the familiar and novel chamber during the second portion of the 3-chamber social interaction test. n=14 (control), 17 (Prosapip1 cKO). (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 four training trials a day over the course of 4 days, separated by an inter-trial interval of 30 min. 24 hr 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 min. (F) Average distance traveled from start point to exit during the Barnes maze training trials. n=9 (control), 12 (Prosapip1 cKO). (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 (control), 12 (Prosapip1 cKO). (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 control (I) and Prosapip1 cKO (J) mice during Barnes maze training. (K–L) Time spent in exit-associated quartile during the probe trial and associated heatmaps (L). n=9 (control), 12 (Prosapip1 cKO). 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.

Figure 4—source data 1

File containing raw data for Figure 4.

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Figure 4—figure supplement 1
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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 (control), 12 Prosapip1 cKO. (B) To assess anxiety, mice were placed on the light side of a light/dark box apparatus and allowed to explore for 10 min. Time spent in the light and dark chambers during the test. Data represented as mean ± SEM and analyzed using a two-way ANOVA (Table 1). ****p<0.0001. n=15 (control), 16 (Prosapip1 cKO). (C) Mice were placed in the center of a plus-shaped maze and allowed to explore two closed and two open arms for 5 min 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 an unpaired two-tailed t-test (Table 1). ns, non-significant. n=7 (control), 17 (Prosapip1 cKO).

Figure 4—figure supplement 1—source data 1

File containing raw data for Figure 4—figure supplement 1.

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Figure 5
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Prosapip1 in the dHP contributes to recognition, social, and spatial memory.

(A) Images of adeno-associated virus (AAV)-GFP (green) and AAV-Cre (red) overexpression in the dHP of Lzts3fl/fl 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 min. 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 min. Time spent in the dark and light chambers 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 hr. 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 min 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 four training trials per day for 4 days, with 30 min 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 min. (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.

Figure 5—source data 1

File containing raw data for Figure 5.

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Figure 5—source data 2

Original files for western blot analysis displayed in Figure 5.

https://cdn.elifesciences.org/articles/100653/elife-100653-fig5-data2-v1.zip
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Tables

Table 1
Statistics.
FigurenSex differenceStatistical testEffectResultp-valuePost-hocPost-hoc comparisonp-value
Figure 1E77D'Agostino & Pearson testNormalityK2=5.4800.0646
Figure 1H10 (Cre- Male), 5 (Cre- Female), 11 (Cre +Male), 11 (Cre +Female)YesThree-way ANOVAGenotypeF(1,33)=0.78420.3823
SexF(1,33)=23.94<0.0001
TimeF(5,158)=588.4<0.0001
Genotype x SexF(1,33)=1.3450.2545
Genotype x TimeF(5,158)=0.14760.9805
Sex x TimeF(5,158)=2.3450.0437
Genotype x Sex x TimeF(5,158)=0.37640.8643
Figure 1I22 (Cre-), 21 (Cre+)NoUnpaired t-testGenotypet(41)=1.4600.1518
Figure 1J22 (Cre-), 21 (Cre+)NoMann-Whitney TestGenotypeU=2200.8007
Figure 2B5 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.963)=1.4270.1968
Figure 2C5 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(5.880)=5.1830.0022
Figure 2F5 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.932)=1.3450.221
Figure 2G5 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.720)=0.13060.8999
Figure 2H9 (Cre-), 10 (Cre+)Unpaired t-testGenotypet(16.38)=0.55880.5839
Figure 2I9 (Cre-), 10 (Cre+)Unpaired t-testGenotypet(16.44)=6.095<0.0001
Figure 2L4 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(4.429)=0.10500.921
Figure 2M4 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.689)=0.17580.8656
Figure 2N4 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.700)=1.6750.1398
Figure 2O4 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.332)=5.5240.0012
Figure 2P4 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.956)=0.18310.8599
Figure 2Q4 (Cre-), 5 (Cre+)Unpaired t-testGenotypet(6.060)=0.85480.4252
Figure 3D10/6 (Cre-), 9/5 (Cre+)Unpaired t-testGenotypet(17)=3.9330.0011
Figure 3E14/4 (Cre-), 11/4 (Cre+)Two-way ANOVAGenotypeF(1,3)=9.4410.044Tukey0.2 (Cre- vs. Cre+)0.345
IntensityF(1,3)=9.441<0.0010.4 (Cre- vs. Cre+)0.025
Genotype x IntensityF(1,3)=1.3760.2580.6 (Cre- vs. Cre+)0.034
0.9 (Cre- vs. Cre+)0.046
Figure 3F3/4 (Cre-), 11/4 (Cre+)Unpaired t-testGenotypet(22)=3.0940.00529
Figure 3G11/3 (Cre-), 13/4 (Cre+)Unpaired t-testGenotypet(22)=2.0990.0475
Figure 4A22 (Cre-), 20 (Cre+)NoTwo-way ANOVAGenotypeF(1,40)=0.3079P=0.5821ŠidákFamiliar vs. Novel (Cre-)<0.0001
ObjectF(1,40)=34.32<0.0001Familiar vs. Novel (Cre+)0.4871
Genotype x ObjectF(1,40)=18.370.0001
Figure 4B22 (Cre-), 19 (Cre+)NoUnpaired t-testGenotypet(26.08)=5.434<0.0001
Figure 4D14 (Cre-), 17 (Cre+)NoTwo-way ANOVAGenotypeF(1,29)=5.4190.0271ŠidákEmpty vs. Social (Cre-)0.0006
SocialF(1,29)=34.30<0.0001Empty vs. Social (Cre+)0.0005
Genotype x SocialF(1,29)=0.056550.8137
Figure 4E14 (Cre-), 17 (Cre+)NoTwo-way ANOVAGenotypeF(1,29)=1.4630.2362ŠidákFamiliar vs. Novel (Cre-)0.0008
Social NoveltyF(1,29)=17.600.0002Familiar vs. Novel (Cre+)0.1451
Genotype x Social NoveltyF(1,29)=2.9470.0967
Ext. Figure 4A6 (Cre-), 12 (Cre+)NoUnpaired t-testGenotypet(16)=0.26350.7955
Figure 4F9 (Cre-), 12 (Cre+)NoTwo-way ANOVAGenotypeF(1,19)=102.0<0.0001ŠidákTrial 2 (Cre- vs. Cre+)<0.0001
TrialF(15,285)=7.899<0.0001Trial 3 (Cre- vs. Cre+)0.0067
Genotype x TrialF(15,285)=1.4010.1454Trial 4 (Cre- vs. Cre+)0.0018
Trial 5 (Cre- vs. Cre+)0.0002
Trial 6 (Cre- vs. Cre+)0.002
Trial 7 (Cre- vs. Cre+)0.0002
Trial 8 (Cre- vs. Cre+)0.0016
Trial 9 (Cre- vs. Cre+)<0.0001
Trial 10 (Cre- vs. Cre+)0.0006
Trial 11 (Cre- vs. Cre+)0.0049
Trial 12 (Cre- vs. Cre+)0.0169
Trial 13 (Cre- vs. Cre+)0.0098
Figure 4G9 (Cre-), 12 (Cre+)NoThree-way ANOVAGenotypeF(1,19)=100.7<0.0001
TrialF(15,289)=7.289<0.0001
Error TypeF(0.4468, 8.489)=4.5540.0772
Genotype x TrialF(15,289)=1.5290.094
Genotype x Error TypeF(1,19)=15.090.001
Trial x Error TypeF(6.157, 117)=2.8650.0115
Genotype x Trial x Error TypeF(15,285)=1.8310.0303
Figure 4K9 (Cre-), 12 (Cre+)NoMann-Whitney TestGenotypeU=80.0004
Ext. Figure 4B15 (Cre-), 16 (Cre+)NoTwo-way ANOVAChamberF(1,29)=92.69<0.0001ŠidákLight vs. Dark (Cre-)<0.0001
GenotypeF(1,29)=0.95040.3377Light vs. Dark (Cre+)<0.0001
Genotype x ChamberF(1,29)=0.096450.7584
Ext. Figure 4C7 (Cre-), 17 (Cre+)NoMann-Whitney TestGenotypeU=420.2878
Figure 5C19 (AAV-GFP), 15 (AAV-Cre)NoUnpaired t-testTreatmentt(29.59)=1.9610.0594
Figure 5D19 (AAV-GFP), 15 (AAV-Cre)NoUnpaired t-testTreatmentt(29.72)=1.0660.2951
Figure 5E19 (AAV-GFP), 15 (AAV-Cre)NoTwo-way ANOVATreatmentF(1,32)=2.3610.1342ŠidákLight vs. Dark (AAV-GFP)<0.0001
ChamberF(1,32)=46.74<0.0001Light vs. Dark (AAV-Cre)0.0003
Treatment x ChamberF(1,32)=0.15880.6929
Figure 5F16 (AAV-GFP), 17 (AAV-Cre)NoTwo-way ANOVATreatmentF(1,31)=3.0690.0897ŠidákFamiliar vs. Novel (AAV-GFP)<0.0001
ObjectF(1,31)=36.30<0.0001Familiar vs. Novel (AAV-Cre)0.9965
Treatment x ObjectF(1,31)=35.05<0.0001
Figure 5G18 (AAV-GFP), 15 (AAV-Cre)NoUnpaired t-testTreatmentt(26.04)=3.7770.0008
Figure 5H18 (AAV-GFP), 15 (AAV-Cre)NoTwo-way ANOVATreatmentF(1,31)=0.00039570.9843ŠidákEmpty vs. Social (AAV-GFP)<0.0001
SocialF(1,31)=31.60<0.0001Empty vs. Social (AAV-Cre)0.0062
Treatment x SocialF(1,31)=0.77810.3845
Figure 5I18 (AAV-GFP), 15 (AAV-Cre)NoTwo-way ANOVATreatmentF(1,31)=0.20450.6543ŠidákFamiliar vs. Novel (AAV-GFP)0.0303
Social NoveltyF(1,31)=9.7770.0038Familiar vs. Novel (AAV-Cre)0.1319
Treatment x Social NoveltyF(1,31)=0.11310.7389
Figure 5J8 (AAV-GFP), 6 (AAV-Cre)NoTwo-way ANOVATreatmentF(1,12)=22.950.0004ŠidákTrial 5 (AAV-GFP vs. AAV-Cre)<0.0001
TrialF(15,180)=6.246<0.0001Trial 9 (AAV-GFP vs. AAV-Cre)0.0026
Treatment x TrialF(15,180)=2.0330.0153Trial 10 (AAV-GFP vs. AAV-Cre)<0.0001
Trial 14 (AAV-GFP vs. AAV-Cre)0.0475
Figure 5K8 (AAV-GFP), 6 (AAV-Cre)NoMann-Whitney TestTreatmentU=30.0047
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Biological Sample (Mus musculus)Single cell C57BL/6 J embryosJackson LaboratoryElectroporated with gRNA to create new mouse strain
Strain, strain background (Mus musculus, female)CD-1 IGS MouseCharles RiverStrain Code 022Pseudopregnant recipient female
Strain, strain background (Mus musculus, female)C57BL/6 JJackson Laboratory000664
RRID:IMSR_JAX:000664		Mating with first Lzts3fl/fl offspring
Strain, strain background (Mus musculus, male and female)B6.Cg-Tg(Syn1-cre)671Jxm/JJackson Laboratory003966
RRID:IMSR_JAX:003966		Mouse line
Strain, strain background (Mus musculus, male and female)Lzts3fl/flFirst described in this paperMouse line.
Available upon request to Dorit Ron
Strain, strain background (Mus musculus, male and female)Lzts3fl/fl;Syn1-CreFirst described in this paperMouse line.
Syn1-Cre available at Jackson Laboratory
Sequence-based reagentAlt-R CRISPR-Cas9 crRNAsIDT DNAThis paperGenerated by Gregg E. Homanics and available upon request.
Sequence-based reagentlong PAGE-purified Ultramer single-stranded DNA oligosIDT DNAThis paper140 nt, target loci in intron 2 and exon 5
Generated by Gregg E. Homanics and available upon request.
Sequence-based reagentIntron 2 lox insertion FThis paper (materials and methods section)PCR primersAGAGAAGTCTACGCTGTAGTCAG
Generated by Gregg E. Homanics and available upon request.
Sequence-based reagentIntron 2 lox insertion RThis paper
(materials and methods section)		PCR primersAAGCGGGAAGGTAGAGAGGT
Generated by Gregg E. Homanics and available upon request.
Sequence-based reagentExon 5 lox insertion FThis paper
(materials and methods section)		PCR primersTGCACAACCTTCTGACACGT
Generated by Gregg E. Homanics and available upon request.
Sequence-based reagentExon 5 lox insertion RThis paper
(materials and methods section)		PCR primersAGGGCACAGACAGTAGCACT
Generated by Gregg E. Homanics and available upon request.
Transfected construct (M. musculus)AAV8-Ef1a-mCherry-IRES-CreAddgene55632-AAV81×1013 vg/ml. AAV to infect mouse brain.
Transfected construct (M. musculus)AAV2-CMV-EGFPAddgene105530-AAV21×1013 vg/ml, AAV to infect mouse brain
Antibodyanti-SPAR (SIPA1L1) (Rabbit, polyclonal)

ProteinTech25086–1-AP
RRID:AB_2714023
WB (1:500)
Antibodyanti-Prosapip1 (Rabbit, polyclonal)ProteinTech24936-1-AP
RRID:AB_2714022		WB (1:2000)
Antibodyanti-Shank3 (Mouse, monoclonal)AbcamAb93607
RRID:AB_10563849		WB (1:500)
Antibodyanti-GluN2B (Rabbit, monoclonal)Cell Signaling4212
RRID:AB_2112463
WB (1:1000)
Antibodyanti-GluA1 (Rabbit, monoclonal)Cell Signaling13185 S
RRID:AB_2732897		WB (1:1000)
Antibodyanti-CREB (Rabbit, monoclonal)Cell Signaling9197
RRID:AB_2800317		WB (1:500)
Antibodyanti-GluN2A (Goat, polyclonal)Santa CruzSC-1468
RRID:AB_2630886		WB (1:500)
Antibodyanti-GAPDH (Mouse, monoclonal)Sigma-AldrichG8795
RRID:AB_1078991
WB (1:10,000)
Antibodyanti-PSD-95 (Mouse)Millipore05–494
RRID:AB_2315219
WB (1:100,000)
Antibodyanti-rabbit horseradish peroxidase (Donkey, polyclonal)Jackson ImmunoResearch711-035-152
RRID:AB_10015282
WB (1:5,000)
Antibodyanti-goat HRP (Donkey, polyclonal)Jackson ImmunoResearch705-035-003
RRID:AB_2340390		WB (1:5,000)
Antibodyanti-mouse HRP (Donkey, polyclonal)Jackson ImmunoResearch715-035-150
RRID:AB_2340770
WB (1:5,000)
Commercial assay or kitPierce bicinchoninic acid (BCA) protein assay kitThermo Scientific23225
Commercial assay or kitNuPAGE Bis-Tris precast gelsLife TechnologiesNP00321BOX
Chemical compound, drugEnhance Chemiluminescence reagent (ECL)MilliporeWBAVDCH01
Chemical compound, drugcOmplete, Mini, EDTA-free Protease Inhibitor CocktailRoche11836170001

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  1. Zachary W Hoisington
  2. Himanshu Gangal
  3. Khanhky Phamluong
  4. Chhavi Shukla
  5. Jeffrey J Moffat
  6. Alexandra Salvi
  7. Gregg Homanics
  8. Jun Wang
  9. Yann Ehinger
  10. Dorit Ron
(2025)
Prosapip1 (encoded by the Lzts3 gene) in the dorsal hippocampus mediates synaptic protein composition, long-term potentiation, and spatial memory
eLife 13:RP100653.
https://doi.org/10.7554/eLife.100653.3