Isoform-specific roles for AKT in affective behavior, spatial memory, and extinction related to psychiatric disorders
- Institute for Behavioral Genetics, University of Colorado, United States
- Linda Crnic Institute, Anschutz Medical Center, United States
- Department of Integrative Physiology, University of Colorado, United States
Figures
Figure 1 with 1 supplement
Akt deficiency affects the expression of anxiety-related behavior in an isoform- and sex-specific fashion.
Male and female mice with single-isoform deletions of Akt1, Akt2, or Akt3 were assessed for anxiety-related behaviors in the open field arena (OFA) and elevated plus maze (EPM) tests. (A–B) OFA …
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Figure 1—source data 1
Figure 1 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Schematic representation of the experimental timeline.
Figure 2
Akt deficiency affects spatial memory formation in an isoform- and sex-specific fashion.
Spatial learning and memory were assessed using the Morris Water Maze (MWM) in male and female mice with single-isoform deletions of Akt1, Akt2, or Akt3. Left graph: Learning measured by latency of …
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Figure 2—source data 1
Figure 2 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig2-data1-v2.xlsx
Figure 3
Single Akt isoform deficiency does not impact cued fear LTM but Akt2 deficiency affects contextual fear LTM in males.
Associative fear conditioning acquisition and long-term memory (LTM) in Akt isoform mutant male and female mice shown as percent time spent freezing. In training, freezing behavior was assessed …
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Figure 3—source data 1
Figure 3 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig3-data1-v2.xlsx
Figure 4
AKT1 deficiency enhances fear extinction learning and AKT2 removal enhances fear renewal.
Extinction training to the CS was performed over three days (D1–D3) with 20 CS presentations each day. Extinction learning in mice measured as percent freezing during the baseline period prior to …
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Figure 4—source data 1
Figure 4 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Restoration of AKT1 expression in the PFC normalizes extinction learning and LTM in Akt1 KO males.
(A) Experimental design to test the function of AKT1 in the PFC. AKT1 expression was restored using AAV-mediated pan-neuronal (hSyn) co-expression of AKT1 and green fluorescent protein (GFP) in the …
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Figure 5—source data 1
Figure 5 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Restoration of neuronal AKT1 expression in the PFC of Akt1 KO male mice.
(A) Representative image of AKT1 and green fluorescent protein (GFP) co-expression in the PFC of Akt1 KO male mice injected with AAV-hSyn::AKT1-GFP. Staining for AKT1 (red) confirms AKT1 expression …
Figure 6 with 1 supplement
Conditional Akt1 deficiency in excitatory neurons impairs contextual fear LTM but does affect anxiety-related behaviors or fear extinction processes.
Behavior of conditional Akt1 KO (Akt1 cKO) male mice generated by removal of Akt1 with a forebrain excitatory neuron-specific Cre driver in floxed Akt1 mice. (A) OFA activity, measured as percent …
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Figure 6—source data 1
Figure 6 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
AKT1 expression in conditional Akt1 KO (Akt1 cKO) male mice under the excitatory neuron-specific T29-1 Camk2α-Cre driver.
(A) Western blot analysis shows reduced AKT1 levels in the amygdala (amy) and prefrontal cortex (PFC) of Akt1 cKO mice compared with WT controls while AKT3 levels are not affected. β-actin, loading …
Figure 7
Simultaneous removal of AKT1 and AKT3 isoforms from the brain promotes memory deficits in both male and female mice.
Male and female mice with conditional Akt1 removal in the background of Akt3 deficiency (Akt1 cKO Akt3 KO) were assessed in anxiety-related behavior, spatial memory, and associative fear memory …
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Figure 7—source data 1
Figure 7 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig7-data1-v2.xlsx
Figure 8
AKT expression under normal and deficiency conditions are regulated similarly between sexes.
(A) Western blot analysis of AKT isoforms individually, detected with isoform-specific AKT antibodies, and all together, detected with pan-AKT antibodies, in the hippocampus of male versus female WT …
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Figure 8—source data 1
Figure 8 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig8-data1-v2.xlsx
Figure 9 with 3 supplements
Examination of Akt isoform deficiency effects on neuromolecular signaling pathways.
Upstream signaling of AKT was assessed by measuring phosphorylation levels of phosphoinositide-dependent protein kinase 1 (PDK1) at serine 241. Downstream signaling of AKT was assessed by measuring …
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Figure 9—source data 1
Figure 9 source data.
- https://cdn.elifesciences.org/articles/56630/elife-56630-fig9-data1-v2.xlsx
Figure 9—figure supplement 1
Schematic of AKT-dependent and interacting signaling pathways.
Candidate pathways (boxed) upstream (PDK1), downstream (GSK3β), and parallel (ERK) to AKT were examined by western blot analysis to assess the impact of Akt isoform deficiency on signaling in the …
Figure 9—figure supplement 2
Akt3 deficiency does not alter GSK3β activation in the hippocampus of female mice.
Western blot analysis of GSK3β phosphorylation at serine 9 (pGSK3β) normalized by total GSK3β levels reveals no difference in the hippocampus between female Akt3 KO and WT mice. GAPDH, loading …
Figure 9—figure supplement 3
Interneuron expression in the PFC of male and female WT mice.
(A) Representative immunostaining for the interneuronal markers parvalbumin (PV, red) and glutamate decarboxylase 67 (GAD67, green) in the prelimbic region of the PFC of male and female WT mice. …
Tables
Table 1
Summary of behavioral impacts of Akt isoform deficiency.
| Behavioral assay | Genotype and/or viral expression manipulation | |||||
|---|---|---|---|---|---|---|
| Akt1 KO | Akt2 KO | Akt3 KO | Akt1 KO + virally expressed PFC AKT1 (male only) | Akt1 cKO (male only) | Akt1 cKO Akt3 KO | |
| OFA | reduced center time in males | no effect | no effect | n/a | no effect | no effect |
| EPM | reduced open arm time in males | reduced open arm time in males | no effect | n/a | no effect | no effect |
| MWM | reduced platform crossings during probe test in males | no effect | no effect | n/a | no effect | reduced quadrant time and platform crossings during probe test in males and females |
| Contextual fear LTM | no effect | impaired in males | no effect | no effect | impaired | impaired in males and females |
| Cued fear LTM | no effect | no effect | no effect | no effect | no effect | impaired in males and females |
| Fear extinction learning | enhanced | no effect | no effect | restored to WT levels | no effect | n/a |
| Fear extinction LTM | reduced in males | no effect | no effect | restored to WT levels | no effect | n/a |
| Fear extinction renewal | reduced in males | enhanced in males | no effect | no statistical difference from WT | no effect | n/a |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-AKT1 (Rabbit monoclonal) | Cell Signaling | Cat# 2938; RRID:AB_915788 | WB (1:2000) |
| Antibody | anti-AKT1 (Rabbit monoclonal) | Cell Signaling | Cat# 75692; RRID:AB_2716309 | IHC (1:100) |
| Antibody | anti-AKT1 phospho-S473 (Rabbit monoclonal) | Cell Signaling | Cat# 9081; RRID:AB_11178946 | WB (1:1000) |
| Antibody | anti-AKT2 (Rabbit monoclonal) | Cell Signaling | Cat# 2964; RRID:AB_331162 | WB (1:1000); IHC (1:100) |
| Antibody | anti-AKT2 phospho-S474 (Rabbit monoclonal) | Cell Signaling | Cat# 8599; RRID:AB_2630347 | (1:1000) |
| Antibody | anti-AKT3 (Mouse monoclonal) | Cell Signaling | Cat# 8018; RRID:AB_10859371 | WB (1:2000) |
| Antibody | anti-AKT3 (Rabbit monoclonal) | Cell Signaling | Cat# 14982; RRID:AB_2716311 | IHC (1:100) |
| Antibody | anti-pan-AKT (Rabbit monoclonal) | Cell Signaling | Cat# 4685; RRID:AB_2225340 | WB (1:5000) |
| Antibody | anti-pan-AKT phospho-S473 (Rabbit monoclonal) | Cell Signaling | Cat# 4058; RRID:AB_331168 | WB (1:2000) |
| Antibody | anti-GAPDH (Rabbit monoclonal) | Cell Signaling | Cat# 5174; RRID:AB_10622025 | WB (1:20000) |
| Antibody | anti-β-actin (Mouse monoclonal) | Cell Signaling | Cat# 3700; RRID:AB_2242334 | WB (1:20000) |
| Antibody | anti-PDK1 phospho-S241 (Rabbit polyclonal) | Cell Signaling | Cat# 3061; RRID:AB_2161919 | WB (1:5000) |
| Antibody | anti-PDK1 (Rabbit polyclonal) | Cell Signaling | Cat# 3062; RRID:AB_2236832 | WB (1:1000) |
| Antibody | anti-GSK3β phospho-S9 (Rabbit monoclonal) | Cell Signaling | Cat# 5558; RRID:AB_10013750 | WB (1:5000) |
| Antibody | anti-GSK3β (Rabbit monoclonal) | Cell Signaling | Cat# 9315; RRID:AB_490890 | WB (1:5000) |
| Antibody | anti-ERK1/2 phospho-T202/Y204 (Rabbit polyclonal) | Cell Signaling | Cat# 9101; RRID:AB_331646 | WB (1:2000) |
| Antibody | anti-ERK1/2 (Rabbit polyclonal) | Cell Signaling | Cat# 9102; RRID:AB_330744 | WB (1:7500) |
| Antibody | anti-NeuN (Mouse monoclonal) | Novus | Cat# NBP1-92693; RRID:AB_11036146 | IHC (1:1000) |
| Antibody | anti-GAD67 (Mouse monoclonal) | Millipore | Cat# MAB5406 RRID:AB_2278725 | IHC (1:1500) WB (1:10000) |
| Antibody | anti-paravalbumin (Mouse monoclonal) | Millipore | Cat# MAB1572; RRID:AB_2174013 | IHC (1:1000) |
| Antibody | anti-rabbit Cy3 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 711-165-152; RRID:AB_2307443 | (1:250) |
| Antibody | anti-mouse IgG2b Alexa 647 (Goat polyclonal) | Invitrogen | Cat# A-21242; RRID:AB_2535811 | (1:500) |
| Antibody | anti-mouse IgG1 Alexa 488 (Goat polyclonal) | Invitrogen | Cat# A-21121; RRID:AB_2535764 | (1:500) |
| Antibody | anti-mouse HRP (Goat polyclonal) | Promega | Cat# W4021; RRID:AB_430834 | (1:5000–20000) |
| Antibody | anti-rabbit HRP (Goat polyclonal) | Promega | Cat# W4011; RRID:AB_430833 | (1:5000–20000) |
| Other | Hoechst | Thermo Fisher Scientific | Cat# H3569; RRID:AB_2651133 | (1:3000) |
| Genetic reagent (Mus musculus), both sexes | Akt1tm1Mbb, C57BL/6 (Akt1 KO) | Jackson Laboratory | Stock # 004912; RRID:IMSR_JAX:004912 | |
| Genetic reagent (Mus musculus), both sexes | Akt2tm1.1Mbb, C57BL/6 (Akt2 KO) | Jackson Laboratory | Stock # 006966; RRID:IMSR_JAX:006966 | |
| Genetic reagent (Mus musculus), both sexes | Akt3tm1.3Mbb, C57BL/6 (Akt3 KO) | Easton et al., 2005; PMCID:PMC549378 | MGI Cat# 3804003, RRID:MGI:3804003 | Obtained from Birnbaum lab (UPenn) |
| Genetic reagent (Mus musculus), both sexes | Akt1tm2.2Mbb,C57BL/6 (Akt1fl/fl) | Jackson Laboratory | Stock #026474; RRID:IMSR_JAX:026474 | |
| Genetic reagent (Mus musculus), females only | Tg(CamkIIa-Cre)T29Stl, C57BL/6 (Camk2a-Cre) | Hoeffer et al., 2008; PMCID:PMC2630531 | MGI Cat# 6273652, RRID:MGI:6273652 | Obtained from Kelleher lab (MIT) before the Tonegawa lab submitted a strain to Jax with same name; not same strain as listed at JAX. Expression properties for this line in Hoeffer et al., 2008. |
| Recombinant DNA reagent | hSyn-hAKT1-hSyn-eGFP (AAV) | Vector Biolabs | RRID:SCR_011010 AKT1:Genbank RefSeq# BC000479.2 | Commercially produced custom construct |
| Recombinant DNA reagent | hSyn-eGFP-Cre (AAV) | Penn Vector Core | RRID:Addgene 105540 | |
| Software, algorithm | IBM SPSS Statistics | IBM Analytics | RRID:SCR_002865 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | ImageQuant TL | GE Healthcare | RRID:SCR_014246 | |
| Software, algorithm | Icy | Institut Pasteur and France-BioImaging | RRID:SCR_010587 | Open source image processing |
Additional files
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Supplementary file 1
Statistical analysis using t-tests.
- https://cdn.elifesciences.org/articles/56630/elife-56630-supp1-v2.docx
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Supplementary file 2
Outliers and justification.
- https://cdn.elifesciences.org/articles/56630/elife-56630-supp2-v2.docx
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Supplementary file 3
Statistical analysis using ANOVAs.
- https://cdn.elifesciences.org/articles/56630/elife-56630-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56630/elife-56630-transrepform-v2.docx
