Fig 2: AKT3 is widely expressed in neurons but is not expressed in interneurons or astrocytes of area CA1. (A) Representative image of AKT3 (red) staining in the hippocampus and cortex of coronal mouse brain sections. AKT3 is present throughout the brain, showing strong staining in both the cell body and neuropil layers of the hippocampus, especially in area CA3 cell bodies. (B, C) Higher magnification images of area CA1 corresponding to the white square in (A), showing AKT3 (red) costaining with (B) the interneuronal marker PV (green) and neuronal marker NeuN (blue) or (C) the astrocytic markers ALDH1L1 (green) and GFAP (blue). (D) Higher magnification of SP in (B) shows that AKT3 staining overlaps with NeuN but not PV+ cells. (E) Profile views of the Z-stack images through a PV+ cell (green) in area CA1 SP confirms the absence of AKT3 immunostaining in the interneuron cell body. (F) Higher magnification of SP and SR in (C) shows that AKT3 does not have a similar staining pattern to ALDH1L1 or GFAP. (G) Profile views of the Z-stack images through an ALDH1L1 and GFAP double-positive cell (arrow) in area CA1 SR confirms the absence of AKT3 immunostaining in the astrocyte cellbody.

Fig 3: AKT isoform expression in human brain tissue. Immunofluorescent staining using isoform-specific AKT antibodies and cell-type-specific markers in human brain tissue isolated from the temporal cortex shows an expression pattern like our mouse brain staining. AKT1 (red, left column) shows colocalization with the neuronal marker NeuN (green), AKT2 (red, middle column) shows colocalization with the astrocytic marker GFAP (white), and AKT3 (red, right column) shows diffuse staining in neuronal cell bodies and neuropil throughout the tissue. Bottom row: Human cortical tissue costained for NeuN (green) and GFAP (white) with AKT isoform-specific primary antibodies omitted but the secondary antibody included (red) to demonstrate the specificity of AKT isoform staining. Hoechst (blue), nuclear stain. Specimens were obtained from four subjects (N = 2/sex) considered as controls by the brain bank with a mean age of 58.75 years (Table 1), and images are representative of AKT isoform staining patterns for each subject.

Fig 4: AKT isoform expression in response to genetic deletion of the other two isoforms. (A) Western blot analysis of AKT isoform levels in hippocampal lysates prepared from Akt mutant mice. Cre-mediated removal of floxed Akt2 under the early neural progenitor Nes promoter in an Akt3 KO background (n2F3K) led to near-complete removal of both AKT2 and AKT3 in the hippocampus (AKT2: t(5) = 12.07, P < 0.001; AKT3: t(5) = 40.15, P < 0.001). This genetic manipulation also led to a significant increase in the AKT1 levels in n2F3K mice when compared with WT controls (t(5) = 6.471, P = 0.001). Cre-mediated removal of floxed Akt1 under the excitatory neuron Cam2α promoter in an Akt3 KO background (c1F3K) led to a small reduction in AKT1 levels in the hippocampus (t(5) = 4.310, P = 0.008) with complete AKT3 loss (t(5) = 16.99, P < 0.001) but no change in AKT2 levels (t(5) = 0.340, P > 0.05). Nes-Cre-mediated removal of floxed Akt1 and Akt2 (n1F2F) resulted in near-total removal of AKT1 (t(6) = 15.45, P < 0.001) and AKT2 (t(6) = 17.80, P < 0.001) but no significant change in AKT3 expression (t(6) = 0.297, P > 0.05). N = 3–4/genotype. (B) AKT1 staining in area CA1 of n2F3K mice shows a similar pattern to WT controls except AKT1 immunoreactivity is increased in cell bodies, including that of PV+ interneurons, within the n2F3K hippocampus. AKT1 (red), PV (white). (C) Higher magnification of AKT1 costaining with the astrocytic marker GFAP (white) in area CA1 shows that AKT1 expression is not detected in astrocytes (yellow arrows) of both n2F3K mice and WT controls. Hoechst (blue), nuclear stain. *P < 0.05.

Fig 5: Cell-type-specific expression of AKT isoforms in hippocampal area CA1.(a). AKT1 was mainly expressed in neuronal cell bodies, indicated by co-localization with NeuN. Certain neurons in the pyramidal layer, stratum oriens and molecular layer showed greater expression levels of AKT1 (yellow arrows). AKT2 was specifically expressed in astrocytes, shown by co-localization with the astrocyte marker GFAP. AKT3 co-localized with NeuN like AKT1 but was also expressed in the stratum radiatum, most likely within dendrites. (b) Higher magnification showed no specific co-localization of AKT1 with GFAP and that certain neurons in the stratum oriens expressed high levels of AKT1 (yellow arrows). AKT2 was mainly expressed in the cell bodies of astrocytes. AKT3 showed high expression levels in neuronal cell bodies and dendrites and some expression in astrocytes.