Conditional inactivation of Akt three isoforms causes tau hyperphosphorylation in the brain

Background Tau hyperphosphorylation plays a critical role in neurodegenerative diseases [EMBO Mol Med. 6:1142-60, 2014; Annu Rev Neurosci. 24:1121-59, 2001]. Recent evidence has shown that Akt is down-regulated in AD [J Pathol. 225:54-62, 2011]. However, it remained unknown which pathological process, e.g. tau pathology or neuron death, Akt may contribute to. In this study, Cre-loxP technique was employed to generate a viable Akt three isoforms conditional knockout (Akt cTKO) mouse in which total Akt levels were dramatically reduced in the adult brain. Results Significantly increased levels of tau phosphorylated (p-tau) at various sites were observed in Akt cTKO mice as compared to age-matched littermate controls. Increased levels for phosphorylated GSK3α and phosphorylated PKA substrates were detected in Akt cTKO brains. In contrast, no significant changes on p-tau levels were found in Akt1−/−, Akt2−/− or Akt3−/− mice. Conclusions Akt may regulate tau phosphorylation in the adult brain by affecting activities for PKA and GSK3α. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0030-y) contains supplementary material, which is available to authorized users.


Generation of Akt1 f/f and Akt cTKO mice.
Since Akt1 -/mice are perinatal lethal [1,2], we generated floxed Akt1 mice. The gene targeting strategy was shown in Fig. S1A. Two loxP sites were inserted into introns 2 and 11 separately. After obtaining Akt1 f/+ , we intercrossed them to get Akt1 f/f . To generate viable Akt cTKO mice, first, we crossed Akt2 -/- [3] with Akt3 -/- [4] to obtain Akt2 +/-;Akt3 +/-. The latter were bred with Akt1 f/f to get Akt1 f/+ ;Akt2 +/-;Akt3 +/-, which were bred with a CAG-CreER to get Akt1 f/+ ;Akt2 +/-;Akt3 +/-;CAG-CreER. All the mice were bred in an SPF room in the core animal facility of the Model Animal Research Center (MARC) at Nanjing University. The room temperature was 25ºC and the light-cycle was automatically controlled. The genetic background of the mice used in this study is C57BL/6. The protocol for the study has been approved by the institutional Animal Care and Use Committee of the MARC.

Treatment of mice with tamoxifen.
To induce Cre-mediated gene recombination, Akt1 f/f ;Akt2 -/-;Akt3 -/-;CAG-CreER and controls were treated with tamoxifen for 5 consecutive days. Tamoxifen was purchased from Sigma-Aldrich (catalogue No. T5648), and was freshly prepared in each injection day. The treatment was conducted by intraperitoneal (i.p.) injection of the drug at the concentration of 20mg/kg. Akt1 f/f ;Akt2 -/-;Akt3 -/-;CAG-CreER mice receiving 5 doses of tamoxifen injections were designated as Akt cTKO mice. The number of animals used for the Akt cTKO study was as follows: control=7, Akt cTKO=9. For Akt single isoform KO studies, the number was 3-4 per genotypic group.

Genotyping.
To detect the floxed Akt1 allele, the following primers were used: GGGATCAGCAG-TTGAAGGACAGA and GCCAGGAATACAGCATGAGCCAC. The PCR products for the WT and the floxed allele are 196bp and 302 bp, respectively. To detect Akt1 ∆/∆ allele, primers of AGACTCTGAGCATCATCCCTGGG, CGTCTGGCCTTCCTGTA-GCCAG and TGAAGCAGGCCTAGAGCCCCATG were used. The PCR product for the WT allele is 300bp and that for the ∆ allele is 200 bp. To identify Akt2 ∆/∆ allele, primers of CTCAGGGACACCCATGTGTGGCTGC, GCTGCCTCGTCCTGCAGT-TCATTC and CCACAGGCAGCAGAAAGGAA were used. The PCR size for the WT allele is 360bp and that for the ∆ allele is 600 bp. For Akt3 ∆/∆ mice, primers used were: GGTTCTGTGGGAGGTAGTTCTC, GCAATCCATCTTGTTCAATGGCCG and CCATCGGTCGGCTACGGCTTGG. The PCR band for the WT allele is 500bp and that for the ∆ allele is 350 bp.

Nissl staining.
Sagittal brain sections (10µm) were de-paraffinized, ethanol dehydrated, and were then washed using distilled water. Sections were treated with 0.5% cresyl-violet for 10 minutes and then washed with PBS. Sections were incubated in a solution containing 1% glacial acetic acid and 16% ethanol. Sections were dehydrated using an ascending series of ethanol (70%, 90%, 95% and 100%), and were then placed in toluene. Slides were coverslipped using neutral resin.

Brain lysates preparation.
The mice for experiments were euthanized by CO 2. For each Akt isoform KO line, mice were sacrificed at 2-3 months of age. For the Akt cTKO study, control and mutant mice were sacrificed 3 weeks after the tamoxifen treatment. After euthanasia, different brain areas such as the cortex and the cerebellum were quickly dissected out and were immediately put into liquid nitrogen. Brain samples were stored at -80℃ until use. Mice cortices were homogenized in cold radio immunoprecipitation assay lysis buffer [consisting of the following (in mM): 20mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] containing protease and phosphatase inhibitors (Thermo). Lysates were cleared by centrifugation (14,000 rpm for 20 min). Protein concentration was analyzed using a standard BSA method [5,6].

Immunoblotting.
Normalized volumes of samples (30µg total protein) were resolved in 10% SDS-PAGE (invitrogen), transferred to nitrocellulose membrane. After blocking with 5% (w/v) dry milk for 1h, membranes were incubated with primary antibodies overnight and detected using infrared dye-coupled secondary antibodies (goat anti-rabbit IRdye800, goat anti-rabbit IRdye680, goat anti-mouse IRdye800 and goat anti-mouse IRdye680).
Membranes were scanned and data were quantified using Odyssey Infrared Imaging System (Li-Cor). Antibody against total Akt (t-Akt) (1:1000) was made by the Z.Y.

Supplementary Figure 1. Molecular characterization of tamoxifen-induced
(B)Immunohistochemistry of GFAP. There was no detectable astrocytosis in the cortex (e), hippocampal CA1(f), hippocampal CA3 (g) and the dentate gyrus (h) of Akt cTKO mice, as compared to the control (a-d). (C)Immunohistochemistry of Iba1. There was no significant activation on microglia in the brain (e-h) of Akt cTKO mice. Scale bar=20μm.