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

Mice with global deletion of Akt three isoforms (Akt TKO or Akt1 ^?/? ;Akt2 ^?/? ;Akt3 ^?/?
) were early embryonic lethal 1], precluding the possibility to study its in vivo function. To obtain viable Akt conditional TKO (cTKO) mouse, we generated floxed Akt1 (Additional file 1: Figure S1A). After several steps of crossing, Akt1 ^f/f ;Akt2 ^?/? ;Akt3 ^?/? ;CAG-CreER mice were obtained and became Akt cTKO after the treatment with tamoxifen. To examine Cre-mediated recombination efficiency
in the brain, biochemical analyses were conducted. Dramatic reduction on levels for
total Akt (t-Akt) was observed in the brain of cTKO mice (Additional file 1: Figure S1B). Levels of p-Akt
^Ser473
, an activated form of Akt, were markedly decreased in Akt cTKO (Additional file 1: Figure S1B). Immunohistochemistry (IHC) results showed very weak immuno-reactivity
of t-Akt in the brain of Akt cTKO mice (Additional file 1: Figure S1C-d-f, a-c for controls).

Increased levels of tau phosphorylated (p-tau) at the Thr205, Thr231 and Ser396 epitopes
have been widely reported in Alzheimer’s brain and mouse models 2]. Our Western results showed highly increased levels for p-tau
^T205
, p-tau
^T231
and p-tau
^S396
in Akt cTKO mice (Fig. 1a). Moreover, the position of p-tau bands at the 60, 64 and 68 kDa in the SDS gels
was consistently higher in Akt cTKOs than that in controls (Fig. 1a), likely due to the slow migrating rate caused by tau hyperphosphorylation. We conducted
additional control experiments and used two groups of Akt2/3 double KO mice, Akt1 ^f/f ;Akt2 ^?/? ;Akt3 ^?/? ;CAG-CreER and Akt1 ^f/f ;Akt2 ^?/? ;Akt3 ^?/?
, which were age- matched littermates to Akt cTKOs. We found that Akt DKO mice did not show significant changes on p-tau levels (Additional file 1: Figure S2). Strong p-tau
^Thr205
immuno-reactivity was clearly seen in the cytoplasm of neurons in various brain sub-areas
of Akt cTKOs but not controls (Fig. 1b). Increased p-tau
^Ser396
immuno-reactivity was largely detected in glial-like cells in the brain of Akt cTKOs (Additional file 1: Figure S3). Immunostaining on p-tau
^Thr231
showed strong signals in neuronal branches of the cTKO mice (Additional file 1: Figure S4), and double-staining with MAP2 suggested dendritic localization for p-tau
^Thr231
(Additional file 1: Figure S4C: a-d).

Fig. 1. Tau hyperphosphorylation in the brain of Akt cTKO mice. a Western blotting of p-tau using cortical and cerebellar samples. Antibodies against
tau phosphorylated at the Thr205, Thr231 and Ser396 epitopes were used. The migration
of three p-tau bands (60 kDa, 64 kDa and 68 kDa) in SDS gels was slower in Akt cTKO (Akt1 ^f/f ;Akt2 ^?/? ;Akt3 ^?/? ;CAG-CreER with tamoxifen treatment) than in control mice (Akt1 ^f/f ;Akt2 ^+/+ ;Akt3 ^+/+
and Akt1 ^f/+ ;Akt2 ^+/+ ;Akt3 ^+/+
). Intensities of the p-tau bands were dramatically increased in Akt cTKO mice. Tau5 antibody was used to detect total tau (t-tau). b Immunohistochemistry of p-tau
^Thr205
in the brain. Strong immuno-reactivity of p-tau
^Thr205
was seen in the cortex (e), hippocampal CA3 (f), the hilus of the dentate gyrus (g)
and the cerebellum (h) of Akt cTKO mice. Weak immuno-reactivity of p-tau
^Thr205
was observed in different brain sub-areas of control mice (a-d)

TUNEL assay was performed to examine cell death. No difference in the total number
of TUNEL+ cells was observed in brains of Akt cTKO and control mice (Additional file 1: Figure S5), suggesting no significant change in apoptosis. Markers for neurons,
astrocytes and microglia were used for biochemical and morphological analyses. First,
there was no significant reduction on cortical levels for NeuN, GFAP and Iba1 in Akt cTKO mice (Additional file 1: Figure S6A). Second, there was comparable immuno-reactivity for NeuN, GFAP or Iba1
(Additional file 1: Figure S6B-C) in control and Akt cTKO mice. Therefore, deletion of Akt does not significantly affect the survival
of cortical cells.

To determine the role of Akt single isoform in tau phosphorylation in the brain, we
analyzed p-tau levels using cortical lysates from three lines of Akt single isoform
KO mice including Akt1 ^?/?3], Akt2 ^?/?4] and Akt3 ^?/?5]. Nissl staining revealed comparable brain structure between Akt1 ^?/?
, Akt2 ^?/?
, Akt3 ^?/?
and their age-matched WT littermates (Fig. 2). Consistent with previously published observations 5], 6], Akt3 ^?/?
mice displayed smaller brain than WT (Fig. 2e). However, no significant changes in p-tau levels were observed in Akt1 ^?/?
, Akt2 ^?/?
and Akt3 ^?/?
mice (Fig. 2b,d,f: ps??0.05), suggesting that tau hyperphosphorylation shown in Akt cTKO mice is unlikely to be caused by loss of a single Akt isoform.

Fig. 2. Unchanged levels of p-tau in Akt single isoform KO mice. a Nissl staining for the brain of Akt1^?/?
mice. Normal brain structure was observed. b Western analyses on p-tau levels using cortical samples of Akt1^?/?
mice. There was no significant difference on relative levels of p-tau
^Thr205
, p-tau
^Thr231
and p-tau
^Ser396
in Akt1^?/?
mice. c Nissl staining for the brain of Akt2^?/?
mice. No abnormal brain structure was detected. d Western analyses on p-tau levels using cortical samples of Akt2^?/?
mice revealed no significant difference. e Nissl staining for the brain of Akt3^?/?
mice. There was a smaller brain in Akt3^?/?
mice than in WT animals. f Western analyses on p-tau levels showed no significant difference in Akt3^?/?
mice (NS?=?not significant)

To dissect molecular pathways involved, we conducted Western blotting using an antibody
against phosphorylated Akt substrates. We found increased intensities for several
bands including those for GSK3?/3? in Akt cTKO mice (Additional file 1: Figure S7A). Since GSK3 is a substrate of Akt, it is expected that deletion of Akt
may cause reduced levels for phosphorylated GSK3?/3? 7]. However, we found that relative levels for p-GSK3?
^Ser9
were significantly increased in Akt cTKO mice (Additional file 1: Figure S7B) and those for p-GSK3?
^Tyr279
were also increased (Fig. 3a), suggesting inhibition of GSK3? but activation of GSK3?. Since ?-catenin is a well-known
substrate of GSK3 8], 9], p-?-catenin
^{Ser33/Ser37/Thr41}
was examined and showed significantly increased levels in Akt cTKOs (Additional file 1: Figure S7C), suggesting elevated GSK3? activity. Moreover, we examined several other
tau kinases. First, although levels for p-Cdk5
^Ser159
were increased, those for p25 were decreased (Fig. 3b, p??0.05). Second, no significant changes on levels for p-Erk1/2 and p-p38 were
detected in Akt cTKO mice (Fig. 3c-d, ps??0.05). Overall, activities for GSK3?, Cdk5, Erk and MAPK p38 were not enhanced.

Fig. 3. Increased levels for an activated form of GSK3? in the brain of Akt cTKO mice. a Western blotting on activated forms of GSK3? and GSK3?. Quantitative analysis showed
significantly increased levels for p-GSK3?
^Tyr279
(*, p??0.05) but unchanged levels for p-GSK3?
^Tyr216
(NS, p??0.05) in Akt cTKO mice. b Western blotting on p-Cdk5, p35 and p25. Relative levels for p-Cdk5
^Ser159
were significantly increased (*, p??0.05), but those for p25 were significantly decreased
(*, p??0.05) in the cortex of Akt cTKO mice. GAPDH served as the loading control. c Western blotting on MAPK Erk1/2. Total Erk1/2 levels in Akt cTKO mice were not different from those in control mice. ?-actin served as the loading
control. Ratio of p-Erk1/2 to the total Erk1/2 in Akt cTKO mice was also not different from that in control mice (p??0.05). d Western blotting on phosphorylated MAPK p38 (p-p38). Levels of total p38 did not
differ between Akt cTKO and control mice (p??0.05). Ratio of p-p38 to total p38 in Akt cTKO mice was not changed (NS?=?not significant)

It has been shown that GSK3? is phosphorylated at the Ser9 site not only by Akt but
also by PKA 10]. The tyrosine phosphorylation of GSK3 requires the cAMP-PKA signaling 11], 12]. Moreover, it is known that PKA is a kinase to phosphorylate tau at the sites of
Thr205, Thr231 and Ser396 2], 13]. To test the possibility that PKA is involved, we performed the following experiments.
First, an antibody against phosphorylated PKA substrates was used to conduct Western
blotting. Increased levels for several bands with a wide range of molecular weights
were observed (Fig. 4a), suggesting increased PKA activity. Second, we examined VASP, a well-known PKA substrate
14], 15], and observed highly increased p-VASP
^Ser157
levels (Fig. 4b). In contrast, expression levels of PKA regulatory subunits, 1? and 1?, were unchanged
(Fig. 4b). Third, since the Ser214 16] and Ser356 17], 18] sites of tau are phosphorylated by PKA as well, we analyzed p-tau
^Ser214
and p-tau
^Ser356
, which exhibited increased levels in Akt cTKO mice (Fig. 4c).

Fig. 4. Increased levels for phosphorylated PKA substrates in Akt cTKO mice. a Western blotting for phosphorylated PKA substrates using cortical samples of Akt cTKO mice. Levels for several phosphorylated PKA substrates with molecular weights
at about 100 kDa, 60 kDa and 40 kDa were increased. b Western analyses on VASP and PKA subunits. P-VASP
^Ser157
levels in Akt cTKO mice were highly increased but total VASP levels were not changed. Relative
levels for PKA regulatory subunits, 1? and 1?, were not changed. GAPDH served as the
loading control. c Western blotting on p-tau
^Ser214
and p-tau
^Ser356
. Ratios of p-tau to total tau were significantly increased in Akt cTKO mice, as compared to control animals (*, p??0.05)