GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease

Abstract

Based on the protein kinase A (PKA)/GSK3β interaction protein (GSKIP)/glycogen synthase kinase 3β (GSK3β) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3β-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3β with PKA but not Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer's disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA-Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APP^WT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3β, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3β function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.

Keywords: Alzheimer’s disease; PKA/GSKIP/GSK3β/Tau axis; SH-SY5Y; cerebrospinal fluid; iPS cells.

Figure 1

Tau interacts with glycogen synthase kinase 3β (GSK3β), GSK3β interaction protein (GSKIP), and protein kinase A (PKA) in HEK293 cells. pEGFP-C1-Tau, pET32a-HA-GSKIP (L130P), or pET32a-HA-GSKIP (V41/L45P) transfected cells were collected, and total lysates were subjected to IP using anti-GFP antibody. The resulting precipitates were then analyzed through immunoblotting with anti-GFP, GSK3β, HA, and PKA antibodies.

Figure 2

GSKIP through GSK3β-mediated anchoring to modulate Tau phosphorylation by PKA kinase. (A) pEGFP-C1 or pEGFP-C1–GSKIP wt and mutants were transiently expressed in SH-SY5Y cells. The cells were untreated (left four lanes) or incubated with 25 μM forskolin (FSK) for 1 h (right four lanes). (B) Kinase-inactive pET32a-HA-GSK3β-K85R or -K85M mutants were transiently expressed in SH-SY5Y cells. pET32a-HA-GSK3β-K85R mutant retained the capacity to bind GSKIP, but such capacity was not evident in pET32a-HA-GSK3β-K85M. Cells were incubated with 25 μM FSK for 2 h and were then analyzed through Western blotting. (C) Knockdown of GSK3β but not GSK3α expression altered phosphorylation of dynamin related protein 1 (Drp1) Ser637 and Tau Ser409.

Figure 3

Site-specific phosphorylation of Tau by PKA or GSK3β and Ca2+/calmodulin-dependent protein kinase II (CaMKII). (A) Site-specific effects of prephosphorylation of Tau by PKA on the subsequent phosphorylation by GSK-3β. Recombinant Tau was phosphorylated by GSK3β and PKA for 3 h (lines 1, 3, respectively); line 2 shows the control-treated Tau. Tau was first phosphorylated by PKA for 1 h and then incubated with GSK3β kinase for 120 min (line 4); then, it was phosphorylated by GSK3β for 1 h, followed by incubation with PKA kinase for 120 min (line 5). (B) Site-specific effects of prephosphorylation of Tau by PKA on the subsequent phosphorylation by CaMKII.

Figure 4

Western blot of Tau46 (total Tau) and phosphorylated Tau by GSK3β and PKA kinases from cerebrospinal fluid (CSF) sampling of four groups: normal, Alzheimer’s disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). (A) Western blotting with Tau 46, Tau Ser205, Tau Ser231, and Tau Ser396 by GSK3β or Tau Ser214, Tau Ser262, and Tau Ser409 by PKA antibodies. Albumin was used as an internal control. (B) Statistical analysis. Bar graphs represent the mean ± SD of triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Figure 5

Tau phosphorylation in a patient-derived Alzheimer’s disease induced pluripotent stem cell (AD-iPSC) line with amyloid precursor protein (APP^WT/D678H) mutation cultured at 29 days. Isogenic control (^WT/WT) and isogenic mutant (^D678H/D678H) were generated by CRISPR/Cas9 gene editing and examined in parallel. (A) The diagram presents APP protein and the ^D678H mutation within amyloid-β (Aβ) region in AD-iPSC (upper panel). Direct sequencing of APP exon 16 PCR products derived from the patient and from healthy controls revealed a GAC-to-CAC nucleotide substitution in Aβ region of the patient’s APP gene (in 678th amino acid using APP⁷⁷⁰ numbering or in 7th amino acid using Aβ numbering) (lower panel). (B) Western blotting with 6E10 antibody, which recognizes residues 3-8 of Aβ, was used to detect the expression of APP protein in D29 iPSC-derived neurons. GAPDH was used to confirm similar protein loading across samples. (C) Western blotting with antibodies of Tau 5, Tau Ser409, Tau Ser231, Tau Ser396 by GSK3β or Tau Ser214, Tau Ser262 and Tau Ser409 by PKA. Tau 5 (Total Tau) was used as an internal control compared with the control group. β-actin was used as an internal control. (D) Statistic analysis for (C). (E) Western blotting with GSKIP antibody. (F) Statistic analysis for (E). Bar graphs represent the mean of triplicates ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Schematic of GSKIP, GSK3β, PKA RII, and Tau complex. While activating cAMP signaling, GSKIP wt conjugated with activated PKA to facilitate GSK3β Ser9 phosphorylation. GSKIP negatively regulates GSK3β activity (previously characterized in [1,6,7,9]); resulting in double inhibition of GSK3β activity and mediation of Tau phosphorylation by active catalytic form of PKA (red triangle). GSKIP anchors Tau through L130 by tethering GSK3β for PKA-mediated phosphorylation of Ser214, Ser262, and Ser409. This is thought to cause neuron tangles through Tau hyperphosphorylation. R, regulatory subunit of PKA; C, catalytic subunit of PKA.