Study of a cyclic nucleotide-mediated mechanism that could favor an antiaggregant conformation of tau protein

Tau is a protein which normally participates in the assembly and stability of microtubules. However, under pathological conditions, it can form aggregated and hyperphosphorylated structures that are associated with the pathogenesis of Alzheimer’s disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP-dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU-S214), an event that exerts a protective effect against the assembly of the protein. The activation of PKA by cAMP also sustains long-term potentiation (LTP), a form of synaptic plasticity, through the stimulation of Aβ production. In a similar manner, cGMP was found to boost Aβ levels and to favour LTP and memory, but an effect of cGMP on tau phosphorylation has never been reported. To investigate this issue, we first verified the effect of cAMP-enhancing treatments on pTAUS214 and then evaluated the possibility of a PKG-induced phosphorylation of tau. Different cAMP and cGMP enhancing strategies were used in different model systems: neuro N2a cells, rat hippocampal slices and adult male C57BL/6 mice. Phosphorylation of tau was analyzed by immunoblotting, gene silencing, in vitro enzymatic assays and nano-HPLC mass spectrometry. Aggregation of tau was evaluated by gel electrophoresis and thioflavin T-binding assay. Our data confirm that cAMP stimulates the phosphorylation of tau at Ser214, as expected, but not at Ser202, which is considered one of the earliest markers of tau aggregation. Furthermore, we report for the first time that the cGMP-activated PKG phosphorylates tau at Ser214 and other 7 Ser/Thr tau residues, but not Ser202. Also, preliminary results indicate that PKG phosphorylation could reduce the aggregating capacity of tau. Taken together, our results demonstrate the existence of a PKG-mediated mechanism that might shift tau from a pro-aggregant to an anti-aggregant conformation, which has been reported to exert neuroprotective functions.