The Role Of Adenosine A2A Receptors Activation In Hyperphosphorylation Of Tau Protein And Cognitive Dysfunction Induced By Traumatic Brain Injury

With the fast development of society, there appears an increasing number of traumaticbrain injury and related cognitive disfunction patients. Traffic accidents, warfare and fierceantagonistic sports are of the most common causes. It is well known that tau protein’shyperphosphorylaton is closely related to cognitive disfunction in Alzheimer’s disease.Recent studies have shown that TBI can cause hyperphosphorylation of tau protein andlong-term cognitive dysfunction. PKA and GSK-3β are both very important tau kinase inhuman brain. Adenosine is one of the most important neurotransmitter in the centralnervous system. Increased adenosine produced from cells or directly from ATP breakdownactivate the Adenosine A2A receptor(A2AR) after TBI. A2AR plays an important role inregulating neurotransmitter release, neuron excitability, synaptic plasticity, glial cells andcognitive function. Meanwhile, A2AR defciency can alleviate the TBI-induced cognitivedysfunction. But we don’t know whether this effect of A2AR is related to tau protein’shyperpho-sphorylation and if so,which signaling pathway is of the most significance inthis process.In our study, controlled cortical impact (CCI) method was used in the same place ofthe left parietal cortex to reproduce the moderate TBI model constructed in our lab’sprevious study by testing brain water content and neurological deficits. Thephosphorylation level of tau protein at Thr205, Ser262and Ser404sites in different brainregions was tested by using immunohistochemistry and western blot methods24h, and7dafter TBI. We found that compared with the sham group tau protein’s phosphorylaytionlevel at Ser404site significantly increased after TBI in ipsilateral parietal cortex, ipsilateralhippocampus and contralateral dentate gyrus and was accompanied by GFAP antibodylabeled gliosis, NeuN antibody labeled neuron lost and amyloid precursor protein (APP)antibody labeled axon damage. Then we tested whether inhibition of A2AR could reduce tau protein’s phosphorylaytion level and alleviate cognitive dysfunction after by usingselective A2AR antagonist ZM24138515min after TBI or A2AR knock out (KO) mice. Wefound that A2AR inhibiton after TBI could significantly reduce the phosphorylation level oftau at Ser404site of C-terminal and alleviates TBI-induced reference memory and workingmemory dysfunction at the time point of7d and4w. Then we stimulated cultured rathippocampal neurons and SH-SY5Y cells with selective A2AR agonist CGS21680,antagonist ZM241385, and H-89(selective protein kinase A inhibitor),SB216763(non-selective GSK-3β inhibitor) and tested the phosphorylaytion level of tauprotein at Ser404site to find the possible cell signalling passway how the activation ofA2AR caused tau protein’s hyper phosphorylaytion and cognitive disfunction.Main results and conclusions:1. We found hyperphosphorylated tau at Ser404site of its C-terminal and there was nosignificant change of phosphorylation level at Thr205site (in tau’s projection domain,) andSer262site(in tau’s microtubule binding domain)24h,7d and4w after TBI. We foundhyperphosphorylated tau at Ser404site in injury cortex, hippocampus and contralateraldentate gyrus (DG)24h after TBI. There was still hyperphosphorylated tau at Ser404site incontralateral DG7d and4w after TBI with most of the ipsilateral cortex and hippocampuslost. These tau abnormalities was accompanied by gliosis, neuron lost and axon damage.The above results indicated that C-terminal of tau protein is the main phosphorylation siteafter TBI and there is a different time and spatial distribution feature of tau abnormalitydifferent from other neurodegeneration diseases. Those phenomenon was accompanied withgliosis, neuron lost and axon damage, it may be the main cause of cognitive disfunctionafter TBI.2. Morris water maze (MWM) and open field test was performed after using selectiveA2AR agonist and antagonist. We found that: A2AR deficiency can alleviate the cognitivedysfunction after TBI. We found those results were not influenced by motor functiondamage by comparing swimming speed between different groups. Meanwhile,phosphorylation level of tau protein at Ser404site was also decreased obviously byinhibiting A2AR. We found similar phenomenon in cultured rat hippocampal neurons andSH-SY5Y cells. The above results indicate that tau protein’s hyperphosphorylation atSer404site is closely related with the activation of A2AR. Reducing tau protein’s phosphorylation level in DG region by inhibiting the activation of A2AR may be the mainmechanism of the cognitive function protective effect. The activation of A2AR also inducedmitochondria axonal transportation defect in primary hippocampal neurons. These resultsfurther confirms the neurotoxicity role of A2AR in tau hyperphosphorylation and axonaltransportion defect.3. By using selective PKA inhibitor and non-selective GSK-3β inhibitor respectivelyand simultaneously after the activation of the A2AR in SH-SY5Y cells, we found tauprotein’s phosphorylation level decreased obviously. By analysising our result with otherresearch about the interaction between PKA and GSK-3β we believe the activation ofA2AR may increase the phosphorylation level of tau protein through the activation of PKAand then facilitating the function of GSK-3β to the C-terminal of tau protein.In summary, we demonstrated that the activation of A2AR is a main cause of tauprotein’s hyperphosphorylation and cognitive disfunction through activating PKA and thenfacilitating the phosphorylating function of GSK-3β to the C-terminal of tau. And tauprotein’s hyperphosphorylation may be a main cause of axonal transportation deficit oreven neuron death after TBI. These findings may provide some experimental evidence forthe therapy strategy of cognitive impairment after TBI.