The Mechanism Of Aberrant A_2AR Signaling Induced Accumulation Of Hyperphosphorylated Tau Protein In Various Brain Regions And Spatial Memory Dysfunction After TBI

Traumatic brain injury（TBI）which is characterized by a high rate of mortality and sever sequela in the chronic phase post injury has been widely concerned in modern soci ety.Spatial memory dysfunction is one of the most predominant aspects of cognitive impairment after TBI.Hyperphosphorylated tau protein（p-tau）as well as oligomers and tangles composed of p-tau are defined as tau pathology and the appearance of tau pathology is a typical feature of Alzheimer’s disease（AD）which is mainly characterized by cognitive dysfunction.Thus,neurodegenerative diseases containing tau pathology including chronic traumatic encephalopathy are defined as tauopathy.It has been demonstrated in recent studies on AD that the propagation of tau pathology in brain is an important mechanism of disease deterioration.However,tau pathology in TBI patients with spatial memory dysfunction occurs much earlier than other neurodegenerative diseases and in its unique manner.Whether this should be blamed for the early occurred cognitive dysfunction after TBI remains unclear.It has been reported that adenosine A_2A receptors(A_2ARs)were activated after TBI due to the increased adenosine level.Inactivation of A_2ARs was shown to be closely associated with the amelioration of p-tau and amyloid β（Aβ）accumulation,and cognitive dysfunction in AD mouse models.In addition,"glymphatic system" was a recently defined brain wide waste clearing system.The function of glymphatic system is mediated by astroglial water transport via AQP4,which is regulated by reactive astrogliosis and phosphorylation.A_2AR can regulate the activation of astrocytes and cAMP/PKA signaling as reported previously.Thus,we hope to explore whether the cognitive protective effect of A_2AR is related to the alleviation of cerebral tau pathology after TBI,and the cellular mechanism of this phenomenon.In the present study,we use controlled cortical impact（CCI）method to produce a moderate TBI model.We evaluated the temporal,spatial and cellular pattern of p-tau accumulation 24 h,7 d and 4 w after TBI by using immunohistochemistry and western blot method.Then,the A_2AR induced p-tau accumulation and neuronal damages were evaluated in vivo and in vitro through pharmacological blockade or genetic knockout of A 2AR.Further,we explored the influence of A_2AR overexpression on the brain regional specific impairment of glymphatic AQP4 polarity impairment and extracellular p-tau clearance.Finally,we evaluated the influence of A_2AR activation on the propagation of tau pathology from hippocampus to prefrontal cortex after TBI through overexpression of human P301 S tau protein or brain region specific injection of A_2AR agonist in mild TBI model.Western blot and co-immunoprecipitation tests were performed to explore the underlying molecular mechanism and its relationship with the fast deteriorating cognitive impairment after TBI.The main results and conclusions are as follows:1.In mouse model of moderate TBI,phosphorylation of tau at Thr205,Ser262 and Ser404 was measured 24 h,7 d and 4 w post injury.Cellular localization of tau was detected through double-labeling with neuronal marker,namely NeuN.Our results indicated TBI can induce an increase of p-tau level at Ser404,which mainly locates in bilateral cortex,hippocampus and thalamus 24 h after TBI.At 7 day after TBI,p-tau level continues to increase ipsilateral cortex,thalamus as well as contralateral parietal cortex and hippocampus.In addition,p-tau level was shown to increase in the contralateral prefrontal cortex（PFC）at this time point.At 4 w after TBI,p-tau mainly locates in ipsilateral thalamus and contralateral hippocampus and PFC.Increased p-tau was shown to be closely related to axonal and dendritic impairment through amyloid precursor protein（APP）and Golgi staining.Behavioural test results indicated that spatial reference memory impairment occured 7d after TBI and returned to normal level 4w after TBI.Spatial working memory impairment was observed at 7d and 4w after TBI.This part of result indicates that TBI can induce an immediate increase and enduring accumulation of p-tau in spatial working memory circuit related brain regions including parietal cortex,hippocampi,prefrontal cortex and thalamus.The appearance of spatial memory dysfunction is in accordance with p-tau accumulation and neuronal impairment.These phenomena may lead to the spatial memory impairment after TBI.2.The molecular mechanism of A_2AR regulated tau phosphorylation and the protect effect of A_2AR inactivation on cognitive function were evaluated by pharmacologically or genetically blockade of A_2AR in vivo and in vitro.Our results indicated that A_2AR knockout can alleviate the increased p-tau level and spatial memory dysfunction after TBI.Caffeine ingestion can mimic the neural protective effect of A_2AR knockout.A_2AR regulates p-tau level through two main mechanisms:（1）A_2AR activates PKA and thus facilitates the phosphorylation of tau by GSK-3β;（2）A_2AR knockout can modulate the phosphorylation pattern of GSK-3β directly and inhibit the activation of GSK-3β.Caffeine consumption alleviated the impairments in dendritic spine morphology and density 4 w after TBI.This part of result indicates that TBI can induce A_2AR activation in brain regions of cognitive neuronal circuits leading to an increase in intracellular p-tau level and may contribute to the formation of "seeds" for propagation of tau pathology.3.Recent studies have demonstrated that clearance of extracellular solutes by glymphatic system depends on the perivascular localization of AQP4 which can be disrupted by reactive astrogliosis.Our results indicated that TBI triggered an enduring increase in the expression level of A_2AR in the contralateral hippocampus till 4 w after TBI and peaked at 3 d after the injury.At 7 d after TBI,the increased A_2ARs locate in neurons and astrocytes.At 4 w after TBI,the increased A_2ARs were found mainly located in the reactive astrogliosis.AQP4 polarization in the perivascular astrocytic end feet was impaired after TBI,which was most prominent in the ipsilateral brain tissue surrounding the directly impacted region and the contralateral hippocampal CA1 area and was accompanied by increased local p-tau,changes in dendritic spine density and morphology,and up-regulation of the adenosine A_2ARs.The critical role of the A_2AR signaling in these pathological changes was confirmed by alleviation of the impairment of AQP4 polarity and accumulation of p-tau in the contralateral CA1 area in A_2AR knockout mice.Our results indicate that TBI can induce an overexpression of A_2AR in astrocytes which is related to the regional disruption of AQP4 polarity in the hippocampal CA1 area,the impairment of extracellular p-tau clearance and neuronal damages.4.Overexpression of human P301 S tau in hippocampus of non-injury mouse brain can induce an increase in the phospho-tau（Ser404）level in the surrounding areas as well as the contralateral hippocampus and bilateral PFC.In mild mouse model of TBI,A_2AR agonist was injected to contralateral hippocampus for 7 days continuously from 7 d after TBI.A significant increase in p-tau level were observed in the injected region as well as in the PFC compared to SHAM group and TBI + vehicle group.These results indicate the spread of tau pathology along neuronal projections may help to explain the reason why p-tau appeared in PFC which was not directly injured.Expression of kinesin heavy chain member 2A（KIF2A）,which is closely associated with depolymerization of microtubules was found increased immediately at 24 h after TBI and last until 7 d post injury.The increased KIF2 A mainly locates in the ipsilateral hemisphere and the contralateral hippocampus.Protein interactions between KIF2A/translin-associated protein X（TRAX）and A_2AR were found increased 24 h and 4 w after TBI.Our results indicate the activation of A_2AR may facilitate the propagation of p-tau from contralateral hippocampus to PFC through C terminal mediated TRAX and KIF2 A interaction.Up-regulated KIF2 A facilitates the spread of tau pathology in neuronal circuits via accelerating the depolymerization of microtubules.In summary,we demonstrated that TBI triggers activation of A_2AR signaling in the contralateral hippocampus and induced an increase in intracellular p-tau level via PKA/GSK-3β pathway,leading to the formation of "seeds" for tau pathology propagation.Meanwhile,A_2AR facilitates the spread of p-tau from hippocampus to PFC via TRAX/KIF2 A signaling resulting in the increase of p-tau in various brain regions and the early occured spatial memory impairment.In addition,astrocytic A_2ARs can regulate AQP4 polarization and the extracellular p-tau clearance by glymphatic system,which may be a risk factor for tau pathology spread to neighboring cells after TBI.These findings provide experimental basis for a promising therapeutic and prophylactic strategy for the progressive cognitive impairment after TBI by targeting aberrant A_2AR signaling through preventing tau hyperphosphorylation in different processes.