Construction Of Light-switchable A_2AR Antagonistic Tool And The Role Of A_2AR In The Impulsive Behavior

G-protein coupled receptors(GPCR)is a superfamily of large amounts of membrane receptors.They play important roles inside the human beings.Adenosine 2A receptor(A_2AR)belongs to the A class GPCR,it can be found in many tissues and organs in the body.A_2AR is widely expressed in the central neural system(CNS),especially in hippocampus and striatum.Stimulus of traumatic brain injury(TBI)and inflammation can cause A_2AR over expression.In addition,the expression of A_2AR is increased in neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease and Huntington's disease,and the increase of A_2AR will lead to corresponding behavioral changes.This suggests that A_2AR plays an important role in the central nervous system related diseases.At present,the regulation methods of A_2AR include the use of natural or synthetic compounds,biomacromolecules,genetic methods or Optogenetic methods.Optogenetic technology has the advantage of high spatial-temporal specificity in the study of GPCR function in the CNS.It can effectively reveal the role of GPCR including A_2AR in neural circuits and the relationship between GPCR and behavior.At present,there are active and inhibitory A_2AR optogenetic tools,but these inhibitory optogenetic tools are based on the chemical synthesis of drugs,the principle is the ligand and receptor binding,so it is difficult to avoid the problem of drug receptor selection specificity.Previous studies have shown that the antagonism of A_2AR plays a neuroprotective role in many central related diseases.Therefore,effective and specific inhibition of A_2AR is an important way to reveal the function of A_2AR.However,due to the lack of inhibitory optogenetic tools,it is difficult to further study the role of A_2AR in specific circuit in the CNS diseases.So,can we construct a highly A_2AR specific optogenetic tool that not bases on the ligand-receptor interaction,and use this tool to further study the role of A_2AR?Impulsive behavior is a behavioral that can occur under normal physiological conditions,but is likely to occur after traumatic brain injury(TBI).Patients with increased impulsive behavior are likely to have a significant impact on their lives.The amygdala and cortex are brain regions highly related to impulsive behavior.The connection between the two regions plays an important role,and the circuit connecting the brain regions is the basis of this connection.After TBI,A_2AR is highly expressed in many brain regions,and some studies have shown that A_2AR participates in the regulation of impulsive behavior.However,the current studies have not revealed the role and mechanism of A_2AR in the impulsive behavior after TBI,especially whether there are functional differences between A_2AR pre and post of the synapses of the circuit.So,can we explore the effect of A_2AR at different positions on impulsive behavior and the mechanism that leads to these effects by using the tools of light controlled A_2AR inhibition?In this study,we synthesized an Anti A_2AR peptide with the function of antagonizing A_2AR by using the fragments of A_2AR functional antibody.Through enzyme linked immunosorbent assay,immunohistochemistry,western blotting and animal behavior,we proved that the constructed Anti A_2AR can effectively express in vitro and in vivo and specifically block A_2AR downstream signal.Based on the constructed Anti A_2AR,a tool LOV-Anti A_2AR,which can achieve high selectivity and high spatial-temporal specific inhibition of A_2AR,was designed by using the light,oxygen and voltage sensing domain(LOV).Finally,by expressing LOV-Anti A_2AR in amygdala and observing the mice behavior,we revealed the role and mechanism of A_2AR in impulsive behavior.The main results and conclusions are as follows:1.Several Anti A_2AR peptides were constructed from the heavy chain of the functional antibody Fab2838.The constructed peptide was successfully expressed in HEK293T cells and co-expressed with A_2AR.The results showed that the antibody against A_2AR 213-230 could not detect the expression of A_2AR,but the antibody against 373-391 amino acids could detect the expression of A_2AR,and Anti A_2AR could also block A_2AR in immunofluorescence staining.Anti A_2AR(29)can effectively antagonize the phosphorylation levels of cAMP and its downstream signal PKA,ERK and CREB,and can also effectively block the increase of cAMP in the presence of CGS21680.As the ratio of Anti A_2AR to A_2AR increased,the antagonistic effect of Anti A_2AR on A_2AR increased gradually.When the ratio reached 400:1,the antagonistic effect did not increase.The levels of IP1 and c GMP were not affected by Anti A_2AR,suggesting that Anti A_2AR did not affect the downstream signal changes of other G proteins.In primary cultured cortical neurons,the expression of Anti A_2AR can inhibit the increase of calcium signal.In the striatum,Anti A_2AR is co-expressed with Neu N but not GFAP,suggesting that Anti A_2AR was specifically expressed in neurons.Immunofluorescence staining also showed that the phosphorylation levels of PKA and CREB downstream of A_2AR were decreased.After Anti A_2AR was expressed in striatum,the activity of mice in open field and home cage was increased,and the number of open arm entrances and duration in elevated-plus maze increased,which indicated that antagonizing A_2AR in striatum resulted in anxiolytic behavior.These results suggest that Anti A_2AR can specifically antagonize A_2AR in vivo and in vitro.2.Based on the constructed peptide Anti A_2AR which can antagonize A_2AR function,LOV-Anti A_2AR,a spatial-temporal specific photo-controlled A_2AR tool,was constructed by connecting with LOV.Five LOV-Anti A_2AR peptides with different length were constructed.The expression of LOV-Anti A_2AR in HEK293T cells co-expressed with A_2AR.LOV-Anti A_2AR with 177 amino acids showed the strongest cAMP inhibition effect under blue light irradiation,and the inhibitory effect was also obvious after treatment with agonist CGS21680.Compared with the control group,LOV-Anti A_2AR had no effect on cAMP level without light,suggesting that LOV-Anti A_2AR had no activity under the dark condition.The results of different intensity blue light stimulation showed that the antagonistic effect of LOV-Anti A_2AR enhanced with the increase of light intensity and reached light saturation at 1 m W/cm².The time-effect curve of cAMP level decreased after light stimulation and increased after light withdrawal,indicating that LOV-Anti A_2AR has the characteristics of instant on-off.The activation of LOV-Anti A_2AR had no effect on the levels of c GMP and IP1.Western blot and immunofluorescence results showed that light activated LOV-Anti A_2AR could significantly reduce the phosphorylation levels of PKA,ERK and CREB.LOV-Anti A_2AR was expressed in vivo and light activated LOV-Anti A_2AR could reduce the firing frequency of neurons without affecting the amplitude.When LOV-Anti A_2AR was injected into the striatum and activated by light,the mice showed anxiolytic behavior in open field and elevated-plus maze tests.The results of immunofluorescence staining also showed that LOV-Anti A_2AR was neuron specific and could inhibit PKA phosphorylation after illumination.These results suggest that LOV-Anti A_2AR can effectively inhibit the expression of A_2AR in vivo and in vitro,and this antagonism is spatial-temporal specific.3.The 5-Choice serial reaction time task(5-CSRTT)device was assembled to establish a model for detecting impulsive behavior.In this device,the wild-type C57 mice were trained and tested.The results show that the device can effectively detect the impulsive behavior of mice.After establishing closed-TBI model,mice were trained with 5-CSRTT.Compared with the sham group,TBI mice needed more training times to complete each stage,and the premature response rate of TBI mice was increased.These results indicated that A_2AR of amygdala was involved in the occurrence of amygdala hijack.Mice expressing LOV-Anti A_2AR in amygdala showed a decrease in the premature response rate after TBI,indicating a decrease in impulsive behavior.These results suggest that the activation of A_2AR plays an important role in the impulsive behavior of mice with TBI.Photoinhibition of A_2AR in amygdala can inhibit the occurrence of impulsive behavior.Above all,our research shows that Anti A_2AR is a peptide that can effectively antagonize A_2AR function in vivo and in vitro;LOV-Anti A_2AR constructed on the basis of Anti A_2AR not only with A_2AR selectivity,but also spatial-temporal specificity,and this kind of inhibition is reversible.We have successfully constructed LOV-Anti A_2AR to explore the role of A_2AR on amygdala neurons in impulsive behavior after TBI,and lay a foundation for further exploring the mechanism of A_2AR in it.