Behavioral Characterization Of SAPAP4 Knock-out Mice

Understanding the molecular mechanism of postsynaptic proteins functions is of great significance not only to clarify how neurons communicate in the nervous system,but also to provide a theoretical basis for the treatment of mental disorders caused by synaptic dysfunction.SAPAP(SAP90/PSD-95-associated protein)family proteins are postsynaptic scaffolding proteins at excitatory synapses.In postsynaptic density(PSD),SAPAPs interact with the PSD95 and Shank to form a key scaffolding comlex that regulate the trafficking and targeting of neuronotransmitter receptors as well as signaling molecules to the postsynaptic membrane.Thus,it is believed to play an important role in maintaining synaptic structure and advanced cognitive function.The SAPAPs family is composed of four highly homologous subtypes SAPAP1,2,3 and 4,which are encoded by 4 different genes.Distinct mRNA and protein expression in the central nervous system had been well studied among the four isoforms.It has high expression in many brain regions related to advanced cognitive function,such as cortex,hippocampus,thalamus and locus coeruleus.At present,limited studies focused on SAPAP4 function in vivo.In this study,we inverstigated SAPAP4 function by using SAPAP4 knock-out mice as an animal model.1.Physiological characteristics of SAPAP4 knock-out miceSAPAP4 knock-out mice were initially generated by homologous recombination,by which the sequence containing 3-6 exons of SAPAP4 gene was replaced by YFP.Heterozygotes and homozygotes of SAPAP4 knock-out mice were identified by PCR.Compared to black wild-type C57BL/6 mice,brown SAPAP4 knock-out mice had YFP expressed in different brain regions,including coertex,striatum,thalumas,cerebellum,hippocampus,locus coeruleus and olfactory bulb etc.There were no significant differences of morbidity and ablactation survival rate comparing koock-out mice with wild-type littemates.Similar brain weight and size were observed between wildtype mice and SAPAP4 knock-out mice.No obvious developmental disorders was abserved.2.Behaviral characteristics of SAPAP4 knock-out miceA variety of abnormal behavioral phenotypes in SAPAP4 gene knockout mice were observed in a series of behavioral tasks,including open field test,pre-pulse inhibition test,passive avoidance test,rotarod and 3-chamber social interaction task.Compared to wild-type littermates,SAPAP4 knock-out mice showed hyperactive performance in open-field testh but without apparant anxiety-like behavior.The knock-out mice also demonstrated significant pre-pulse inhibition deficits in PPI test as well as fear memory deficits in passive avoidance test,although with normal motor coordination and balance.However,the knock-out mice exhibited normal social interaction behavior as tested by 3-chamber social interaction task.Hyperactivity and pre-pulse inhibition deficits are two characteric symptoms of ADHD(attention deficit/hyperactivity disorder)symptoms,which suggests that SAPAP4 knock-out mice may serve as a potential animal model of ADHD.3.Effects of atomoxetine on SAPAP4 knock-out mouse behaviorSince SAPAP4 highly expressed in the locus coeruleus,where is the primary region for norepinephrine synthesis and projects to the whole brain.Atomoxetine is a norepinephrine reuptake inhibitor approved for the treatment of ADHD and it could be used for testing predictive validity of candidate ADHD animal models.To further explore the mechanism underlying the abnormal behavior of SAPAP4 knock-out mice,we adminnistered animals with atomoxitine for 28 days.We found out that long-term atomoxetine(3mg/kg)treatment reversed hyperactivity to normal level and attenuated PPI deficits of SAPAP4 knock-out mice.However,low-dose atomoxetine(0.3mg/kg)treatment only partially reduced hyperactive level and had no significant effect on PPI performance of SAPAP4 knock-out mice.The learning and memory deficits of SAPAP4 knock-out mice could not reversed by long-term atomoxetine(3mg/kg)treatment.4.Effects of fluoxetine on SAPAP4 knock-out mouse behaviorFluoxetine,a selective serotonin reuptake inhibitor(SSRI),mainly used for the treatment of major depression and other disorders.In several reports,fluoxetine was considered as an alternative treatment for ADHD.To further investigate the mechanism underlying the abnormal behavior of SAPAP4 knock-out mice,we treated animals with fluoxetine and found that acute 20mg/kg injection reversed SAPAP4 knock-out mice hyperactive locotmotion activity.However,long-term 5mg/kg fluoxetine treatment or long-term 10mg/kg fluoxetine treatment only partially reduced hyperactive level and partially improved PPI performance of SAPAP4 knock-out mice.Similar as atomoxetine,the fear memory deficits of knock-out mice was not rescued by fluxetine.5.Effects of SAPAP4 gene knock-out on SHANK and mGluR5 distribution in mPFC PSDIn the post-synaptic dense region(PSD),SAPAP4 is indirectly associated with the metabolic glutamate receptor 5(mGluR5)on the PSD membrane via SHANK and HOMER proteins.SAPAP4 knockout mice had hyperactive symptoms and pre-pulse inhibition deficits.These phenotypes were similar to those of animal models with mGluR5 dysfunction in some extent.In order to further identify the effects of gene knockout on mGluR5 function and further analyze the possible mechanisms of the abnormal behavior,we extracted PSD protein from mouse medial prefrontal cortex(mPFC)to detect the changes of SHANK protein and mGluR5 receptor level.We found that the SAPAP4 gene knockout had no significant effect on the distribution change of SHANK protein in mPFC PSD,but it significantly decreased the distribution of mGluR5 in mPFC PSD.This finding suggests that the expression alternation of mGluR5 in PSD may be caused by SAPAP4 loss,and it could be one of the main factors that lead to abnormal behavior.6.Effects of atomoxetine on neurotransmitters contents in mPFC of SAPAP4 KO miceThe use of atomoxetine hydrochloride,norepinephrine reuptake inhibitor and fluoxetine,5-serotonin transporter inhibitor,can reverse or attenuate hyperactivity symptoms and prepulse inhibition defects of the SAPAP4 gene knockout mice.Both of the two drugs may related to dopamine,5-hydroxy tryptamine and norepinephrine neurotransmitter system adjustment.To further analyze which neurotransmitter was affected by SAPAP4 ablation,we measured the three neurotransmitters levels in the mPFC by HPLC.We found that: 1)all the three neurotransmitters levels in mPFC of SAPAP4 knock-out mice were similar to wild type mice;2)3mg/kg atomoxetine treatment can significantly increase the concentration of dopamine and norepinephrine in mouse mPFC but not 5-HT.These findings suggests that the effect of atomoxetine on hyperactivity may due to the regulation fo DA and NE system.In conclusion,we found that SAPAP4 knock-out in mice leads to hyperactivity,PPI disruption and fear memory deficits.Atomoxetine or fluoxetine treatment could totally or partially rescue hyperactivity and PPI disruption in SAPAP4 knock-out mice.We speculate that SAPAP4 ablation affects the expression of mGluR5 in PSD and the function of norepinephrine or dopamine transmitter system.The decrease of mGluR5 and the dysregulation of the transmitter system may be some of the key factors that cause abnormal behavior in mice.This study suggeted that SAPAP4 knockout mice have the potential to be an ADHD animal model.SAPAP4 may be a key protein to maintain a variety of physiological functions,such as normal locomotion,learning and memory,sensory gating mechanism and so on.It is an important functional component of the nervous system.Our findings provide a preliminary basis for further research on mechanisms of SAPAP4 dysfunction in abnormal behaviors,such as hyperactivity.