Involvement Of Aberrant ACC Wnt-glycolysis Signaling In The Defects Of ASD Excitatory Synapse And Social Function

【Background】Social impairment is one of the two core symptoms of autism spectrum disorder（ASD）,which seriously affects the quality of life and career development of patients.Because the pathogenesis of ASD is unclear,there lacks early diagnosis and effective pharmaceutical therapy in clinical practice.In the past decade,approximate 1000 risk genes and numerous environmental factors have been identified to be associated with the development of ASD.Therefore,elucidating the convergent molecular mechanisms of related social dysfunction is crucial for developing effective treatments to benefit more patients.Electrophysiological studies have shown that synaptic dysfunction,particularly impairment of excitatory synaptic transmission or an imbalance in the excitation-inhibition ratio of synaptic transmission,is a major pathological change in multiple ASD mouse models and may account for the ASD social dysfunction.It was found in previous studies that an increased proportion of silent synapses in the anterior cingulate cortex（ACC）involved in connecting social information and regulating social functions,that is,the proportion of immature synapses,may be responsible for the social impairment of Shank3 knockout mice,suggesting that abnormality of ACC excitatory synapses plays a fundamental role in ASD social dysfunction.However,whether a convergent molecular mechanism exists for such synaptic deficits associated with social dysfunction remains questionable.Maintaining synaptic transmission in neurons generally requires a high energy supply.Abnormal synaptic transmissions usually can be detected in electrophysiological studies among ASD patients.Metabolic abnormalities are often reported in clinical studies for children with ASD.Mitochondrial dysfunction has been found in 30%to 80%of blood samples from ASD patients by various research teams,suggesting that imbalances in energy metabolism may contribute to cellular dysregulation and disease progression.Increased lactate/pyruvate ratios were observed in serum biomarker studies for patients with ASD.In addition,a ketogenic diet has also been recommended as a nutritional intervention to alleviate ASD-related behavioral problems,suggesting that there may be abnormalities in glycolysis in ASD patients.In addition to metabolic abnormalities,mutations in Wnt signaling components are frequently detected in ASD patients.For example,multiple micro-deletions and micro-duplications of Wnt signaling components have been found in ASD patients.A patient-based exome sequencing study mapped 39%of the most destructive or severe mutations to aβ-catenin-associated network.Recent network studies on ASD patients have shown that Wnt/β-catenin signaling pathway,RAS-ERK and PI3K-AKT are the three major abnormal signaling pathways in the development of ASD.Given that Wnt signaling plays an important role in multiple aspects of neural development,and synaptic transmission requires high energy supply,Wnt signaling and its metabolic abnormalities may be involved in the synaptic defects of ASD neurons.In cancer cells,the Wnt signaling pathway promotes tumor cell proliferation by driving the Warburg effect（aerobic glycolysis）.Whether/how Wnt-glycolytic cascade signaling is involved in ASD neuron synaptic abnormalities and causes ASD social dysfunction should be investigated in detail.Our research aimed to address this issue as the followings.【Methods】1)Phenotype analysis.Shank3^-/-mice was first adopted.Considering Shank3 as a post-synaptic scaffolding protein,the proteins interacting with SHANK3 were analyzed and verified by IP-MS technology to find its possible interactions with Wnt signaling and glycolysis-related proteins.Then,antibody blocking experiments,nuclear membrane protein isolation and Western-blotting are used to observe the influence of Shank3^-/-on Wnt signaling in vitro and in vivo,followed by using transcriptomics,LC-MS and Seahorse detection techniques to study the influence of Shank3^-/-on energy metabolism,particularly on glycolysis.Finally,the expression profiles of Wnt signaling and glycolysis changes in Shank3^-/-mice during development are described,so as to provide evidence for the postnatal intervention time window.2)Signal intervention.At first,usingβ-catenin EX3^loxp/loxpmice,through Golgi staining,patch clamp and Seahorse and other techniques,under the induction of CaMKII-cre,the impact of over-activation of Wnt signaling in ACC on synaptic structure and function was observed.Its impacts on social,stereotyped,and anxiety behaviors,as well as the impact on glycolysis were observed.Secondly,using wild-type mice,the impact of knocking down Axin2 in ACC on social behavior wasevaluated.Finally,glycolysis was systematically inhibited in Shank3^-/-mice,and its impact on synaptic maturation and social behavior was observed,in order to understand the underlying mechanism by which glycolysis affected synaptic maturation.3)Mechanism elucidation.Firstly,the effects of two Wnt signaling inhibitors（ICG001 and XAV939）on glycolysis and oxidative phosphorylation of Shank3^-/-ACC neurons were observed respectively.Using Co-IP,immunofluorescence staining,PLA,and other technologies,the key protein of Wnt-glycolysis interaction and its intracellular location were detected.Secondly,following the use of a chemical compound to interfere with interacting proteins,through Golgi staining,patch clamp and cellular calcium techniques,its long-term and short-term effects on the synaptic structure and function in Shank3^-/-mice,as well as the impact on social functions were observed.At the same time,the impact of manipulating non-canonical Wnt signaling regulation and the biosafety of this compound on wild-type mice were explored.4)Human neuron validation.Firstly,the Shank3-mutated H8-h ESC line was established and verified by using CRISPR-Cas9 technique,Western-blotting and DNA sequencing,and differentiated into human neurons through a mature neural induction protocol;and then using Western-blotting,Co-IP,Seahorse and other technologies,the changes of Wnt-glycolysis signaling in human Shank3^-/-neurons were observed to clarify the inhibitory effect of XAV939.5)Environmental factor model validation.By establishing valproic acid（VPA）treated mice and VPA-treated human neurons and using Western-blotting,LC-MS,Seahorse,Co-IP and other techniques,changes in Wnt signaling and glycolysis levels in the VPA model were observed.The aim is to find the time window for abnormal Wnt-glycolysis during the development of the model,so as to find the common time point for postnatal intervention in the ASD models.The final step is to clarify the rescue effect of Wnt-glycolysis intervention in the VPA model.【Results】1)Canonical Wnt signaling was over-activated and levels of glycolysis were elevated in the ACC of Shank3^-/-mice.The results of IP-MS suggested that Wnt signaling and glycolysis-related proteins（such asβ-catenin and ENO1）possibly interacted with SHANK3.The results of WB showed that the levels ofβ-catenin and TCF7L1 in ACC nucleoprotein of Shank3^-/-mice significantly increased,and the expression ofβ-catenin on the membrane significantly decreased;the lactate/pyruvate ratio was elevated in the ACC,and the basal and maximal glycolytic values were abnormally elevated in the ACC neurons.At the same time,it was found that the abnormal time points of Wnt signaling and glycolysis in ACC of Shank3^-/-mice were mostly detected around P14.2)Over-activation of canonical Wnt signaling within ACC excitatory neurons in wild-type mice was sufficient to induce synaptic and social impairment and upregulate glycolytic activity.Sholl and spine analysis revealed that the dendrite branch and mature dendritic spine density in ACC neurons ofβ-catenin overexpressed mice were both reduced.The average amplitude and frequency of m EPSCs were significantly lower than those of the control group.The results of behavioral experiments suggested that the social preference ofβ-catenin overexpressed mice was significantly reduced.ECAR experiments showed that excessive activation of Wnt signaling significantly increased the basal and maximal glycolysis in ACC neurons.3)Inhibiting glycolysis partially rescued Shank3^-/-mice from synaptic and social abnormalities.Systemic administration of 2-DG in Shank3^-/-mice significantly increased the unit density of dendritic spines,and passed through the Rho A/Rock/Cofilin signaling cellular membrane transport of Glu R1 in Shank3^-/-neurons.In addition,2-DG treatment significantly improved social preference and social interaction in Shank3^-/-mice,which had no significant effect on their social novelty.4)The Axin2/ENO1 interaction could be a key node for the cross-linking of Wnt signaling and glycolysis in Shank3^-/-neurons.ECAR results showed that XAV939 significantly increased the maximum respiratory capacity and ATP production of Shank3^-/-neurons.Co-IP experiments revealed the basal level of Axin2/ENO1 interaction in WT neurons.The interaction was significantly enhanced in Shank3^-/-neurons.XAV939 inhibited the Axin2/ENO1 interaction and the activity of ENO1.5)Stabilizing Axin2 effectively rescued Shank3^-/-mice from abnormal glycolysis,synaptic defects and social dysfunction.Administration of XAV939 via ACC implantation significantly increased the branch number and average length of Shank3^-/-ACC neuron basal dendrites,especially increasing mature dendritic spines.After treatment,both the average frequency and amplitude of mEPSCs were effectively restored,and the response amplitude and rising time of cytoplasmic Ca²⁺were effectively alleviated;expression of membrane-bound Glu R1 was increased.In addition,social preference,social novelty and social interaction were effectively improved in Shank3^-/-mice.6)Wnt signaling and glycolysis levels in human Shank3^-/-neurons were abnormally elevated,and stabilizing Axin2 effectively improved both conditions.In human neurons with neural inducted Shank3mutation,the levels of p-GSK3β（S9）and TCF7L1 in the canonical Wnt signaling pathway were significantly increased,the glycolytic activity was abnormally increased,and the expression of synaptic proteins such as PSD95 was very low,and XAV939 effectively improved these conditions,which was similar to the effects previously mentioned in the Shank3^-/-mice.7)Wnt signaling and glycolysis were abnormally elevated in VPA-induced ASD models,which was effectively alleviated by stabilizing Axin2.Higher levels of p-GSK3β（S9）,Axin2,and nuclearβ-catenin expression,higher levels of lactate and pyruvate,and higher levels of glycolytic activity were detected in VPA-treated mouse ACC and human neurons.Moreover,by inhibiting the Axin2/ENO1 interaction,XAV939 reduced the abnormally activated Wnt-glycolysis signal so to improve social impairment.【Conclusion】By using two widely adopted ASD mouse models(Shank3^-/-and VPA-treated mice)and their corresponding human neurons,we found abnormal activation of canonical Wnt signaling and abnormally increased levels of glycolysis within the ACC.Overexpression ofβ-catenin in ACC resulted abnormal glycolysis and social impairment in wild-type mice.Blocking glycolysis in ASD mice partially rescued synaptic phenotype and social function.Axin2,a key inhibitory molecule in the Wnt signaling pathway,interacted with enolase（ENO1）of the glycolytic pathway in ASD neurons to activate glycolysis.It is worth noting that the Axin2 stabilizer XAV939 effectively blocked the Axin2/ENO1 interaction,inhibited Wnt signaling and glycolysis at the same time,shifted the balance of energy supply from glycolysis to oxidative phosphorylation,and promoted synaptic maturation and improved social phobia.This study unexpectedly revealed that abnormally activated Wnt-glycolytic signaling in neurons was an important underlying mechanism for synaptic deficits in ASD.Our findings suggest that Axin2,as a link of Wnt signaling and glycolytic activity in ASD neurons,may serve as a potential therapeutic target of those Wnt-glycolysis abnormal ASD patients to improve social dysfunction.