Study Of The Rescue Effect Of Selective Activation Of MeCP2 In Cholinergic Neurons On Rett Syndrome-like Phenotypes And Its Mechanism In Mice

Rett syndrome(RTT)is an X-linked neurodevelopmental disorder predominently affecting the female and is a major genetic cause of mental retardation in females.RTT is characterized by growth arrest after 6-18 months’ normal development followed by the regression of acquired motor skills speech and intelligence,the occurrence of stereotypic hand movement and autism features of social dysfunction,commonly accompanied by respiratory abnormality and seizures.Patients usually lose locomotor function at the late stage of RTT.MeCP2 mutations had been identified as the cause of RTT.MeCP2 gene encodes methyl-CpG-binding protein 2(MeCP2),which functions as a transcriptional regulator and a modulater of chromatin structure.MeCP2 is critical for maintaining the brain function through regulating the expression of genes involved in neuronal maturation and function maintenance.It is important to explore the pathophysiological mechanism of MeCP2 mutations to cause RTT.Studies of conditional knockout of MeCP2 suggested that different types of neurons,including excitatory neurons,GABAergic interneurons,catecholaminergic neurons,and so on,play different roles in different aspects of RTT symptoms,indicating the neuronal-specificity of RTT pathogenesis,supporting the possibility of therapies targeting a certain type of neurons.Studies based on MeCP2-mull model,which is similar to the condition of patients are more important for exploring the improvement effect of function enhancement of certain neurons on the RTT-like phenotypes,providing more direct evidence for the targeted treatment.As an important modulating system in brain,cholinergic neurons widely project to many brain regions to modulate other nocholinergic neuron types including those involved in RTT mentioned above(GABAergic interneurons,catecholaminergic neurons,and so on),playing critical roles in keeping the excitation/inhibition balance of brain and in local dopaminergic microcircuits etc..Hence,we select chonlinergic neurons as a candidate neuron type to study its specific role in RTT-like phenotypes and the underlying mechanism in mice.Objective:1 To activate MeCP2 selectively in cholinergic neurons in otherwise MeCP2-null mice.2 To detect the effects of selective activation of cholinergic MeCP2 on RTT-like phenotypes in mice for understanding the mechanism of RTT-like phenotypes in mice:1)Exploring whether selective activating MeCP2 in cholinergic neurons can rescue most of the RTT-like symptoms effectively to demonstrate the importance of cholinergic MeCP2 in contributing to and in rescuing the RTT-like phenotypes in mice.2)Further exploring the molecular mechanism of the effects of selective activation of cholinergic MeCP2 on RTT-like phenotypes in mice.Methods:1 We use Chat-Cre;MeCP2lox-stop breeding system to selectively delete the Lox-Stop-Lox sequence in MeCP2 gene in cholinergic neurons to allow MeCP2 be expressed solely in cholinergic neurons in otherwise MeCP2-null mice.2 For study the roles of cholinergic MeCP2 in RTT-like phenotypes,1)a variety of behavioral tests were used to systemetically detect the RTT-like phenotypes of the cholinergic MeCP2 activation model,predominantly including locomotor function(Open field test),forelimb grip strength,anxiety-like behavior(Open field test and Elevated plus maze),social behavior(Three-chamber test),sensory-motor gating function(Prepulse inhibition test),and the general condition assay(Phenotypic severity scoring,etc.).2)Western blot was done to detect the alteration of the key candidate molecules,Chat and Chrna7,of cholinergic system in the cholinergic MeCP2 activation mice.Results:1 Confirmation of the validity and efficiency of the mouse model:1)Genotyping of the male offsprings of Chat-Cre;MeCP2lox-stop breeding system was done to group the test male mice into 4 cohorts:WT controls,Cre controls,MeCP2-Stop and Cholinergic MeCP2 rescue mice.2)We also used immunofluorescent staining for expression of MeCP2 and the cholinergic neuron marker Chat to confirm the validity of the cholinergic MeCP2 preservation.It was proved that most of the Chat positive cells showed MeCP2 expression in Rescue mice and the mouse model could be used for further studies of the rescue effect and its mechanism of cholinergic MeCP2 on aspects of RTT-like symptoms in mice.2 Assays of RTT-like phenotypes of mice:1)Test for locomotor ability of the test mice:(1)The open field test for 15 min(6-7 weeks old),(2)Forelimb grip strength measurement(8-9 weeks old).The results manifested that all parameters showing decreased locomotor activity in Stop mice were significantly reversed in the Rescue group without significant differences from the controls,including distance traveled per 5 min,inactive duration,distance traveled at large speed(10cm/s),and the total distance traveled.Consistently,the decreased level of forelimb grip strength in Stop mice was reversed in the Rescue group,reaching to the control level.2)Tests for anxiety-like behavior:1)First 5 min’s performance in the open field apparatus(6-7 weeks old),2)Elevated plus maze(EPM)(6-7 weeks old).The results showed the increased anxiety level of Stop mice in open field was well rescued in the Recue group including the decreased duration and the reduced distance traveled in the central zone of the open field.However,both the Stop and the Rescue mice performed decreased anxiety level in the EPM reflected by increased duration in the open arms and the percentage of time in open arms.3)3-chamber test was used to detect the social behaviors of the mice.Both the interaction time and the corresponding preference index of the Stop mice with the introduced strange partner or the later-introduced novel stranger were showed to be increased.And the Rescue group performed similarly without significant difference from the Stop mice.4)Prepulse inhibition test was also done to detect the sensory-motor gating fuction of the test mice.The result showed there wasn’t significant alteration in stop mice.And chonlinergic MeCP2 rescue had no effect on the sensory-motor gating function of the Stop mice.5)Assessments for the abnormal phenotypes of general condition:(1)Body weight(6-15 weeks,once a week),(2)Severity scoring(6-15 weeks,once a week),(3)Heart rate(8 weeks),(4)Brain weight(8 weeks),(5)Lifespan.It was showed that the phenotypes of lower body weight,increasing severity score,decreased heart rate,lower brain weight and early death in the Stop mice were not improved in the cholinergic MeCP2 rescue group.3 Exploration of the molecular mechanism of the symptomatic improvement effect of selectively activating MeCP2 in cholinergic neuronsWe also attempted to pinpoint some underlying molecular mechanism of the rescue effect by Western blot,finding that the whole brain levels of two candidate molecules,cholinergic marker Chat and the acetylcholine receptor Chrna7 which had been reported to be associated with RTT,didn’t showed significant alterations in both the Stop and the Rescue mice.It was indicated that cholinergic MeCP2 might not affect the cholinergic neuronal function contributing to RTT-like phenotypes by regulating the synthesis of cholinergic transmitter acetylcholine and the level of the RTT-associated acetylcholine receptor α7(Chrna7).Conclusion&Discussion:1 MeCP2 in cholinergic neurons alone was sufficient to reverse the hypoactivity caused by systematic loss of MeCP2.It was suggested that cholinergic MeCP2 was sufficient to maintain normal locomotor function of mice.2 Selective activation of cholinergic MeCP2 could reverse the anxiety-like behavior of the Stop mice.However,it was not enough for cholinergic MeCP2 alone to rescue the decreased axiety level of Stop mice in EPM.It was suggested that cholinergic MeCP2 might downregulate anxiety level,but cholinergic MeCP2 alone is not sufficient to keep the proper anxiety level in mice.Hence,non-cholinergic neurons were also required to normalize the anxiety-like behavior in mice.3 Selective activation of cholinergic MeCP2 couldn’t rescue the hypersocial abnormality of the Stop mice.It was indicated that cholinergic function might be dispensible for mediating the social behavior in mice,which might be attributed to non-cholinergic function.4 Cholinergic MeCP2 appeared to be not essencial for rescuing the symptoms of the underweight,lower brain weight,lower heart rate,increasing severity score and short lifespan showed in Stop mice.It was suggested that the relatively small number of cholinergic MeCP2 was not sufficirnt to maintain the normal general condition in mice.Although cholinergic function widely regulates several types of non-cholinergic neurons,MeCP2 in the relatively small number of cholinergic neurons in brain couldn’t compensate the non-cholinergic dysfuctions which might be responsible for the RTT-like abnormalities of general condition of mice.The results also further support that neuronal MeCP2 primarily functions in a cell-autonomous manner.5 Cholinergic MeCP2 might not mediate cholinergic function through regulating the levels of the key enzyme Chat of acetylcholine synthesis and the RTT-associated acetylcholine receptor Chrna7 to contribute to RTT-like phenotypes.Our study first use the mouse model of selective activation of cholinergic MeCP2 for further investigating the role of cholinergic MeCP2 in the RTT-like phenotypes and in the rescue of RTT.The results further demonstrate the cholinergic role in RTT and add to the supporting evidence for targeted cholinergic treatment for poor motor function and anxiety of RTT patients.Future researches of the underlying neural circuit and molecular mechanism remain to be done.