微管相关蛋白1B在Fmr1基因敲除小鼠脑组织内的表达变化(英文)

目的探讨脆性X智能低下蛋白(fragile X mental retardation protein,FMRP)对微管相关蛋白1B(microtubuleassociated protein 1B,MAP1B)是否具有调控作用。方法应用免疫组化、免疫印记和原位杂交的方法,对1周龄和6周龄的Fmr1基因敲除型(KO)和同龄野生型(WT)小鼠脑组织MAP1B及MAP1B mRNA进行分析。结果免疫组化的结果显示 :6周龄KO小鼠各个脑区MAP1B的平均光密度值(MOD)值均显著低于同龄WT小鼠(P < 0.05),1周龄KO小鼠仅在小脑和海马显著降低(P < 0.01) ;各脑区MAP1B的MOD值在6周龄小鼠均比同基因型的1周龄小鼠显著降低(P < 0.05)。免疫印记和原位杂交结果分别显示MAP1B及MAP1B mRNA在KO小鼠的海马组织均显著降低(P <0.05)。结论MAP1B和MAP1B mRNA在Fmr1基因敲除小鼠脑组织的表达均显著减少,提示FMRP 可能正性调节MAP1B的表达。     

Fmr1基因敲除小鼠脑组织神经调节蛋白1表达的改变及其意义

目的 明确神经调节蛋白1 (NRG1)在Fmr1基因敲除(KO)小鼠中的变化,探讨其在脆性X综合征发病机制中的作用. 方法 应用免疫组织化学染色法检测FVB近交系雄性2周龄Fmr1 KO小鼠(KO2w)、4周龄Fmr1 KO小鼠(KO4w)和同龄野生型(WT)小鼠大脑皮层及海马CA1区、CA3区、齿状回中神经调节蛋白1的阳性神经元的数量;Western blotting检测上述小鼠大脑皮层和海马组织NRG1蛋白的含量. 结果 与同龄WT小鼠相比,KO2w、KO4w小鼠大脑皮层、海马CA1和CA3区NRG1阳性神经元的数量明显减少,在海马齿状回却明显增多,差异均有统计学意义(P＜0.05);KO2w、KO4w小鼠大脑皮层、海马中NRG1含量(相对分子质量为55 000亚型)分别较同龄的WT小鼠明显减少,差异亦有统计学意义(P＜0.05). 结论 Fmr1 KO小鼠大脑皮层和海马组织NRG1阳性神经元及NRG1蛋白表达明显减少,NRG1可能参与脆性X综合征发病机制.     

代谢性谷氨酸受体在脆性X综合征树突棘发育中的作用

目的探讨代谢性谷氨酸受体-Ⅰ(mGluR-Ⅰ)在脆性X综合征发病中的作用和mGluR-Ⅰ拮抗剂治疗的机制。方法培养FMR-1基因敲除小鼠(KO)和野生(WT)小鼠海马神经元,给予mGluR-Ⅰ激动剂和拮抗剂进行处理,Western-Blot法检测处理前后脆性X智力低下蛋白(FMRP)和微管相关蛋白1B(MAP1B),共聚焦显微镜观察树突棘的长度和密度。结果KO小鼠海马神经元树突棘长度(1·32±0·79)和MAP1B含量显著高于WT小鼠(1·00±0·62)。mGluR-Ⅰ激动剂干预后,KO和WT小鼠树突棘长度(1·60±0·88和1·33±0·80)和MAP1B均显著增加。mGluR-Ⅰ拮抗后,KO小鼠树突棘长度(0·95±0·57)和MAP1B均显著降低,在WT小鼠未见明显变化。结论脆性X综合征模型鼠由于缺乏FMRP的负性调节作用,导致mGluR-Ⅰ激活的MAP1B过度表达,可能是树突棘异常的主要原因。mGluR-Ⅰ拮抗剂逆转树突棘缺陷可能参与治疗机制。     

MicroRNA-134在FMR1基因敲除鼠脑组织中的表达及意义

目的 观察脆性X综合征(FXS)模型小鼠不同发育时期脑组织中microRNA-134(miR-134)的表达,明确miR-134的表达特点及脆性X智力低下蛋白(FMRP)缺失是否导致miR-134转录的改变. 方法 应用荧光实时定量PCR检测FVB近交系雄性0 d、4、6周(W)龄FMR1基因敲除型(KO)(KO0d、KO4w、KO6w)和同龄野生型(WT)(WT0d、WT4w、WT6w)小鼠脑组织中miR-134的表达(n=5). 结果同龄KO与WT小鼠miR-134的转录表达量差异无统计学意义(P＞0.05);KO6w小鼠脑组织miR-134的转录表达量低于KO0d和KO2w小鼠,WT6w小鼠脑组织miR-134的转录表达量也低于WT0d和WT2w小鼠,差异均有统计学意义(P＜0.05). 结论 FMRp的缺失并未影响miR-134的转录;miR-134的转录表达量在神经系统发育期保持着较高水平,至成年期则下降,提示其在调控神经系统发育中可能起着重要作用.     

CaMKⅡα在FMR1基因敲除小鼠脑组织中的表达

目的 观察FMRI基因敲除型(KO)小鼠脑组织中钙/钙调素依赖蛋白激酶Ⅱα(CaMKⅡα)表达的改变,探讨CaMKⅡα是否为脆性X综合征相关蛋白(FMRP)的下调蛋白. 方法 PCR鉴定FVB近交系小鼠的基因型,按基因型的不同分为KO组和野生型(WT)组,每组10只.免疫组化染色检测KO及WT小鼠脑组织CaMKⅡα的表达与分布,用图像分析仪分别采集不同脑区免疫信号的吸光度(A)值进行比较. 结果 免疫组化染色检测显示KO与WT小鼠各个脑区普遍存在阳性信号;神经元胞浆尤其是靠近胞体的近端突起上信号呈强阳性,树突中亦有阳性信号,轴突上信号较弱;KO小鼠各脑区CaMKⅡα阳性信号的A值均较WT小鼠显著增高,差异有统计学意义(P＜0.05). 结论 CaMKⅡα在成年KO小鼠各脑区的表达均显著增多,提示FMRP负性调节CaMKⅡα的表达.     

LIMK1mRNA及其蛋白在脆性X综合征小鼠大脑皮层中的表达

目的 观察LIMK1 mRNA及其蛋白在脆性X综合征(FXS)小鼠大脑皮层中的表达,探讨其在FXS发病机制中的作用.方法 选择雄性FMR1基因敲除型FVB近交系新生鼠和2、4、6周小鼠做为实验组(记为 KO0d、KO2W、KO4W和KO6W),雄性同龄野生小鼠(WT)作为对照组(记为WT0d、WT2W、WT4W和WT6W),每组每时间点9只.取小鼠单侧大脑皮层行LIMK1 mRNA实时荧光定量PCR分析,取另一侧大脑皮层行LIMK1蛋白Western blotting分析.结果 (1)KO6W组小鼠LIMK1 mRNA含量较同龄组WT小鼠及KO0d、KO2W和KO4W组小鼠明显升高,WT0d组小鼠LIMK1mRNA含量明显高于WT2W、WT4W和WT6W组小鼠,差异有统计学意义(P<0.05).(2)同龄KO、WT小鼠大脑皮层中LIMK1蛋白含量差异无统计学意义(P>0.05);KO0d组小鼠LIMK1蛋白含量明显低于KO2W、KO4W和KO6W组,WT0d组小鼠大脑皮层中LIMK1蛋白含量明显低于WT2W、WT4W和WT6W组,差异有统计学意义(P<0.05).结论 LIMK1蛋白的翻译过程在6周时受到明显抑制,反馈性调节转录过程使LIMK1 mRNA表达急剧升高.KO鼠LIMK1蛋白表达下降,从而影响树突棘的骨架蛋白重构,影响树突棘的功能改变,可能是FXS神经系统改变的重要机制之一.

Abstract：

Objective To observe the mRNA and protein expressions of LIMK1 in the cerebral cortex of mice with FMR1 gene knockout, and explore the roles of LIMK1 mRNA and LIMK1 protein inthe pathogenic mechanism of fragile X syndrome (FXS). Methods FVB strain male mice with FMR1 gene knockout (KO, n=36, experimental group) and their wild type (WT, n=36, control group) were equally divided into 8 counterpart subgroups (WT0d, WT2W, WT4W, WT6W, KO0d, KO2W, KO4W and KO6W),respectively, according to different ages. LIMK1 mRNA expression in the left sides of the cerebral cortex were analyzed with RT-qPCR and protein expression of LIMK1 in another side with Western blotting.Results No significant differences of LIMK1 mRNA expression in the cerebral cortex of KO0d, KO2Wand KO4W subgroups were noted as compared with that of respective age-matched WT mice (P>0.05);but that of KO6W subgroup was significantly increased as compared with that of WT6W subgroup, and KO0d,KO2W and KO4W subgroups (P<0.05);the level of LIMK1 mRNA in WT0d subgroup was obviously higher than that of WT2W, WT4W and WT6W subgroups (P<0.05). No statistic differences of LIMK1 protein between the same-age KO and WT mice were noted (P>0.05);significantly lower level of LIMK1 protein in KO0d subgroup was found as compared with that of KO2W, KO4W and KO6W subgroups (P<0.05);that of WT0d subgroup was lower than that of WT2W, WT4W and WT6W subgroups (P<0.05). Conclusion The translation process of LIMK1 protein is significantly inhibited at 6 weeks and LIMK1 mRNA expression increased sharply based on the feedback adjustment of LIMK1 protein expression declining;once this translation process is inhibited or interrupted, it will affect the dendritic spines skeleton protein reconstruct and lead to the dendritic spines function deficient;the inhabitation of translation process might probably play an important role in the process of dendritic spines maturation and should be an important pathogenic mechanism of FXS.     

FMR1基因敲除小鼠脑组织微白蛋白表达的改变及其意义

目的 探讨微白蛋白(PV)阳性中间神经元在脆性X综合征(FXS)癫痫易感性增加中的作用. 方法 应用免疫组织化学染色检测FVB近交系雄性2、4、6 W龄FMR1基因敲除型(KO)(KO2W、KO4W、KO6W)和同龄野生型(WT)(WT2W、WT4W、WT6W)小鼠大脑纹状皮质、颞听皮质、梨状皮质及海马CA1区、CA3区、齿状回中PV的表达(n=6);应用Western blot法检测上述小鼠大脑皮层、海马组织PV的含量(n=6). 结果 KO2W、KO44W小鼠的大脑纹状皮质、颞听皮质、梨状皮质、海马CA1和CA3区PV阳性中间神经元的数量分别较WT2W、WT4-小鼠减少,差异有统计学意义(P<0.05);KO2W和KO4W小鼠大脑皮层、海马中PV含量分别较WT2W、WT4W小鼠减少,差异有统计学意义(P<0.05). 结论 PV阳性中间神经元及PV含量的减少.可能是引起FXS模型鼠癫痫易感性增加的主要原因.     

FMR1基因敲除鼠感觉皮质功能柱树突棘修剪发育的研究

目的研究脆性X智力低下蛋白（FMRP）缺失对感觉皮质功能柱神经可塑性的影响。方法从纯合子鼠血液提取DNA，对FMR1基因片段进行扩增，电泳观察结果。并选用子代幼龄FMR1基因敲除及野生型小鼠共16只，按基因型和年龄分为1周龄FMR1基因敲除型组（KO^1），1周龄野生型组（WT^1），4周龄FMR1基因敲除型组（KO^4），4周龄野生型组（WT^4），每组各4只。采用快速Golgi染色法分别观察不同发育时间段感觉皮质功能柱区域神经元的功能柱中心和周边两个方向上树突分支、树突棘密度和长度。结果与对照组相比，幼龄FMR1基因敲除鼠树突棘密度显著增高（P〈0．05）而长度无变化，感觉皮质功能柱区域中心朝向的树突棘密度无显著性差异，而周边朝向的树突棘密度KO^1、KO^4分别高于WT^1、WT^4，并且主要表现在距胞体10～80μm段的密度异常（P〈0．01）。结论FMRP缺失导致树突棘密度增高和感觉皮质功能柱区域树突棘修剪异常，并且树突棘修剪和树突修剪的机制不同。     

钙结合蛋白Calbindin在FMR1基因敲除小鼠脑组织中的表达

目的探讨脆性X综合征中钙结合蛋白Calbindin在神经元树突棘形态异常中的作用。方法我们选用FVB品系的FMR1基因敲除型(KO)和野生型(WT)小鼠,分别取新生1、3、5、7、10、14d,及成年(6周)的KO小鼠以及WT小鼠用免疫组织化学方法检测钙结合蛋白Calbindin在脑组织中的分布和表达情况,分别对大脑纹状皮质、海马、颞叶听区、梨状皮质、丘脑及小脑的免疫阳性显色细胞进行检测。结果在新生1d龄WT型及KO型小鼠中Calbindin免疫阳性细胞首先出现于梨状皮质和小脑皮质中,随着天龄的增长脑内其他各区逐渐出现Calbindin免疫阳性细胞的表达,且≤10dKO型小鼠Calbindin免疫阳性细胞平均光密度均显著高于WT型小鼠(P<0.05)。结论FMRP通过负性调节脑内钙结合蛋白Calbindin的表达,这推测与FMR1基因敲除小鼠神经元树突和树突棘形态异常有关。     

GSK3抑制剂氯化锂对脆性X综合征模型小鼠避暗行为的干预作用

目的探讨糖原合成酶激酶3(glycogen synthase kinase3,GSK3)抑制剂氯化锂对脆性X综合症小鼠模型的避暗行为的干预作用及机制。方法通过对30日龄脆性X综合症小鼠连续腹腔注射不同剂量氯化锂5 d,用药第4天和第5天进行避暗实验;同时用免疫印迹技术检测Fmr1 knockout(KO)及wild type(WT)小鼠的海马和皮层总GSK 3β和磷酸化GSK 3β(P-GSK3β)的变化。结果在避暗实验中,KO鼠与WT鼠,两者潜伏期及错误次数分别为(56±32)s,(83±24)s;(7±3)次,(3±2)次;免疫印迹实验结果:KO鼠皮层及海马P-GSK3β表达平均灰度值分别为69,63;WT鼠皮层和海马均为100。注射氯化锂后,KO鼠和WT鼠总GSK3β无明显改变,而KO鼠60 mg/kg,120 mg/kg,200 mg/kg组皮层P-GSK3β表达平均灰度值分别为:147,151,234;海马P-GSK3β分别为108,111,146,较空白组增多;P<0.05。WT鼠用氯化锂后,潜伏期和错误次数以及P-GSK3β表达变化无统计学意义。结论氯化锂能改善KO鼠的学习记忆能力,可能与氯化锂导致的P-GSK3β的表达增加有关,对脆性X综合征基因敲除小鼠有治疗作用。     

Spatiotemporal expression of PSD‐95 in Fmr1 knockout mice brain

To investigate and compare the spatial and temporal expression of post‐synaptic density‐95 (PSD‐95) in Fmr1 knockout mice (the animal model of fragile X syndrome, FXS) and wild‐type mice brain, on postnatal day 7 (P7), P14, P21, P28 and P90, mice from each group were decapitated, and three principal brain regions (cerebral cortex, hippocampus and cerebellum) were obtained and stored for later experiments. PSD‐95 mRNA in the three brain areas was analyzed with quantitative RT‐PCR. PSD‐95 protein was measured by immunohistochemical staining and Western blot. In the three principal brain areas of Fmr1 knockout mice and wild‐type mice, the expression of PSD‐95 mRNA and protein were detected at the lowest levels on P7, and then significantly increased on P14, reaching the peak levels in adolescents or adults. Moreover, it was found that PSD‐95 mRNA and protein in the hippocampus were significantly decreased in Fmr1 knockout mice during the developmental period (P7, P14, P21 and P28) as well as at adulthood (P90) (P < 0.05, and P < 0.01, respectively). However, there was no significant difference of expression of PSD‐95 in the cortex and cerebellum between Fmr1 knockout and wild mice. The expression of PSD‐95 in the hippocampus might be regulated by fragile X mental retardation protein (FMRP) during mice early developmental and adult periods. It is suggested that impairment of PSD‐95 is possibly involved in hippocampal‐dependent learning defects, which are common in people with FXS.     

Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines

Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of functional fragile X mental retardation protein (FMRP). FXS is associated with elevated density and length of dendritic spines, as well as an immature-appearing distribution profile of spine morphologies in the neocortex. Mice that lack FMRP (Fmr1 knockout mice) exhibit a similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a morphology profile that, while essentially opposite of that described in the Fmr1 knockout neocortex, appears similarly immature. This finding strongly suggests that FMRP is required for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.     

Neuropeptide Release Is Impaired in a Mouse Model of Fragile X Mental Retardation Syndrome

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FMRP Sustains Presynaptic Function via Control of Activity-Dependent Bulk Endocytosis

Synaptic vesicle (SV) recycling is essential for the maintenance of neurotransmission, with a number of neurodevelopmental disorders linked to defects in this process. Fragile X syndrome (FXS) results from a loss of fragile X mental retardation protein (FMRP) encoded by the FMR1 gene. Hyperexcitability of neuronal circuits is a key feature of FXS, therefore we investigated whether SV recycling was affected by the absence of FMRP during increased neuronal activity. We revealed that primary neuronal cultures from male Fmr1 knock-out (KO) rats display a specific defect in activity-dependent bulk endocytosis (ADBE). ADBE is dominant during intense neuronal activity, and this defect resulted in an inability of Fmr1 KO neurons to sustain SV recycling during trains of high-frequency stimulation. Using a molecular replacement strategy, we also revealed that a human FMRP mutant that cannot bind BK channels failed to correct ADBE dysfunction in KO neurons, however this dysfunction was corrected by BK channel agonists. Therefore, FMRP performs a key role in sustaining neurotransmitter release via selective control of ADBE, suggesting intervention via this endocytosis mode may correct the hyperexcitability observed in FXS.SIGNIFICANCE STATEMENT Loss of fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), however whether its loss has a direct role in neurotransmitter release remains a matter of debate. We demonstrate that neurons lacking FMRP display a specific defect in a mechanism that sustains neurotransmitter release during intense neuronal firing, called activity-dependent bulk endocytosis (ADBE). This discovery provides key insights into mechanisms of brain communication that occur because of loss of FMRP function. Importantly it also reveals ADBE as a potential therapeutic target to correct the circuit hyperexcitability observed in FXS.     

氯化锂可恢复脆性X综合征小鼠记忆行为和糖原合成酶激酶3β的活性

In the present study,Fmr1 knockout mice (KO mice) were used as the model for fragile X syndrome.The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts,indicating a learning and memory disorder.After treatment with 30,60,90,120,or 200 mg/kg lithium chloride,the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated,in particular,the 200 mg/kg lithium chloride treatment had the most significant effect.Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta,an inactive form of glycogen synthase kinase 3 beta,in the cerebral cortex and hippocampus of the Fmr1 KO mice.These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice,possibly by inhibiting glycogen synthase kinase 3 beta activity.     

Corticosterone response to acute stress in a mouse model of Fragile X syndrome

Fragile X syndrome (FXS), the most common form of inherited mental retardation, results from the silencing of the Fmr1 gene that encodes the Fragile X mental retardation protein (FMRP). Because (1) mRNA for the glucocorticoid receptor is bound by FMRP and (2) the response to acute stress is elevated in children with FXS, we examined whether this heightened response is characteristic of a mouse model of FXS. Fmr1 knockout (KO) and wildtype (WT) control mice were exposed to 30 min of acute restraint; serum corticosterone levels were assayed from unstressed animals and those examined either immediately following stress or after a 15 or 60 min recovery period. Under unstressed conditions, KOs and WTs did not differ in serum corticosterone, although both genotype and sex affected corticosterone levels observed following exposure to acute stress. Similar to FXS patients, serum glucocorticoid levels of KO mice exhibited a protracted return to baseline following acute stress. This suggests that the stress response is misregulated in Fmr1 KO mice as in FXS patients and provides the first evidence for a link between a particular FMRP-binding mRNA and a functional phenotype of FXS (impaired glucocorticoid negative feedback).