Q241R mutation of Braf gene causes neurological abnormalities in a mouse model of cardio-facio-cutaneous syndrome independent of developmental malformations

Constitutively active mutants of Braf cause cardio-facio-cutaneous (CFC) syndrome, which is characterized by growth and developmental defects, cardiac malformations, characteristic facial features, cutaneous manifestations, and mental retardation. An animal model of human CFC syndrome, a systemic BrafQ241R/+ mutant mouse has been reported to exhibit multiple CFC syndrome-like phenotypes. In this study, we analyzed the effects of Braf mutation on neural functions separately from their effects on developmental processes. To this end, we generated Braf mutant mice that express BrafQ241R specifically in mature excitatory neurons (n-BrafQ241R/+). We found no growth retardation or cardiac malformations in n-BrafQ241R/+ mice, indicating normal development of these mice. Behavioral analysis revealed that the mutant mice exhibited decreased locomotor activity and exploratory behavior, enhanced auditory startle response, and impaired hippocampus-dependent learning, many of which were similar to the systemic BrafQ241R/+ mouse. In the hippocampus of n-BrafQ241R/+ mice, the long-term potentiation of synaptic transmission was enhanced, and active forms of ERK1/2 were increased. Transcriptome analysis of hippocampal tissue revealed significant changes in the expression of genes involved in synaptic function and memory learning, as well as increased expression of Dusp6 and Spred3, suppressors of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. These data suggest that the neuronal dysfunction observed in the systemic CFC mouse model is due to the disruption of homeostasis of RAS/MAPK signaling pathway by the activated Braf mutant after maturation, rather than abnormal development of the brain, and a similar mechanism may be possible in human CFC syndrome. Overall design: To investigate the effect of hippocampal function on BrafQ241R mutant expression, we generated mice expressing BrafQ241R specifically in mature excitatory neurons.

Braf的组成型激活突变体可引发心面皮肤综合征（cardio-facio-cutaneous syndrome，CFC），该综合征以生长发育缺陷、心脏畸形、特征性面部表型、皮肤损害以及智力障碍为主要特征。已有研究报道，系统性BrafQ241R/+突变小鼠作为人类CFC综合征的动物模型，可呈现多种CFC综合征样表型。本研究中，我们将Braf突变对神经功能的影响与其对发育过程的影响分离开来进行分析。为此，我们构建了仅在成熟兴奋性神经元中表达BrafQ241R的Braf突变小鼠（命名为n-BrafQ241R/+）。我们观察到n-BrafQ241R/+小鼠未出现生长迟缓或心脏畸形，表明其发育状态正常。行为学分析结果显示，该突变小鼠表现出运动活性与探索行为降低、听觉惊反射增强以及海马依赖型学习能力受损，其中多种表型与系统性BrafQ241R/+小鼠相似。在n-BrafQ241R/+小鼠的海马组织中，突触传递的长时程增强（long-term potentiation，LTP）效应增强，且活化型细胞外信号调节激酶1/2（extracellular signal-regulated kinase 1/2，ERK1/2）的水平升高。对海马组织进行转录组分析后发现，参与突触功能与记忆学习的基因表达发生显著改变，同时RAS/丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路的抑制因子Dusp6与Spred3的表达水平上调。上述结果表明，系统性CFC小鼠模型中观察到的神经元功能异常，是由成熟后活化的Braf突变体扰乱RAS/MAPK信号通路稳态所导致，而非大脑发育异常所致；这一机制或许同样适用于人类CFC综合征。总体实验设计：为探究BrafQ241R突变表达对海马功能的影响，我们构建了仅在成熟兴奋性神经元中表达BrafQ241R的小鼠。