The molecular mechanism and behavioral abnormality in autism models

树突丝状伪足在突触快速发生期间最为丰富，但此后丝状伪足数量减少。当丝状足接触突触前部位并形成突触时，丝状足转化为树突棘。正常的脊柱形成可能是学习和记忆功能的基础，而异常的脊柱形成可能与智力迟钝和/或自闭症的发病机制有关。结节性硬化症复合体(TSC)-2突变导致遗传性结节性硬化症自闭症。TSC2对小GTP酶rheb具有GAP活性，TSC2通过刺激GTP水解rheb而拮抗mTOR通路。我们分析了Eker大鼠的杂合tsc2突变，以检查TSC通路在突触发生中的参与。TSC2突变引起培养海马神经元脊柱形成的抑制。在用gtp结合形式的rheb表达的野生型神经元中也观察到类似的脊柱形成异常。因此，rheb的过度激活可能引起树突棘发生的紊乱。接下来，我们用mTOR抑制剂雷帕霉素减弱了rheb-mTOR通路的过度活性。雷帕霉素抑制mTOR下游p70 S6激酶的磷酸化，但不增加tsc2破坏神经元的脊柱形成，表明mTOR途径可能不参与脊柱形成。为了确定rheb的另一个下游靶点，我们进行了酵母双杂交筛选，发现了rheb结合蛋白（RBP）。RBP下调可诱导tsc2突变神经元的脊柱形成。相反，RBP的过表达会阻止野生型神经元向棘的成熟。此外，显性阴性rheb的表达减少了RBP的数量，并增加了tsc2突变神经元的树突脊髓发生。这些结果表明，TSC2可能通过控制神经元RBP的数量以一种依赖于rheg的方式调节树突棘的形成。

Dendritic filopodia are most abundant during periods of rapid synaptogenesis, but the number of filopodia declines thereafter. When filopodia contact presynaptic sites and form synapses, filopodia convert into dendritic spines. Normal spine formation may underlie learning and memory function, and abnormal spine formation may be associated with the pathogenesis of mental retardation and/or autism.Mutation of tuberous sclerosis complex (TSC)-2 causes a hereditary autistic disorder in tuberous sclerosis. TSC2 has GAP activity towards small GTPase rheb, and TSC2 antagonizes mTOR pathway by stimulation of GTP hydrolysis of rheb. We analyzed Eker rats heterozygous for a mutation in the tsc2 to examine an involvement of TSC pathway in synaptogenesis. TSC2 mutation caused an inhibition of spine formation in cultured hippocampal neurons. A similar abnormality in spine formation was also observed in wild-type neuron expressed with a GTP-bound form of rheb. Thus, the excessive activation of rheb may cause the disturbance of the dendritic spinogenesis. We next attenuated such excessive activity of rheb-mTOR pathway with an mTOR inhibitor rapamycin. Rapamycin suppressed a phosphorylation of mTOR-downstream p70 S6 kinase, but did not increase the spine formation in tsc2-disrupted neurons, indicating that mTOR pathway may not be involved in spinogenesis. To identify another downstream target of rheb, we performed yeast two-hybrid screening and found a rheb-binding protein (RBP). Knockdown of RBP induced the spine formation in tsc2-mutated neurons. Conversely, overexpression of RBP abolished the maturation to spines in wild-type neurons. Furthermore, the expression of dominant-negative rheb reduced the amount of RBP, and increased dendritic spinogenesis in tsc2-mutated neurons. These results suggest that TSC2 may regulate the dendritic spine formation by controling neuronal RBP amount in a rheb-dependent manner.