Defining the Molecular Origins of Developmental Brain Disorders

项目总结/摘要 只有一小部分自闭症谱系障碍似乎有明确的遗传病因（约25%）， 导致大多数案件的起因不明。环境敏感表观遗传标记的改变（例如， DNA甲基化）与包括自闭症在内的精神疾病有关。一种新的表观遗传标记，5- 羟甲基胞嘧啶（5 hmC），在神经元中富集，并与神经元的活性转录有关。 基因.虽然许多研究已经描述了5 hmC，并发现了与神经系统疾病的联系，但很少有研究表明， 一直致力于确定5 hmC的功能作用，因为它涉及基因表达。因此，全 5 hmC修饰的功能潜力尚未在与行为相关的结果中实现。最近的一 自闭症小鼠模型中纹状体5 hmC的全基因组分布（即，Cntnap 2-/-纯合子），其表现出 核心自闭症特征，包括纹状体GABA能信号传导的缺陷，揭示了基因组范围内的破坏， 5 hmC在与人类自闭症有关的大量基因的直系同源物中（N=68/233）。因此这些 数据表明5 hmC介导的表观遗传调节在自闭症发病机制中的作用， 这是理解纯合突变的全基因组分子后果的关键一步， 导致自闭症样表型。值得注意的是，Cntnap 2 +/-杂合突变小鼠缺乏自闭症样特征。 由于5 hmC水平对环境敏感，这些发现导致了Cntnap 2 +/-的假设。 受到早期环境应激的杂合子小鼠会有5 hmC的破坏， 表现出类似于Cntnap 2-/-纯合突变体的自闭症样行为。为了验证这一假设， 在胚胎第12天，使Cntnap 2 +/-杂合小鼠经受7天的产前应激， 随后检查成人行为。与野生型同窝仔不同，产前应激的雌性Cntnap 2 +/- 杂合子小鼠表现出社会缺陷。此外，基因组分析显示5 hmC 在整个纹状体基因组中，这与在Cntnap 2-/-纯合子小鼠中观察到的相似。最后， 上述数据与纹状体基因表达数据的整合显示，差异羟甲基化 与基因转录水平的改变相关，这些基因被证明有助于GABA能信号传导的缺陷， Cntnap 2-/-纯合突变小鼠的社会行为（例如，GABA受体和催产素）。因此这些 数据表明，基因与环境的相互作用导致社会相互作用的缺陷，并表明， 5 hmC水平可能有助于这一结果。在这里，拟议的研究将：1）确定功能 5 hmC在产前压力导致的社会缺陷中的作用; 2）确定 催产素在产前应激后社会缺陷中的作用这些发现将确定与压力有关的 受环境敏感的表观遗传机制影响的大脑中的分子靶点， 与大脑发育障碍的病因学有关。重要的是，该计划建立在申请人的 在基因组学研究的培训，结合基因组学将在分子和行为的方法深入培训。

PROJECT SUMMARY / ABSTRACT Only a small percentage of the autism spectrum disorders appear to have a clear genetic etiology (~25%), leaving a majority of cases with unclear origins. Alterations in environmentally sensitive epigenetic marks (e.g., DNA methylation) have been implicated in mental illness, including autism. A novel epigenetic mark, 5- hydroxymethylcytosine (5hmC), is enriched in neurons and is associated with active transcription of neuronal genes. While numerous studies have profiled 5hmC and found links to neurological disorders, less effort has been invested in determining the functional role of 5hmC, as it pertains to gene expression. As a result, the full functional potential of 5hmC modifications has yet to be realized in outcomes related to behavior. A recent genome-wide profile of striatal 5hmC in an autism mouse model (i.e., a Cntnap2-/- homozygote) that exhibits core autism features, including deficits in striatal GABAergic signaling, revealed a genome-wide disruption of 5hmC in the orthologs of a remarkable number of genes implicated in human autism (N=68/233). Thus, these data suggest a role for 5hmC-mediated epigenetic modulation in the pathogenesis of autism and represent a critical step toward understanding the genome-wide molecular consequences of a homozygous mutation that results in an autism-like phenotype. Notably, Cntnap2+/- heterozygous mutant mice lack autistic-like features. Since 5hmC levels are environmentally sensitive, these findings led to the hypothesis that Cntnap2+/- heterozygous mice subjected to an early environmental stress would have disruptions in 5hmC and may exhibit autistic-like behaviors similar to the Cntnap2-/- homozygous mutant. To test this hypothesis, beginning at embryonic day 12, Cntnap2+/- heterozygous mice were subjected to seven days of prenatal stress and subsequently examined for adult behaviors. Unlike wild-type littermates, prenatally stressed female Cntnap2+/- heterozygous mice exhibited social deficits. In addition, genomic profiling revealed disruptions in 5hmC throughout the striatal genome, which were similar to those seen in Cntnap2-/- homozygous mice. Finally, integration of the above data with striatal gene expression data revealed that differential hydroxymethylation correlated with altered transcript levels of genes shown to contribute to the deficits in GABAergic signaling and social behavior of the Cntnap2-/- homozygous mutant mouse (e.g., GABA receptors and Oxytocin). Thus, these data demonstrate that gene by environment interactions lead to deficits in social interactions and suggest that 5hmC levels likely contribute to this outcome. Here, the proposed studies will: 1) identify the functional role of 5hmC in social deficits resulting from prenatal stress; and 2) identify the contribution of oxytocin in social deficits exhibited following prenatal stress. These findings will identify stress-related molecular targets in the brain that are influenced by environmentally sensitive epigenetic mechanisms and may be involved in the etiology of developmental brain disorders. Importantly, the plan builds on the applicant's training in genomic studies, combining genomics will in-depth training in molecular and behavioral approaches.