Linking genotype to phenotype in autism: mechanisms of cell-type specific presynaptic dysfunction in Chd8 haploinsufficiency

自闭症是一种神经发育障碍，对于需要治疗的人来说，严重缺乏有效的治疗方法。开发新的治疗方法的一个主要问题是我们对其潜在的生物学知识缺乏了解。虽然显然有很强的遗传成分，但我们现在知道，有数百种不同的基因突变会增加患自闭症的风险。这些危险基因中的许多都与突触的功能或形成有关，这表明突触(大脑中神经元之间的交流地点)可能是自闭症生物学的关键部分。然而，一组更大的自闭症风险基因只是调节其他基因的表达。这些基因的下游会发生什么？它们是否也会影响突触的功能或形成？在这项研究中，我们的目标是确定在大脑中众所周知受自闭症影响的一部分--前额叶皮质--其中一个‘调节基因’的基因突变与突触功能变化之间的联系的分子和途径。我们将专注于一种名为CHD8的特定基因，它是自闭症的最高可信风险基因之一，我们已经在前额叶皮质发现了一种强大的突触表型。特别是，越来越多的证据表明，CHD8(和其他自闭症风险基因)副本缺失的影响严重取决于受影响的细胞类型。我们将使用转基因技术选择性地从兴奋性神经元或不同类型的抑制性神经元中删除CHD8的副本，并确切地找出突触功能和/或形成受到影响的原因。然后，我们将收集每种类型的单个细胞-兴奋性和抑制性-并使用单核测序来发现当该类型的细胞缺少CHD8副本时，哪些基因的表达增加或减少。虽然这种方法通常会导致许多不同的基因出现变化，但我们将重点关注那些我们已经知道的与突触功能/形成有关的基因。我们将对潜在的靶点进行广泛的验证，包括测试DISH中皮质神经元中候选基因表达的简单增加或减少是否会导致预期的突触表型变化。最后，我们将利用CRISPR/Cas9技术(所谓的基因剪刀，允许研究人员编辑DNA)的最新进展，将候选基因的表达水平恢复到活着的大脑中的正常水平，并看看这是否可以挽救突触表型。这将使我们能够找出将原始基因突变与细胞和突触功能变化联系起来的分子和途径，从而帮助填补我们对这种疾病理解的一个主要空白。这些分子和途径可能为治疗干预提供新的靶点，可能在努力开发新的治疗方法方面开辟新的途径。

Autism is a neurodevelopmental disorder for which there is a severe lack of effective treatments for those who need them. A major problem in the development of new treatments is our poor understanding of its underlying biology. While there is clearly a strong genetic component, we now know that there are hundreds of different gene mutations that increase the risk of developing autism. Many of these risk genes are involved in synapse function or formation, suggesting that synapses (the site of communication between neurons in the brain) may be a key part of autism biology. However, an even larger group of autism risk genes simply regulate the expression of other genes. What happens downstream of these genes? Might they also affect synaptic function or formation? In this proposal, we aim to identify the molecules and pathways that link gene mutation in one of these 'regulator genes' to changes in synapse function, in a part of the brain that is well known to be affected in autism, the prefrontal cortex. We will focus on a specific gene called Chd8, which is one of the highest confidence risk genes for autism, and where we have already identified a robust synaptic phenotype in prefrontal cortex. In particular, there is increasing evidence that the effects of missing a copy of Chd8 (and other autism risk genes) depends critically on which type of cell is affected. We will use transgenic technology to selectively delete a copy of Chd8 from excitatory neurons or different types of inhibitory neurons and find out exactly how synapse function and/or formation is affected. We will then collect individual cells of each type - excitatory and inhibitory - and use single nuclei sequencing to discover which genes show either increased or decreased expression when that cell type is missing a copy of Chd8. While this kind of approach usually results in many different genes that show changes, we will focus on those that we already know are involved in synaptic function / formation. We will carry out extensive validation of potential targets, including testing whether simply increasing or decreasing expression of the candidate in cortical neurons in a dish leads to the expected alteration to synaptic phenotype. Finally, we will take advantage of recent advances in CRISPR/Cas9 technology (the so-called genetic 'scissors' that allow researchers to edit DNA) to bring expression levels of candidate genes back to normal in the living brain, and see whether this can rescue the synaptic phenotypes.This will allow us to work out the molecules and pathways that link the original genetic mutation to changes in cell and synapse function, thereby helping to fill a major gap in our understanding of this disorder. These molecules and pathways may provide novel targets for therapeutic intervention, potentially opening up new avenues in the effort to develop new treatments.