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，对于那些需要治疗的人来说，严重缺乏有效的治疗方法。开发新疗法的一个主要问题是我们对其潜在生物学的理解不足。虽然很明显有很强的遗传成分，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。这些风险基因中的许多都涉及突触的功能或形成，这表明突触（大脑中神经元之间的交流部位）可能是自闭症生物学的关键部分。然而，更多的自闭症风险基因只是调节其他基因的表达。这些基因的下游发生了什么？它们是否也会影响突触的功能或形成？在这项提议中，我们的目标是确定将这些“调节基因”中的一个基因突变与突触功能变化联系起来的分子和途径，该突触功能位于大脑的一个部位，即前额叶皮层，众所周知，它会影响自闭症。我们将专注于一个名为Chd 8的特定基因，它是自闭症最高置信风险基因之一，我们已经在前额叶皮层中发现了一个强大的突触表型。特别是，越来越多的证据表明，缺失Chd 8（和其他自闭症风险基因）的影响主要取决于受影响的细胞类型。我们将使用转基因技术从兴奋性神经元或不同类型的抑制性神经元中选择性地删除Chd 8的拷贝，并确切地了解突触功能和/或形成是如何受到影响的。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细胞类型缺失Chd 8的拷贝时，哪些基因表现出增加或减少的表达。虽然这种方法通常会导致许多不同的基因发生变化，但我们将重点关注那些我们已经知道参与突触功能/形成的基因。我们将对潜在的靶点进行广泛的验证，包括测试是否简单地增加或减少培养皿中皮层神经元中候选物的表达会导致突触表型的预期改变。最后，我们将利用CRISPR/Cas9技术的最新进展，（所谓的基因“剪刀”，使研究人员能够编辑DNA），使候选基因的表达水平恢复正常的生活大脑，看看这是否可以挽救突触表型。这将使我们能够找出分子和途径，连接原始基因突变的细胞和突触功能的变化，从而有助于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，可能为开发新的治疗方法开辟新的途径。

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.