Investigating chromatin misfolding as a pathogenic mechanism in neurodevelopmental disorders.

大脑是一个复杂的器官，在胚胎中首次发育时需要仔细组织。小错误，如没有使神经元（大脑的电细胞）的正常平衡，可能导致常见和严重的疾病。其中一个例子是癫痫，它影响到英国人口的1%。不幸的是，它与每年1100多人死亡有关，花费NHS约10亿英镑。更令人担忧的是，大约三分之一的癫痫患者具有耐药形式，这使得这些人面临危险的治疗，如脑部手术。为了改善对这些人的护理，我们需要新的治疗或测试来预测谁会发展为严重的形式。通过研究癫痫大脑如何发育（并将其与正常大脑进行比较），我们将能够确定新的药物和测试目标。研究已经为我们提供了癫痫大脑如何不同发育的线索。正常情况下，当我们的大脑形成时，神经元会增殖并分化--一些刺激电活动，另一些抑制电活动。电刺激和抑制神经元的复杂模式对于大脑中协调的电活动非常重要。然而，在癫痫中，人们认为可能有过多的刺激性神经元，可能是因为形成的抑制性神经元数量减少。这会造成协调大脑电活动的困难，并可能导致过度活跃，不协调的大脑活动和癫痫发作。我们开始揭示为什么抑制性神经元数量减少的机制。科学家们已经确定了一系列基因，这些基因突变后会引起耐药性癫痫特别常见的综合征。有趣的是，这些基因中的许多有助于控制DNA如何折叠和储存，这决定了制造蛋白质的基因，从而决定了细胞的指令。当DNA没有正确存储时，主要的指令--告诉神经元是电刺激还是抑制--变得非常混乱。我们相信，DNA折叠基因发生了突变，很少有神经元得到指令变成电抑制神经元。为了研究我们的想法，我们建议研究两个DNA折叠基因（称为FOXG 1和CHD 2），当突变时会引起耐药性癫痫非常常见的综合征。利用可以被引导在成人体内制造任何细胞类型的干细胞，我们将对它们进行遗传修饰，使DNA折叠基因FOXG 1和CHD 2发生突变。然后，我们将这些干细胞培养为3D集合，类似于发育中的人类大脑的部分，其中产生抑制性神经元（一种称为脑类器官的新技术）。这项技术将使我们能够尽可能准确地研究人类大脑的发育，而不需要使用动物。使用脑类器官和干细胞，我们将研究DNA如何在没有FOXG 1或CHD 2的神经元中折叠。如果我们看到异常的DNA折叠模式，我们将对另一组干细胞进行遗传修饰，试图重建异常的DNA折叠模式（但使用正常的FOXG 1和CHD 2蛋白）。如果这种新的细胞系产生了具有少量抑制性神经元的脑类器官，那么这将是非常有力的证据，表明异常DNA折叠是癫痫和神经发育障碍通常可以发展的一种方式。这项研究的发现可能具有强大的科学和医学意义。我们将提供一些急需的见解，了解具有DNA折叠功能的基因如何控制我们的大脑发育。同样，它在医学上也很重要，因为它开辟了一种可能性，即这是一种机制，可以解释各种其他疾病（与胚胎中异常的大脑发育有关）的发展。这反过来又为未来的研究提供了许多靶点，以寻找诊断测试或新治疗药物的新靶点。

The brain is a complicated organ that requires careful organisation when it first develops in embryos. Small mistakes, such as not making a normal balance of neurons (electrical cells of the brain), can lead to common and severe disorders. One example of this is epilepsy, which affects ~1% of the UK population. Unfortunately, it is linked >1100 deaths every year and costs the NHS ~£1 billion. Even more worrying is that around a third of epilepsy patients have drug resistant forms, which leaves these people with risky treatments, such as brain surgery. To improve the care of these people, we need new treatments or tests to predict who will develop severe forms. By studying how epilepsy brains develop (and comparing them to normal ones) we will be able to identify new drug and test targets.Research has already given us clues about how epilepsy brains may develop differently. Normally, when our brain forms, neurons multiply and specialise - some stimulate electrical activity, others inhibit it. The intricate patterns of electrically stimulatory and inhibitory neurons are important for co-ordinated electrical activity in the brain. In epilepsy, however, it is thought that there may be an excess number of stimulatory neurons, likely because there is a reduced number of inhibitory ones forming. This creates difficulties in co-ordinating the electrical activity of the brain and can lead to overactive, uncoordinated brain activity and seizures. We are starting to uncover the mechanisms for why there is a reduced number of inhibitory neurons. Scientists have identified a list of genes that when mutated give rise to syndromes in which drug-resistent epilepsy is particularly common. Interestingly, many of these genes help to control how DNA is folded and stored, which determines the genes that make get to make proteins and thus instructions given to the cell. The main instruction - telling the neuron whether to become electrically stimulatory or inhibitory - gets very confusing when DNA is not stored correctly. We believe that DNA-folding genes are mutated that far fewer neurons get the instruction to turn into electrically inhibitory neurons. To investigate our ideas, we propose to study two DNA-folding genes (called FOXG1 and CHD2) that when mutated give rise to syndromes in which drug-resistant epilepsy is very common. Using stem cells that can be guided to make any cell type in the adult body, we will genetically modify them so the DNA folding genes FOXG1 and CHD2 are mutated. We will then grow these stem cells as 3D collections that resemble parts of the developing human brain where inhibitory neurons are made (a new technology known as brain organoids). This technique will allow us to study human brain development as accurately as is currently possible and without the need to use animals. Using brain organoids and stem cells, we will then look at how DNA is folded in neurons where there is no FOXG1 or CHD2. If we see abnormal DNA folding patterns, we will then genetically modify another group of stem cells to try and recreate the abnormal DNA folding patterns (but with normal FOXG1 and CHD2 protein). If this new cell line creates brain organoids with low numbers of inhibitory neurons, then this will be very strong evidence that abnormal DNA folding is a way in which epilepsy and neurodevelopmental disorders generally can develop.The findings of this study could have strong scientific and medical implications. We will provide some much-needed insight into how genes with DNA folding functions control how our brains develop. Similarly, it will be important medically as it opens up the possibility that this is a mechanism for how a wide range of other conditions (associated with abnormal brain development in embryos) develop. This will in turn provide numerous targets for future research looking to find new targets for diagnostic tests or drugs for novel treatments.