VAMP2 associated SNAREopathies: from mechanism to therapeutic approaches

近年来，人们发现了许多神经发育疾病背后的基因突变，这些疾病虽然个别罕见，但总体上给个人、他们的家庭和社会带来了沉重的负担。这些突变是如何导致学习障碍、自闭症、言语问题和癫痫的逐渐出现，促使人们寻找新的治疗方法来缓解这些症状。然而，有一组重要的疾病在微观层面上的突变后果仍然知之甚少：这些疾病是由突变引起的，这些突变影响神经元向其他神经元或肌肉细胞发出信号的方式。这种信号发生在突触处，是所有大脑功能的基础，也是控制运动和许多其他器官功能的基础。突触交流的核心是精确地释放化学信使(神经递质)。我们现在对所谓的SNARE蛋白如何组装，将这些信息包，即突触小泡，带到膜上，然后在电信号到达时将神经递质释放到突触裂隙有了非常详细的了解。我们将SNARE缺陷引起的神经发育障碍统称为SNAREopathies。我们建议的目的是了解这些缺陷是如何改变神经递质从突触小泡释放的以及对大脑电路功能的影响，并调查这些影响是否可以通过利用强大的新的基因治疗工具来逆转。对许多SNAREopathies来说，一个特别困难的挑战是，它们被怀疑以遗传主导的方式起作用。也就是说，如果编码SNARE蛋白的两个拷贝中只有一个携带突变，它可能会抑制整个SNARE蛋白复合体的功能，包括正常的、未受影响的基因拷贝。我们选择专门关注一种编码基本SNARE蛋白的基因突变，该基因被称为囊泡相关膜蛋白2，或VAMP2。我们的提案汇集了互补的专业知识。我们能够通过剥离神经递质囊泡运输到VAMP2与其伙伴组装的基本步骤以及在信号到达时发生的详细重排来表征SNARE蛋白的分子机制。我们将把这种方法与对单个突触释放神经递质的精确程度以及不同VAMP2突变如何影响这一过程的高灵敏测量相结合。我们还将讨论携带人类突变的小鼠模型中小神经元回路功能的后果。最后，在定量了解每个关键分子相互作用的多少拷贝以支持正常的囊泡运输的指导下，我们将设计一种旨在挽救突变影响的治疗方法，并在小鼠模型中询问疾病的哪些方面可能适合挽救：如果在症状显现后进行治疗，我们是否只能抑制癫痫发作，或者我们还可以改善在症状出现之前的早期发展过程中可能出现的学习和其他缺陷吗？拟议中的研究将为VAMP2突变的机制提供亟需的澄清，建立一个可行的治疗方案，在适当的时候可以转化到临床上。并定义了一条可以应用于其他SNAREopathy的研究管道。

Recent years have seen the discovery of genetic mutations underlying many neurodevelopmental diseases, which, although individually rare, collectively account for a substantial burden to individuals, their families and society. How these mutations result in learning disability, autism, speech problems, and epilepsy is gradually emerging, prompting the search for new treatments to mitigate the symptoms. However, there is an important group of disorders for which the consequences of the mutations at the microscopic level remain very poorly understood: these disorders are caused by mutations that affect how neurons signal to other neurons or muscle cells. Such signalling takes place at synapses and is fundamental for all brain functions, as well as the control of movements and the function of many other organs. At the core of synaptic communication is the precise discharge of packets of chemical messengers (neurotransmitters). We now have a highly detailed understanding of how the so-called SNARE proteins assemble to bring these packets, known as synaptic vesicles, to the membrane and then discharge the neurotransmitter into the synaptic cleft upon arrival of an electrical signal. We refer to the neurodevelopmental disorders caused by defects of SNAREs and functionally associated proteins collectively as 'SNAREopathies'.The goal of our proposal is to understand how these defects alter the discharge of neurotransmitters from synaptic vesicles and the consequences for brain circuit function and also investigate whether these effects could potentially be reversed by harnessing powerful new tools for genetic therapy.An especially difficult challenge for many SNAREopathies is that they are suspected of acting in a genetically dominant manner. That is, if only one of the two copies of the gene encoding a SNARE protein carries a mutation, it may suppress the function of the entire SNARE protein complex, including the normal, unaffected copy of the gene. We have chosen to focus exclusively on mutations of a gene that encodes the essential SNARE protein known as vesicle-associated membrane protein 2, or VAMP2.Our proposal brings together complementary expertise. We are able to characterise the molecular machinery of SNARE proteins by stripping neurotransmitter vesicle trafficking down to the essential steps of assembly of VAMP2 with its partners and the detailed rearrangements that occur upon the arrival of a signal to discharge the vesicle contents. We will combine this approach with highly sensitive measurements of how precisely neurotransmitter is released at individual synapses and how this process is affected by different VAMP2 mutations. We will also address the consequences for the function of small neuronal circuits in a mouse model carrying a human mutation. Finally, guided by a quantitative understanding of how many copies of each of the key molecules interact to support normal vesicle trafficking, we will design a therapy aimed at rescuing the effects of the mutation, and ask which aspects of the disease are potentially amenable to rescue in the mouse model: if the therapy is delivered once symptoms have manifested, can we only suppress seizures, or can we also improve learning and other defects which might have arisen during early development before symptom onset?The proposed research will provide much-needed clarity on the mechanisms of VAMP2 mutations, build towards a viable treatment option which can be translated to the clinic in due course, and define a research pipeline that can be applied to other SNAREopathies.