L-Aspartate signalling in the brain

神经元通过释放兴奋性和抑制性神经递质在突触上进行交流。氨基酸l -谷氨酸长期以来被认为是大脑中普遍存在的兴奋性神经递质。一种非常相似的氨基酸，l -天冬氨酸，也存在于一些神经元的突触中。尽管l -天冬氨酸只能激活l -谷氨酸作用的受体之一，但有证据表明它在大脑中起着信号作用。尽管神经元根据其释放的神经递质被分为兴奋性或抑制性，但l -天冬氨酸（兴奋性）存在于抑制性中间神经元的突触中，这表明一些抑制性中间神经元可以释放l -天冬氨酸来产生额外的兴奋作用。我们发明的l -天冬氨酸生物传感器使我们能够证明l -天冬氨酸在海马突触通路刺激下的调节释放。在探索控制细胞外l -天冬氨酸的机制时，我们阻断了将l -天冬氨酸转化为l -天冬氨酸的细胞内酶——天冬氨酸合成酶（ASNS）。我们发现这增强了l -天冬氨酸生物传感器信号，表明ASNS虽然是细胞内的，但也是调节细胞外l -天冬氨酸水平的途径的一部分。然而，我们也发现了一些完全出乎意料的事情：在ASNS阻断期间，脑组织开始产生类似癫痫发作的电活动，让人想起癫痫期间发生的异常活动。这一观察结果非常重要：影响ASNS酶活性的人类突变可导致小头畸形、认知障碍和癫痫。我们还发现，在人类癫痫患者脑组织和慢性癫痫小鼠模型中，ASNS表达上调。我们认为癫痫大脑中ASNS的上调是一种防御机制，可以减少l -天冬氨酸信号传导和癫痫发作活动的发生率。为了进一步推进我们的研究，我们将确定释放l -天冬氨酸的神经元。我们假设这些是抑制性中间神经元，并将使用现代遗传方法结合l -天冬氨酸生物传感来明确鉴定这些神经元，并探索突触释放l -天冬氨酸所需的关键分子成分。然后我们将确定l -天冬氨酸的神经靶点——它作用于的细胞以及它对这些细胞的作用。我们的最终目标有三个部分。首先，我们将阐明哪些细胞表达ASNS，以及当慢性癫痫期间ASNS上调时，是否有其他细胞类型表达这种酶。其次，我们将测试ASNS的上调是否具有保护性，减少癫痫发作活动。如果是这样的话，我们预计癫痫病患者的大脑（其中ASNS被上调）将比对照组的大脑对ASNS的抑制更为敏感。第三，我们将从基因上删除部分海马体中ASNS的表达，并测试这是否会使大脑更容易产生癫痫发作。最后，我们将把我们的研究扩展到人类的状况。通过使用癫痫患者的脑组织（切除以帮助控制癫痫），我们将测试ASNS在调节癫痫发作活动中的重要性，并检查ASNS和我们在l -天冬氨酸信号通路中识别的其他关键分子成分的表达水平。在英国大约有60万人被诊断患有癫痫。在这些人中，有30%的癫痫发作不受现有药物的控制。我们在基础层面上理解大脑中l -天冬氨酸信号传导的工作，为寻找基于l -天冬氨酸信号传导分子成分的癫痫治疗开辟了一个新的领域。l -天冬氨酸信号失调在偏头痛、中风和创伤性脑损伤等其他神经系统疾病中也可能很重要。

Neurons communicate at synapses via the release of excitatory and inhibitory neurotransmitters. The amino acid L-glutamate has long been accepted as the brain's universal excitatory neurotransmitter. A very similar amino acid, L-aspartate, is also present at the synapses of some neurons. Despite L-aspartate being able to activate just one of the suite of receptors that L-glutamate acts on, there is some evidence that it plays a signalling role in the brain. Although neurons are classified as either excitatory or inhibitory depending on which neurotransmitter they release, L-aspartate (excitatory) is found in the synapses of inhibitory interneurons, suggesting that some inhibitory interneurons could release L-aspartate to produce additional excitatory actions. Our invention of a biosensor for L-aspartate has enabled us to demonstrate the regulated release of L-aspartate in response to stimulation of synaptic pathways in the hippocampus. In exploring the mechanisms that control extracellular L-aspartate, we blocked an intracellular enzyme, asparagine synthetase (ASNS) that converts L-aspartate to L-asparagine. We found that this enhanced the L-aspartate biosensor signals indicating that ASNS, although intracellular, is part of a pathway that regulates extracellular levels of L-aspartate. However, we also found something completely unexpected: during blockade of ASNS the brain tissue started to produce electrical seizure-like activity, reminiscent of the aberrant activity that occurs during epilepsy. This observation is highly significant: human mutations of ASNS that affect its enzymatic activity lead to microcephaly, cognitive impairment, and epilepsy. We have additionally found that ASNS expression is upregulated in brain tissue of human epilepsy patients and in mouse models of chronic epilepsy. We propose that upregulation of ASNS in the epileptic brain is a defensive mechanism that reduces L-aspartate signalling and the incidence of seizure activity.To advance further our studies, we will identify the neurons that release L-aspartate. We hypothesize that these are the inhibitory interneurons and shall use modern genetic methods combined with L-aspartate biosensing to definitively identify these neurons and explore the key molecular components required for synaptic release of L-aspartate. We shall then determine the neural targets of L-aspartate -the cells it acts on and the actions it has on them. Our final aim has three parts. Firstly, we shall elucidate which cells express ASNS and, when ASNS is upregulated during chronic epilepsy, whether additional cell types express this enzyme. Secondly, we shall test whether upregulation of ASNS is protective, reducing seizure activity. If this is the case, we expect that the epileptic brain (where ASNS is upregulated) will be even more sensitive to inhibition of ASNS than the control brain. Thirdly, we shall genetically delete the expression of ASNS in part of the hippocampus and test whether this makes the brain more likely to generate seizures. Finally, we shall extend our studies to the human condition. By using brain tissue from epileptic patients (removed to help control their epilepsy), we shall test the importance of ASNS in regulating seizure activity and examine the expression levels of ASNS and other key molecular components that we identify in the L-aspartate signalling pathway.There are ~600,000 people in the UK living with a diagnosis of epilepsy. In 30% of these people, the seizures are not controlled by current medication. Our work to understand, at a fundamental level, L-aspartate signalling in the brain opens a new area to search for additional treatments for epilepsy based around the molecular components of L-aspartate signalling. Dysregulation of L-aspartate signalling is also likely to be important in other neurological contexts such as migraine, stroke and traumatic brain injury.