L-Aspartate signalling in the brain

大脑中的 L-天冬氨酸信号传导

• 批准号: MR/W028964/1
• 负责人: Nicholas Dale
• 金额: $ 132.66万
• 依托单位: University of Warwick
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FW028964%2F1
• 关键词: Aspartate; signalling; brain

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.

神经元通过释放兴奋性和抑制性神经递质在突触时进行通信。长期以来，氨基酸L-谷氨酸已被接受为大脑的通用兴奋性神经递质。在某些神经元的突触中，也存在非常相似的氨基酸，L-天冬氨酸。尽管L-天冬氨酸能够仅激活L-谷氨酸作用的受体套件之一，但仍有一些证据表明它在大脑中起信号传导作用。尽管神经元被归类为兴奋性或抑制性，具体取决于它们释放的神经递质，但在抑制性神经元的突触中发现L-天冬氨酸（兴奋性），这表明某些抑制性中间神经元可能会释放L-Appartate以产生其他兴奋性作用。我们对L-Hepartate生物传感器的发明使我们能够证明对海马中突触途径的刺激响应的L-天冬氨酸的调节释放。在探索控制细胞外L-天冬氨酸的机制时，我们阻止了一种细胞内酶，天冬酰胺合成酶（ASNS），将L-天冬氨酸转化为L-天冬酰胺。我们发现，这增强了L-天冬氨酸生物传感器信号，表明ASN虽然细胞内是调节细胞外L-天冬氨酸水平的途径的一部分。但是，我们还发现了完全出乎意料的东西：在ASN的封锁期间，脑组织开始产生癫痫发作样活性，让人联想到癫痫期间发生的异常活性。这一观察结果非常重要：影响其酶活性的ASN的人类突变导致小头畸形，认知障碍和癫痫病。我们还发现，在人癫痫患者的脑组织和慢性癫痫的小鼠模型中，ASNS表达上调。我们提出，癫痫大脑中ASN的上调是一种防御机制，可降低L-天冬氨酸信号传导和癫痫发作的发生率。为了进一步进一步提高我们的研究，我们将确定释放L-Appartate的神经元。我们假设这些是抑制性中间神经元，应使用与L-天冬氨酸生物传感结合的现代遗传方法确定识别这些神经元，并探索突触释放L-Haspartate所需的关键分子成分。然后，我们将确定L-天冬氨酸的神经靶标 - 其作用的细胞及其对它们的作用。我们的最终目标有三个部分。首先，我们应阐明哪些细胞表达ASN，以及当ASN在慢性癫痫中上调时，其他细胞类型是否表达该酶。其次，我们将测试ASN的上调是否具有保护性，以减少癫痫发作活性。如果是这种情况，我们期望癫痫大脑（ASN上调的地方）将对ASN的抑制更敏感，而不是对照大脑。第三，我们将在一部分海马中遗传删除ASN的表达，并测试这是否使大脑更有可能产生癫痫发作。最后，我们将把研究扩展到人类状况。通过使用癫痫患者的脑组织（移除以帮助控制癫痫），我们将测试ASN在调节癫痫发作活性中的重要性，并检查ASN和其他关键分子成分的表达水平，在L-spartate信号通路中识别出的ASN和其他关键分子成分。在其中30％的人中，癫痫发作不受当前药物的控制。我们在基本水平上理解大脑中的L-天冬氨酸信号传导为基于L-天冬氨酸信号传导的分子成分寻找其他治疗的新区域打开了新区域。在偏头痛，中风和脑部损伤等其他神经系统环境中，L-天冬氨酸信号传导的失调也可能很重要。