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IMAGING GLUTAMATE IN THE BRAIN USING NOVEL FAST FLUORESCENT PROBES

使用新型快速荧光探针对大脑中的谷氨酸进行成像

基本信息

批准号：
BB/S003894/1
负责人：
Katalin Torok
金额：
$ 78.47万
依托单位：
St George's, University of London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FS003894%2F1
关键词：
IMAGING GLUTAMATE BRAIN USING NOVEL

项目摘要

Glutamate is recognised as a major excitatory neurotransmitter in the central nervous system. Dysregulation of glutamate is involved in neuropsychiatric disorders like anxiety and depression, as well as acute and chronic neurodegenerative disorders such as Huntington's disease. The development of a genetically-encoded glutamate sensor, termed iGluSnFR, which is based on the bacterial glutamate/aspartate binding protein (BP) and circular permuted enhanced green fluorescent protein (cpEGFP) represented a major breakthrough in monitoring glutamate. However, the kinetics of iGluSnFR are too slow to track high-frequency action potential (AP) firing. Our new variant iGluu with 5-fold faster kinetics was fast enough to show glutamate clearance from the synaptic cleft in between AP. In this proposal we will make use of this probe to not only visualize synaptic glutamate release but also uptake into astrocytes and compare the dynamics in health and Huntington disease using a mouse model (in collaboration with Rosemarie Grantyn, Berlin, Germany). While iGluu is a useful tool, it is still too slow to visualize the fast binding of glutamate of the AMPA receptor which is followed by the channel opening on the microsecond time scale. We generated a novel fluorescently labelled glutamate sensor, Fl-GluBP, with sub-millisecon glutamate binding kinetics. We plan to target this sensor to the extracellular site of the membrane to monitor real-time glutamate release in parallel to AMPAR response (collaboration with Thomas Oertner, Hamburg, Germany). Furthermore we will develop similar probes with altered properties (dynamic range, Kd) to match different glutamate levels (astrocytes, synaptic cleft, neurons). The fast clearance of glutamate from the synaptic cleft is mainly done by transporters located in the plasma membrane of surrounding astrocytes. There the glutamate gets converted into glutamine and is subquentially release, taken up by the presynaptic terminal, converted into glutamate and packed into vesicles (glutamate-glutamine cycle). On the basis of the bacterial glutamine binding protein (GlNBP) we will use a similar approach as for Fl-GluBP to generate glutamine sensors (Fl-GlNBP), which will allow us to investigate the different stages of the glutamate-glutamine cycle by simultaneous imaging of astrocytes and neurons in brain cell culture. Additionally, Rosemarie Grantyn (Berlin, Germany) will use our probes to image glutamate and glutamine in astrocytes in the striatum, cerebellar cortex and subthalamic nucleus in HD and normal mice to determine their role in HD.

谷氨酸被认为是中枢神经系统中的主要兴奋性神经递质。谷氨酸的失调涉及神经精神障碍如焦虑和抑郁，以及急性和慢性神经退行性疾病如亨廷顿病。基于细菌谷氨酸/天冬氨酸结合蛋白（BP）和环状排列增强型绿色荧光蛋白（cpEGFP）的遗传编码谷氨酸传感器（称为iGluSnFR）的开发代表了监测谷氨酸的重大突破。然而，iGluSnFR的动力学太慢而不能跟踪高频动作电位（AP）放电。我们的新变体iGluu具有5倍更快的动力学，足以显示谷氨酸从AP之间的突触间隙中清除。在这项提案中，我们将利用这种探针不仅可视化突触谷氨酸释放，而且还摄取到星形胶质细胞中，并使用小鼠模型比较健康和亨廷顿病的动态（与Rosemarie Grantyn，柏林，德国合作）。虽然iGluu是一种有用的工具，但它仍然太慢，无法可视化AMPA受体的谷氨酸盐的快速结合，随后是微秒时间尺度上的通道开放。我们产生了一种新的荧光标记的谷氨酸传感器，Fl-GluBP，具有亚毫秒谷氨酸结合动力学。我们计划将该传感器定位于膜的细胞外位点，以监测与AMPAR反应平行的实时谷氨酸释放（与托马斯奥特纳，汉堡，德国合作）。此外，我们将开发具有改变的特性（动态范围，Kd）的类似探针，以匹配不同的谷氨酸水平（星形胶质细胞，突触间隙，神经元）。谷氨酸从突触间隙的快速清除主要是通过位于周围星形胶质细胞质膜上的转运蛋白完成的。在那里，谷氨酸被转化为谷氨酰胺，随后被释放，被突触前末端吸收，转化为谷氨酸并包装成囊泡（谷氨酸-谷氨酰胺循环）。在细菌谷氨酰胺结合蛋白（GlNBP）的基础上，我们将使用与Fl-GluBP类似的方法来产生谷氨酰胺传感器（Fl-GlNBP），这将使我们能够通过同时成像脑细胞培养物中的星形胶质细胞和神经元来研究谷氨酸-谷氨酰胺循环的不同阶段。此外，Rosemarie Grantyn（柏林，德国）将使用我们的探针对HD和正常小鼠纹状体、小脑皮质和丘脑底核中星形胶质细胞中的谷氨酸和谷氨酰胺进行成像，以确定它们在HD中的作用。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Vesicular release probability sets the strength of individual Schaffer collateral synapses

囊泡释放概率决定了单个谢弗侧支突触的强度

DOI：
10.1101/2020.08.02.232850
发表时间：
2020
期刊：
影响因子：
0
作者：
Dürst C
通讯作者：
Dürst C

Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes

荧光蛋白探针快速谷氨酸传感的动力学机制

DOI：
10.1016/j.bpj.2019.11.006
发表时间：
2020
期刊：
Biophysical Journal
影响因子：
3.4
作者：
Coates C
通讯作者：
Coates C

P-selectin mobility undergoes a sol-gel transition as it diffuses from exocytosis sites into the cell membrane.

DOI：
10.1038/s41467-022-30669-x
发表时间：
2022-05-31
期刊：
Nature communications
影响因子：
16.6
作者：
通讯作者：

Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses.

DOI：
10.1073/pnas.1720648115
发表时间：
2018-05-22
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Helassa N;Dürst CD;Coates C;Kerruth S;Arif U;Schulze C;Wiegert JS;Geeves M;Oertner TG;Török K
通讯作者：
Török K

Single synapse indicators of impaired glutamate clearance derived from fast iGlu u imaging of cortical afferents in the striatum of normal and Huntington (Q175) mice

正常和亨廷顿 (Q175) 小鼠纹状体皮质传入神经的快速 iGlu u 成像衍生的谷氨酸清除受损的单突触指标