Control of AMPA receptor trafficking during hippocampal long-term depression (LTD): the role of the p38 MAPK-MK2-LIMK1-cofilin1 signalling cascade

学习和记忆是与大脑中神经细胞之间的信息流（突触传递）的长期变化相关的过程。信息流减少，被称为长期抑郁（LTD），是由海马中i -代谢型谷氨酸受体（GpI-mGluR）的激活引起的，它通过一系列事件导致突触中AMPA受体（AMPAR）的丢失。因此，了解这些蛋白的身份和功能作用是非常重要的，这些蛋白参与了由位于神经细胞表面的GpI-mGluR激活引发的级联事件，导致位于神经细胞表面的AMPAR的丢失（内化）。这个过程包括几个步骤和不同的蛋白质。已知p38丝裂原活化蛋白激酶（p38 MAPK）参与GpI-mGluR-LTD的诱导。激酶是一种酶，其功能是将磷酸盐添加到另一种蛋白质（底物）上。添加的磷酸盐激活或抑制底物的功能。在GpI-mGluR-LTD期间，p38 MAPK的底物尚不清楚。该建议的目的是使用广泛的技术来测试假设，MK2是p38 MAPK的直接底物，是mglur介导的p38 MAPK激活和mGluR-LTD期间AMPAR内在化之间缺失的环节。在GpI-mGluR-LTD期间，试点数据已经发现cofilin1是p38 MAPK的潜在靶标。此外，mGluR-LTD也观察到p38 MAPK和MK2磷酸化水平升高。为了确认p38 MAPK-MK2-LIMK1-cofilin1信号级联的生物学功能，我们将使用MK2/3和MK2敲除小鼠的原代培养，结合分子生物学技术、免疫细胞化学、共聚焦成像和电生理记录。首先，我们将使用缺乏MK2/3的神经元并诱导mGluR-LTD来解决AMPAR的内在化问题。我们还将cofilin1和LIMK1中的磷酸化氨基酸替换为无活性氨基酸，并确定这种替换是否会影响内源蛋白的功能。例如，如果内源性蛋白质参与了AMPAR的内化，那么被操纵的蛋白质会阻止AMPAR的内化吗？AMPAR在神经元功能和脑功能中起着非常重要的作用。大脑功能的丧失可能由包括正常衰老在内的多种因素引起，与阿尔茨海默病等几种神经系统疾病有关。因此，了解调节AMPAR内化的机制将提供对正常和异常突触机制的深入了解，也可能确定减缓衰老和神经退行性变中认知能力下降的新靶点。

Learning and memory are processes associated with long-lasting change in information flow between nerve cells in the brain (synapse transmission). Decreased information flow, known as long-term depression (LTD), is induced in the hippocampus by the activation of group I-metabotropic glutamate receptors (GpI-mGluR), which leads through a cascade of events to the loss of AMPA receptor (AMPAR) from the synapse. Therefore, it is very important to know the identity and functional role of the proteins that are involved in the cascade of events initiated by the activation of GpI-mGluR located on the surface of the nerve cell leading to the loss (internalisation) of AMPAR also situated on the surface of the nerve cell. This process involves several steps and different proteins. p38 mitogen-activated protein kinase (p38 MAPK) is known to be involved in the induction of GpI-mGluR-LTD. A kinase is an enzyme which function is to add phosphate to another protein (substrate). The added phosphate activates or inhibits the function of the substrate. During GpI-mGluR-LTD, the substrates for p38 MAPK are not yet known. The aim of this proposal is to test the hypothesis that MK2, which is a direct substrate of p38 MAPK, is the missing link between mGluR-mediated p38 MAPK activation and the internalisation of the AMPAR during mGluR-LTD using a broad range of techniques. Pilot data have already found cofilin1 as a potential target of p38 MAPK during GpI-mGluR-LTD. Furthermore, increased levels in p38 MAPK and MK2 phosphorylation was also observed in mGluR-LTD. To confirm the biological function of p38 MAPK-MK2-LIMK1-cofilin1 signalling cascade, we will use primary cultures from MK2/3 and MK2 knockout mice combined with molecular biology techniques, immunocytochemistry, confocal imaging and electrophysiological recordings. First, we will use neurons lacking MK2/3 and induce mGluR-LTD to address the internalisation of AMPAR. We will also replace the phosphorylated amino acids in the cofilin1 and LIMK1 to inactive amino-acids and determine whether the replacement affects the function of the endogenous protein. For example, if the endogenous protein is involved in the internalisation of AMPAR, will the manipulated protein block the internalisation of AMPAR. AMPAR has a very important role in neuronal function and therefore in brain function. Loss of brain function, which can be caused by several factors including normal ageing, has been associated with several neurological disorders such as Alzheimer's disease. Thus, understanding the mechanisms regulating internalisation of AMPAR will provide insight into both normal and abnormal synaptic mechanisms and may also identify novel targets to slow cognitive decline in ageing and in neurodegeneration.