Neuronal function of the endocytic adaptor CALM in the sorting of SNAREs and AMPARs

脑功能在很大程度上依赖于关键膜蛋白的正确分类。例如，神经递质释放后，突触囊泡（SV）蛋白必须从突触前膜中提取，以局部补充SV池，从而维持神经传递。谷氨酸受体需要动态地从突触后膜插入和移除，以允许突触可塑性，从而实现学习和记忆。膜锚定酶，如g-分泌酶及其底物，如淀粉样前体蛋白（APP），必须遵循确定的运输路线，以避免错误处理。因此，分选缺陷会引起严重的神经系统疾病，从癫痫到阿尔茨海默病（AD）。跨膜蛋白的内吞分选通常由内吞适应蛋白调节，内吞适应蛋白将它们与内吞机制连接起来。密切相关的蛋白AP180和CALM就是两个具有重要功能的内吞适应子。在第一个资助期，我们发现AP180对于SV SNARE蛋白Synaptobrevin/VAMP2的高保真检索至关重要，该蛋白是神经传递机制的重要组成部分。AP180的缺失导致VAMP2主要来自抑制性SVs，因为它们的高周转导致兴奋性/抑制性失衡、癫痫发作和过早死亡。虽然CALM在VAMP2分选中发挥重叠作用，但我们的初步实验表明，它的生理作用远远超出了SV循环，这与它与其他VAMPs（例如VAMP7/8）的关联、它在突触前和突触后位点的定位以及它与人类AD的强烈遗传关联一致。我们对神经元特异性CALM敲除（KO）小鼠的初步分析揭示了突出的分选缺陷：(i)水泡SNARE蛋白VAMP7/8的改变，（ii） APP的错误定位，可能是VAMP运输受损的结果，以及（iii）突触后ampa型谷氨酸受体的表面积累。因此，在拟议的研究中，我们的目标是(i)剖析CALM在VAMP7/8运输中的生理作用，并揭示体内VAMP7/8错误分类的生物体后果；（ii）分析在CALM KO小鼠中观察到的APP定位和加工改变的机制，并了解CALM神经元丢失如何影响AD病理；最后（iii）阐明CALM如何在机制上促进ampar的内吞作用，并描述CALM的丧失如何影响突触后可塑性。通过这些研究以及活细胞成像、超分辨率显微镜、生物化学和小鼠遗传学的结合，我们将解决分子神经科学中的重要问题，并证实运输缺陷可能是AD等神经系统疾病的基础这一新兴范式。因此，我们期望我们的研究从长远来看有助于确定治疗脑部疾病的新治疗途径。

Brain function critically relies on the proper sorting of crucial membrane proteins. Synaptic vesicle (SV) proteins, for example, have to be retrieved from the presynaptic membrane after neurotransmitter release to locally replenish the SV pool and thereby sustain neurotransmission. Glutamate receptors need to be dynamically inserted and removed from the postsynaptic membrane to allow for synaptic plasticity and, thus, learning and memory. Membrane-anchored enzymes such as g-secretase and their substrates, e.g. the amyloid precursor protein (APP), must follow defined trafficking itineraries to avoid erroneous processing. Consequently, sorting defects give rise to severe neurological diseases ranging from epilepsy to Alzheimer´s disease (AD). The endocytic sorting of transmembrane proteins is often regulated by endocytic adaptor proteins, which link them to the endocytic machinery. The closely related proteins AP180 and CALM are two such endocytic adaptors with vital functions. During the first funding period we showed that AP180 is crucial for the high-fidelity retrieval of the SV SNARE protein Synaptobrevin/VAMP2, an essential component of the neurotransmission machinery. Deletion of AP180 depletes VAMP2 primarily from inhibitory SVs due to their high turnover causing excitatory/inhibitory imbalance, epileptic seizures and premature death. While CALM fulfills overlapping roles in VAMP2 sorting, our preliminary experiments show that its physiological role extends well beyond SV recycling in line with its association with other VAMPs (e.g. VAMP7/8), its localization at pre- and postsynaptic sites, and its strong genetic association with AD in humans. Our preliminary analysis of neuron-specific CALM Knockout (KO) mice has revealed prominent sorting defects: (i) alterations in the vesicular SNARE proteins VAMP7/8, (ii) mislocalization of APP, possibly as a result of impaired VAMP trafficking, and (iii) a surface accumulation of postsynaptic AMPA-type glutamate receptors. Within the proposed research we therefore aim to (i) dissect the physiological role of CALM in VAMP7/8 trafficking and to unravel the organismic consequences of VAMP7/8 missorting in vivo; to (ii) analyze the mechanism underlying the observed alteration in APP localization and processing in CALM KO mice and to understand how neuronal loss of CALM affects AD pathology; and finally to (iii) elucidate how CALM mechanistically facilitates the endocytosis of AMPARs and to delineate how the loss of CALM impacts postsynaptic plasticity. With these lines of research and a combination of live cell imaging, super-resolution microscopy, biochemistry and mouse genetics we will address important questions in molecular neuroscience and substantiate the emerging paradigm that trafficking defects may underlie neurological diseases such as AD. We therefore expect our research in the long run to contribute to the identification of new therapeutic avenues to treat brain diseases.