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

大脑的功能关键依赖于关键的膜蛋白的正确分类。例如，突触囊泡蛋白在神经递质释放后必须从突触前膜中回收，以局部补充突触囊泡池，从而维持神经传递。谷氨酸受体需要动态地插入和移除突触后膜，以实现突触的可塑性，从而实现学习和记忆。膜锚定的酶，如g-分泌酶及其底物，如淀粉样前体蛋白(APP)，必须遵循规定的运输路线，以避免错误处理。因此，分类缺陷会导致严重的神经系统疾病，从癫痫到阿尔茨海默病S病(AD)。跨膜蛋白的内吞分选通常受内吞适配器蛋白的调节，后者将它们与内吞机制联系起来。密切相关的蛋白AP180和CAMAL就是两个具有重要功能的内吞适配器。在第一个资助期，我们发现AP180对于SV SNARE蛋白Synaptobrevin/VAMP2的高保真恢复至关重要，SNARE蛋白Synaptobrevin/VAMP2是神经传递机制的重要组成部分。AP180的缺失主要是由于其高周转率导致兴奋性/抑制性失衡、癫痫发作和过早死亡而使VAMP2从抑制性SVS中耗尽。虽然Calm在VAMP2分类中发挥了重叠的作用，但我们的初步实验表明，它的生理作用远远超出了SV循环，这与它与其他吸血鬼(例如VAMP7/8)的联系、它在突触前和突触后位置的定位以及它与人类AD的强烈遗传关联是一致的。我们对神经元特异性的CAMP基因敲除(KO)小鼠的初步分析发现了显著的分类缺陷：(I)囊泡圈套蛋白VAMP7/8的变化，(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堆积。因此，在拟议的研究中，我们的目标是(I)剖析Calm在VAMP7/8转运中的生理作用，并揭示VAMP7/8在体内错误分选的组织后果；(Ii)分析在Calm KO小鼠中观察到的APP定位和处理改变的机制，并了解神经元失去平静如何影响AD病理；以及(Iii)阐明平静如何机械地促进AMPAR的内吞作用，并描述失去平静如何影响突触后可塑性。通过这些研究路线以及活细胞成像、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阿尔茨海默病等神经疾病的基础这一新兴范式。因此，我们希望从长远来看，我们的研究将有助于确定治疗脑部疾病的新的治疗途径。

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.