A kinase pathway required for terminal axon branching and presynaptic function

描述（由申请人提供）：大脑中输入和输出连接的精确模式是通过严格调控的发育过程实现的，其损伤对大脑功能有深远影响，并与精神发育迟滞和自闭症谱系障碍等神经发育缺陷有关。在到达它们的目标后，轴突广泛地分支，并且它们的末端分支的大小限定它们的突触输出。在包括皮质投射在内的许多轴突中，轴突分支受活动非依赖性和活动依赖性机制调节。然而，在体内控制哺乳动物神经元轴突分支的分子机制仍然知之甚少。我们最近确定了一个新的激酶通路定义的LKB 1和它的14个直接下游激酶称为NUAK 1作为关键调节皮质轴突分支在体内。我们发现LKB 1-NUAK 1激酶通路通过促进突触前线粒体捕获来调节轴突分支。这些结果提出了一个尚未解决的中心问题：突触前线粒体如何调节轴突分支？更一般地说，很少有人知道突触前线粒体在轴突发育过程中除了作为ATP提供者的功能。我们已经巩固了令人兴奋的新结果，表明突触前线粒体通过线粒体钙单向转运体（MCU）在突触前钙稳态中发挥关键作用。因此，我们强调了LKB 1在调节（1）突触前线粒体捕获和/或（2）突触前线粒体Ca 2+清除能力方面的功能的修改建议。我们提出，在LKB 1和NUAK 1缺失的轴突中可能存在两种表型，它们都有助于轴突分支的减少：（1）在早期发育过程中，大多数新生的突触前位点无法捕获线粒体（这导致缺乏依赖于LKB 1的钙清除）和（2）在少数突触前位点，在LKB 1缺失的轴突中发现线粒体（25%而不是野生型轴突中的70%），这些线粒体具有减少的MCU表达，其与减少的突触前钙清除相关。我们目前的初步结果表明，这种增加突触前钙积累有显着的后果突触前LKB 1无效轴突的释放性能。这项资助的重点是进一步探索LKB 1信号传导，线粒体在突触前钙稳态中的功能，突触前释放特性和轴突分支之间的关系。拟议的实验将提供重要的新见解的分子和细胞机制的发展皮层连接，认知功能的出现的关键一步。

DESCRIPTION (provided by applicant): The precise pattern of input and output connections in the brain is achieved through tightly regulated developmental processes whose impairment have profound effects on brain function, and are linked to neurodevelopmental defects ranging from mental retardation and autism spectrum disorders. Upon reaching their targets, axons branch extensively and the size of their terminal arborization defines their synaptic output. In many axons including cortical projections, axon branching is regulated by both activity-independent and activity-dependent mechanisms. However, the molecular mechanisms controlling axon branching of mammalian neurons in vivo are still poorly understood. We recently identified a new kinase pathway defined by LKB1 and one of its 14 direct downstream kinases called NUAK1 as critical regulators of cortical axon branching in vivo. We found that the LKB1-NUAK1 kinase pathway regulates axon branching through promoting presynaptic mitochondrial capture. These results raise one central unresolved question: how do presynaptic mitochondria regulate axon branching? More generally, little is known about the function of presynaptic mitochondria during axon development beyond their function as ATP provider. We have consolidated exciting new results showing that presynaptic mitochondria play a critical role in presynaptic calcium homeostasis through the Mitochondrial Calcium Uniporter (MCU). Therefore, we have emphasized the revised proposal on studying the function of LKB1 in regulating (1) presynaptic mitochondrial capture and/or (2) the Ca2+ clearance capacity of presynaptic mitochondria. We propose that there are two phenotypes in LKB1 and maybe in NUAK1-null axons that both contribute to the reduction in axon branching: (1) the failure of most nascent presynaptic sites to capture mitochondria during early development (which results in lack of mitochondria-dependent calcium clearance) and (2) at the few presynaptic sites were mitochondria are found in LKB1-null axons (25% instead of 70% in wild-type axons), these mitochondria have reduced MCU expression which correlates with reduced mitochondria-dependent presynaptic calcium clearance. We present preliminary results showing that this increased presynaptic calcium accumulation has drastic consequence on presynaptic release properties in LKB1-null axons. This grant is focusing on further exploring the relationship between LKB1 signaling, mitochondria function in presynaptic calcium homeostasis, presynaptic release properties and axon branching. The proposed experiments will provide important new insights into the molecular and cellular mechanisms underlying the development of cortical connectivity, a critical step for the emergence of cognitive functions.