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.

Public Health Relevance

This proposal is aimed at determining the role of LKB1-NUAK1 kinases during brain development and in particular to study the function of this new kinase pathway in the development of cortical connectivity. Loss-of-function mutations in NUAK1 and other LKB1-dependent kinases have recently been identified in patients diagnosed with Autism Spectrum Disorder, therefore our results will elucidate how genetic mutation of NUAK1 contributes to abnormal brain development in this socially-devastating neurodevelopmental disorder.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS089456-08
Application #
9096274
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Riddle, Robert D
Project Start
2008-03-01
Project End
2018-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
8
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Neurosciences
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Lewis Jr, Tommy L; Kwon, Seok-Kyu; Lee, Annie et al. (2018) MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size. Nat Commun 9:5008
Hirabayashi, Yusuke; Tapia, Juan Carlos; Polleux, Franck (2018) Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo. Sci Rep 8:14491
Lee, Annie; Hirabayashi, Yusuke; Kwon, Seok-Kyu et al. (2018) Emerging roles of mitochondria in synaptic transmission and neurodegeneration. Curr Opin Physiol 3:82-93
Courchet, Virginie; Roberts, Amanda J; Meyer-Dilhet, Géraldine et al. (2018) Haploinsufficiency of autism spectrum disorder candidate gene NUAK1 impairs cortical development and behavior in mice. Nat Commun 9:4289
Hirabayashi, Yusuke; Kwon, Seok-Kyu; Paek, Hunki et al. (2017) ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons. Science 358:623-630
Mountoufaris, George; Chen, Weisheng V; Hirabayashi, Yusuke et al. (2017) Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly. Science 356:411-414
Ebrahimi-Fakhari, Darius; Saffari, Afshin; Wahlster, Lara et al. (2016) Impaired Mitochondrial Dynamics and Mitophagy in Neuronal Models of Tuberous Sclerosis Complex. Cell Rep 17:1053-1070
Kwon, Seok-Kyu; Sando 3rd, Richard; Lewis, Tommy L et al. (2016) LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons. PLoS Biol 14:e1002516
Kwon, Seok-Kyu; Hirabayashi, Yusuke; Polleux, Franck (2016) Organelle-Specific Sensors for Monitoring Ca(2+) Dynamics in Neurons. Front Synaptic Neurosci 8:29
Lewis Jr, Tommy L; Turi, Gergely F; Kwon, Seok-Kyu et al. (2016) Progressive Decrease of Mitochondrial Motility during Maturation of Cortical Axons In Vitro and In Vivo. Curr Biol 26:2602-2608

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