Summary Dendrite arborization and synapse formation are essential for wiring the neural circuitry. The evolutionarily conserved NDR1/2 kinase pathway, important for polarized growth from yeast to mammals, controls dendrite growth and morphology in worm and fly. Whether NDR1/2 kinases regulate dendrite and synapse development in mammals was not known. Nor have their phosphorylation targets been identified. Here we show that expression of dominant negative (kinase dead) NDR1/2 mutants or siRNA increase dendrite length and proximal branching of mammalian pyramidal neurons in cultures and in vivo, whereas expression of constitutively active NDR1/2 has the opposite effects. Moreover, NDR1/2 contributes to dendritic spine development and excitatory synaptic function. We further employed chemical genetics and identified NDR1/2 substrates in the brain, including two proteins involved in intracellular vesicle trafficking: AAK1 (AP-2 associated kinase) and Rabin8, a GDP/GTP exchange factor (GEF) of Rab8 GTPase. We finally show that AAK1 contributes to dendrite growth regulation and Rabin8 regulates spine development.

Dendritic spines are the major sites of excitatory synaptic transmission in the CNS, and their size and density influence the functioning of neuronal circuits. Here we report that NMDA receptor signaling plays a critical role in regulating spine size and density in the developing cortex. Genetic deletion of the NR1 subunit of the NMDA receptor in the cortex leads to a decrease in spine density and an increase in spine head size in cortical layer 2/3 pyramidal neurons. This process is accompanied by an increase in the presynaptic axon bouton volume and the postsynaptic density area, as well as an increase in the miniature excitatory postsynaptic current amplitude and frequency. These observations indicate that NMDA receptors regulate synapse structure and function in the developing cortex.cortex ͉ development ͉ synapse D endritic spines are bulbous membrane protrusions that form the postsynaptic specializations of the vast majority of excitatory synapses in the CNS (1-3). During the first postnatal week, highly motile and short-lived dendritic filopodia are abundant on cortical pyramidal neurons (4). These actin-rich protrusions make immature synapses along their length or tip (5). The dendritic spines that are present during the first two postnatal weeks display an immature morphology (5), and the spine head is highly motile, with protrusions extending and retracting from the head (6). After the second postnatal week, the spine density increases, and spines attain a mature morphology with bulbous heads similar to that seen in the adult (7). Dendritic spines are sites of excitatory synaptic transmission, and their structure and density are important measures of synaptic function.An important feature of dendritic spines is that their volume and density can be dynamically regulated. Stimuli that induce long-term potentiation (LTP) and long-term depression in hippocampal slices lead to rapid changes in spine volume by activation of NMDA receptors (8-10). Several lines of evidence suggest that NMDA receptor signaling might influence spine volume by recruitment of AMPA receptors (AMPARs). Immature synapses in the CNS have a low AMPAR:NMDA receptor (NMDAR) ratio and gradually acquire AMPARs during development (11,12). The increase in the AMPAR:NMDAR ratio at synapses during development is thought to be mediated by NMDAR-induced recruitment of AMPARs (13-16). This increase in surface AMPARs could influence spine volume because it has been reported that overexpression of the GluR2 AMPAR subunit in hippocampal cultures leads to an increase in spine size (17). NMDAR signaling also has been implicated in regulating spine density. In hippocampal slices, LTP-inducing stimuli or glutamate application leads to the rapid induction of new spines/filopodia, which is blocked by NMDAR antagonists (18,19).These observations suggest a model in which NMDARs recruit AMPARs to developing synapses, which drives spine growth and maturation. This model predicts that loss of NMDARs should lead to smaller AMPA currents and smaller spines. Her...

Loss‐of‐function mutations in CDKL5 kinase cause severe neurodevelopmental delay and early‐onset seizures. Identification of CDKL5 substrates is key to understanding its function. Using chemical genetics, we found that CDKL5 phosphorylates three microtubule‐associated proteins: MAP1S, EB2 and ARHGEF2, and determined the phosphorylation sites. Substrate phosphorylations are greatly reduced in CDKL5 knockout mice, verifying these as physiological substrates. In CDKL5 knockout mouse neurons, dendritic microtubules have longer EB3‐labelled plus‐end growth duration and these altered dynamics are rescued by reduction of MAP1S levels through shRNA expression, indicating that CDKL5 regulates microtubule dynamics via phosphorylation of MAP1S. We show that phosphorylation by CDKL5 is required for MAP1S dissociation from microtubules. Additionally, anterograde cargo trafficking is compromised in CDKL5 knockout mouse dendrites. Finally, EB2 phosphorylation is reduced in patient‐derived human neurons. Our results reveal a novel activity‐dependent molecular pathway in dendritic microtubule regulation and suggest a pathological mechanism which may contribute to CDKL5 deficiency disorder.

SummaryMammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.