The exocyst subunit Sec15 is critical for proper synaptic development and function at the Drosophila NMJ.

The exocyst protein complex is important for targeted vesicle fusion in a variety of cell types, however, its function in neurons is still not entirely known. We found that presynaptic knockdown (KD) of the exocyst component sec15 by transgenic RNAi expression caused a number of unexpected morphological and physiological defects in the synapse. These include the development of active zones (AZ) devoid of essential presynaptic proteins, an increase in the branching of the presynaptic arbor, the appearance of satellite boutons, and a decrease in the amplitude of stimulated postsynaptic currents as well as a decrease in the frequency of spontaneous synaptic vesicle release. We also found the release of extracellular vesicles from the presynaptic neuron was greatly diminished in the Sec15 KDs. These effects were mimicked by presynaptic knockdown of Rab11, a protein known to interact with the exocyst. sec15 RNAi expression caused an increase in phosphorylated Mothers against decapentaplegic (pMad) in the presynaptic terminal, an indication of enhanced bone morphogenic protein (BMP) signaling. Some morphological phenotypes caused by Sec15 knockdown were reduced by attenuation of BMP signaling through knockdown of wishful thinking (Wit), while other phenotypes were unaffected. Individual knockdown of multiple proteins of the exocyst complex also displayed a morphological phenotype similar to Sec15 KD. We conclude that Sec15, functioning as part of the exocyst complex, is critically important for proper formation and function of neuronal synapses. We propose a model in which Sec15 is involved in the trafficking of vesicles from the recycling endosome to the cell membrane as well as possibly trafficking extracellular vesicles for presynaptic release and these processes are necessary for the correct structure and function of the synapse.

Drebrin Regulates Collateral Axon Branching in Cortical Layer II/III Somatosensory Neurons.

Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral branches in a laminar-specific manner that dictates synaptic partners and overall circuit organization. Here, we leverage both male and female mouse models, single-cell labeling and imaging approaches to identify intrinsic regulators of laminar-specific collateral, also termed interstitial, axon branching. We developed new approaches for the robust, sparse, labeling of Layer II/III pyramidal neurons to obtain single-cell quantitative assessment of axon branch morphologies. We combined these approaches with cell-autonomous loss-of-function (LOF) and overexpression (OE) manipulations in an in vivo candidate screen to identify regulators of cortical neuron axon branch lamination. We identify a role for the cytoskeletal binding protein drebrin (Dbn1) in regulating Layer II/III cortical projection neuron (CPN) collateral axon branching in vitro LOF experiments show that Dbn1 is necessary to suppress the elongation of Layer II/III CPN collateral axon branches within Layer IV, where axon branching by Layer II/III CPNs is normally absent. Conversely, Dbn1 OE produces excess short axonal protrusions reminiscent of nascent axon collaterals that fail to elongate. Structure-function analyses implicate Dbn1S142 phosphorylation and Dbn1 protein domains known to mediate F-actin bundling and microtubule (MT) coupling as necessary for collateral branch initiation upon Dbn1 OE. Taken together, these results contribute to our understanding of the molecular mechanisms that regulate collateral axon branching in excitatory CPNs, a key process in the elaboration of neocortical circuit formation.SIGNIFICANCE STATEMENT Laminar-specific axon targeting is essential for cortical circuit formation. Here, we show that the cytoskeletal protein drebrin (Dbn1) regulates excitatory Layer II/III cortical projection neuron (CPN) collateral axon branching, lending insight into the molecular mechanisms that underlie neocortical laminar-specific innervation. To identify branching patterns of single cortical neurons in vivo, we have developed tools that allow us to obtain detailed images of individual CPN morphologies throughout postnatal development and to manipulate gene expression in these same neurons. Our results showing that Dbn1 regulates CPN interstitial axon branching both in vivo and in vitro may aid in our understanding of how aberrant cortical neuron morphology contributes to dysfunctions observed in autism spectrum disorder and epilepsy.