Beyond the cytoskeleton: The emerging role of organelles and membrane remodeling in the regulation of axon collateral branches.

The generation of axon collateral branches is a fundamental aspect of the development of the nervous system and the response of axons to injury. Although much has been discovered about the signaling pathways and cytoskeletal dynamics underlying branching, additional aspects of the cell biology of axon branching have received less attention. This review summarizes recent advances in our understanding of key factors involved in axon branching. This article focuses on how cytoskeletal mechanisms, intracellular organelles, such as mitochondria and the endoplasmic reticulum, and membrane remodeling (exocytosis and endocytosis) contribute to branch initiation and formation. Together this growing literature provides valuable insight as well as a platform for continued investigation into how multiple aspects of axonal cell biology are spatially and temporally orchestrated to give rise to axon branches. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1293-1307, 2016.

Synaptotagmin-1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons.

Synaptotagmin-1 (syt1) is a Ca(2+)-binding protein that functions in regulation of synaptic vesicle exocytosis at the synapse. Syt1 is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. To determine if expression of syt1 has a functional role in neuronal development before synapse formation, we examined the effects of syt1 overexpression and knockdown on the growth and branching of the axons of cultured primary embryonic day 8 chicken forebrain neurons. In vivo these neurons express syt1, and most have not yet extended axons. We present evidence that syt1 plays a role in regulating axon branching, while not regulating overall axon length. To study the effects of overexpression of syt1, we used adenovirus-mediated infection to introduce a syt1-YFP construct, or control GFP construct, into neurons. Syt1 levels were reduced using RNA interference. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the number of axonal filopodia and branches. Time-lapse analysis of filopodial dynamics in syt1-overexpressing cells demonstrated that elevation of syt1 levels increased both the frequency of filopodial initiation and their lifespan. Taken together these data indicate that syt1 regulates the formation of axonal filopodia and branches before engaging in its conventional functions at the synapse.

Transport of a synaptotagmin-YFP fusion protein in sympathetic neurons during early neurite outgrowth in vitro after transfection in vivo.

Developing neurons are engaged in neurite outgrowth as well as the synthesis and transport of proteins involved in synaptic transmission. Very little is known about when transport is established in these rudimentary neurites. We used a novel technique to visualize protein transport during the early hours of neurite outgrowth in culture. Recombinant adenoviruses were used to express a synaptotagmin-YFP fusion protein in the superior cervical ganglia of neonatal rats in vivo and protein transport was examined in neuronal cultures established from the superior cervical ganglions (SCGs). We find that, as early as 4h in culture, synaptotagmin-YFP was present in the cytoplasm, lamellipodia, filopodia and growth cones. Protein expression appeared punctate in neurites at 8h in vitro and is consistent with a vesicular localization. These results indicate that the machinery to transport synapse-specific proteins is functional in rudimentary neurites at this time and indicates that this technique can be used to study early neuronal development.