Fusion is the final step of vesicle transport and is mediated by a family of proteins called SNAREs. The specific pairing of v-SNAREs with cognate t-SNAREs forms the “SNARE complex,” which drives the fusion of the vesicle membrane with the target membrane. Over 38 members of the SNARE family have been characterized. They are distributed throughout distinct subcellular compartments and form specific SNARE complexes that mediate various transport events. The SNARE proteins VAMP2/synaptobrevin, SNAP-25, and Syntaxin 1A are core components of the apparatus that mediates neurotransmitter release.
VAMP2-IR and SNAP-25-IR occurred in the center of the Bombyx brain. In the brain of all insects, the central complex (CX) is a unique midline neuropil. The central complex plays a key role in controlling spatial orientation and navigation. CX is instrumental in many phenomena. In addition to sensory-motor integration, Drosophila CX is required for spatial working memory during navigation tasks (Honkanen et al. 2019; Neuser et al. 2008). Changes in the properties of the CX responses according to the behavioral and motivational states of the insect have been found in Drosophila (Weir et al. 2014) and cockroaches (Martin et al. 2015). To control these diverse behaviors, neurotransmitters in the brain are actively released via VAMP2 and SNAP-25.
The central complex is a focus of neuropeptide-containing interneurons in the insect brain (Nässel and Homberg 2006). Neuropeptides, such as tachykinin, short neuropeptide F, and orcokinin, are expressed in the circuits of the central complex and, thus, may modulate the locomotor behavior. The transport of these neuropeptides in the central complex may be related to VAMP2 and SNAP-25.
The expression of VAMP2 is restricted to neurons in the pars intercerebralis (PI), dorsolateral protocerebrum (DL), and the central complex of the Bombyx brain. The VAMP2/n-synaptobrevin (n-syb) of Drosophila is specific to neurons and synapses, and the n-syb mutants have a strictly synaptic phenotype (Deitcher et al. 1998). In B. mori, VAMP2 mainly mediates membrane fusion in the brain.
The PI and DL of insects play important roles in neurosecretion. These regions send major neural processes to the corpora cardiaca and corpora allata, the main neurohemal organ (Hartenstein, 2006). The notion that VAMP2-IRs frequently occur in the neurosecretory center of the insect brain may prevent the fusion of secretory vesicles in this region.
Anti-VAMP2 detected a restricted set of neurons in the CA section. Neuropeptides are synthesized in specific neurosecretory cells, transported along the axons, and secreted as complexes from the CA into the hemolymph. The CA is a neuroendocrine organ that, together with the associated nerves, synthesizes juvenile hormones (Tobe and Pratt 1974). CA functions as a neurohemal organ for the hormones synthesized in neurosecretory cells in the brain. VAMP2 mainly mediates the exocytosis of neuropeptides in CA.
SNAP-25-IR overlapped with VAMP2-IR in the PI and central regions of B. mori but did not occur in DL. In DL, other SNAP proteins may mediate the fusion of secretory vesicles. The SNAP-25 protein subfamily in insects consists of SNAP-25, SNAP-24, SNAP-29, and SNAP-47 (Kádková et al. 2019). Drosophila SNAP-29 is distributed throughout various tissues and is expressed during development (Morelli et al. 2014; Xu et al. 2014). SNAP-24, a protein closely related to SNAP-25, is present in third-instar larval NMJs and in the larval and adult central nervous systems. SNAP-24 can, similar to SNAP-25, form SNARE complexes with syntaxin and neuronal-synaptobrevin (N-syb). (Vilinsky et al. 2002). SNAP-47 plays a role in the proper localization and function of a subset of VAMPs, likely via regulation of their transport through the early secretory pathway (Kuster et al. 2015). These SNAP proteins undergo membrane fusion in place of SNAP-25 in the DL.
An antibody against Syntaxin 1A recognizes a restricted set of neurons in the PI and DL. Syntaxin 1A is a critical component of the SNARE complex and is thought to be essential for synaptic vesicle fusion, but in the central region, the other type of syntaxin undergoes vesicles fusion in the central region and releases neurotransmitters. In insects, Syntaxin 4, Syntaxin 5, Syntaxin 13, and Syntaxin 17 play important roles in membrane fusion. Syntaxin 4 regulates the localization of key synaptic proteins in the postsynaptic compartment of Drosophila NMJs (Harris et al. 2018; Harris et al. 2016). Syntaxin 5 plays an important role in copper regulation and in the development of photoreceptors in Drosophila (Norgate et al. 2010; Satoh et al. 2016). Syntaxin 17 forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS (Takáts et al. 2018). Many neuropeptides and neurotransmitters are synthesized in the PI and DL. Syntaxin 1A-IRs co-localize with VAMP2-IRs in the PI and DL. VAMP2 and Syntaxin 1A mediate membrane fusion of neuropeptide- or neurotransmitter-filled secretory vesicles in the PI and DL.
SNAREs mediate the membrane fusion of secretory vesicles in mammalian neurons, and neuropeptide release is SNARE-dependent (Arora et al. 2017). In insects, SNARE proteins regulate the fusion of neuropeptide-filled secretory vesicles in the brain and CA.
Insulin and ILPs regulate numerous functions in insects, including growth, development, carbohydrate metabolism, and female reproduction (Nassel and Vanden Broeck 2016). SNARE proteins are also involved in insulin secretion by the β-cells of the pancreas (Hou et al. 2009; Wang and Thurmond 2009). We attempted to clarify the relationship between SNARES and bombyxin secretion using double-staining immunohistochemistry of the brain and CA of B. mori. SNAP-25, VAMP2, and Syntaxin 1A were co-localized with bombyxin-IR in the CA and brain. Insulin-like peptides are synthesized in specific neurosecretory cells, transported along the axons, and secreted as complexes from the CA into the hemolymph. SNAP-25, VAMP2, and Syntaxin 1A mediate the membrane fusion of secretory vesicles containing bombyxin in the brain and CA. Rab1-, Rab3-, Rab6-, Rab7-, and RabX4-IRs were co-localized with bombyxin-IR in the brain and CA (Uno et al. 2016). RNAi knockdown of RabX4 affects the content of bombyxin in the brain (Uno et al. 2017). Rabs containing RabX4 mainly regulate the membrane fusion of bombyxin-filled secretory vesicles in the brain and CA via specific effectors.
PTTH is a pivotal regulator of the molting process and metamorphosis. PTTH was identified as a product of two pairs of neurons in the brain. PTTH-IR occurred in VAMP2-IR and Syntaxin-IR neurons in the brain but did not overlap with SNAP-25-IRs. In CA, PTTH-IRs were not co-localized with VAMP2-IRs or Syntaxin-IR. In the brain, VAMP2 and Syntaxin-IR mediate the fusion of PTTH-filled secretory vesicles. Drosophila has two characterized members of this gene family: synaptobrevin (syb) and neuronal synaptobrevin (n-syb). Syb is the primary candidate for mediating the fusion of vesicles with the plasma membrane for cell growth and maintenance. Syb is also widespread within an organism and is not concentrated at the synapse. The loss of syb in a somatic cell clone is lethal (Bhattacharya et al. 2002). Further, Vamp7 is required for autophagosome-lysosome fusion and is a mutually exclusive subunit in the Syx17-Snap29 complex (Takáts et al. 2018). The other synaptobrevin/VAMP may function as a v-SNARE in CA. The release of PTTH into the hemolymph oscillates and is regulated by the circadian rhythm (Vafopoulou et al. 2012; Vafopoulou et al. 2007). In Lepidoptera, PTTH-IR resides in close proximity to period-IRs in the brain (Sauman and Reppert 1996). Further studies are needed to clarify how circadian rhythm-dependent PTTH is regulated by VAMP2-mediated fusion in the brain.