Complex formation of immunoglobulin superfamily molecules Side-IV and Beat-IIb regulates synaptic specificity.

Neurons establish specific synapses based on the adhesive properties of cell-surface proteins while also retaining the ability to form synapses in a relatively non-selective manner. However, comprehensive understanding of the underlying mechanism reconciling these opposing characteristics remains incomplete. Here, we have identified Side-IV/Beat-IIb, members of the Drosophila immunoglobulin superfamily, as a combination of cell-surface recognition molecules inducing synapse formation. The Side-IV/Beat-IIb combination transduces bifurcated signaling with Side-IV's co-receptor, Kirre, and a synaptic scaffold protein, Dsyd-1. Genetic experiments and subcellular protein localization analyses showed the Side-IV/Beat-IIb/Kirre/Dsyd-1 complex to have two essential functions. First, it narrows neuronal binding specificity through Side-IV/Beat-IIb extracellular interactions. Second, it recruits synapse formation factors, Kirre and Dsyd-1, to restrict synaptic loci and inhibit miswiring. This dual function explains how the combinations of cell-surface molecules enable the ranking of preferred interactions among neuronal pairs to achieve synaptic specificity in complex circuits in vivo.

Effect of a Single Dose of Oxaliplatin on the Induction of Peripheral Neuropathy in a Rat Model: An in Vivo Electrophysiological Study.

The anticancer drug oxaliplatin is associated with peripheral neuropathy as a side effect accompanied by mechanical and cold allodynia. Although the superficial layer of the spinal cord dorsal horn is known to receive information primarily from peripheral pain nerves, to our knowledge, no in vivo electrophysiological analyses have been conducted to determine whether oxaliplatin administration increases the excitability of superficial layer neurons. Therefore, in vivo extracellular recordings were performed to measure action potentials in the deep and superficial layers of the spinal cord dorsal horn in rats treated with a single dose (6 mg/kg) of oxaliplatin. Action potentials were produced by mechanical stimulation with von Frey filaments to the hindlimb receptive fields. The results revealed that the firing frequency of action potentials increased relative to the intensity of mechanical stimulation, and that both deep and superficial layer neurons in the spinal cord dorsal horn increased significantly in oxaliplatin-treated compared with vehicle-treated rats, especially in the superficial layer. Several superficial layer neurons showed spontaneous firing that was not seen in vehicle-treated rats. In addition, a clear increase was seen in the firing frequency of neurons in the superficial layer of oxaliplatin-treated rats in response to a cold stimulus (here, the addition of acetone to the hindlimb receptive field). This study suggests that the superficial spinal cord dorsal horn strongly reflects the pain pathophysiology in peripheral neuropathy induced by oxaliplatin administration, and that the superficial layer neurons are useful for in vivo electrophysiological analysis using this pathological model.

Activity-dependent endocytosis of Wingless regulates synaptic plasticity in the Drosophila visual system.

Neural activity contributes to synaptic regulation in sensory systems, which allows organisms to adjust to changing environments. However, little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. Previous studies have shown that following prolonged exposure to natural ambient light, the presynaptic active zone (AZ), an area associated with presynaptic neurotransmitter release in Drosophila photoreceptors, undergoes reversible remodeling. Other studies suggest that the secretory protein Wingless (Wg; an ortholog of Wnt-1) can mediate communication between synaptic cells to achieve synaptic remodeling. However, the source of Wg and the mechanism of Wg signal modulation by neuronal activity remained unclear. Here, we found that Wg secreted from glial cells regulates synaptic remodeling in photoreceptors. In addition, antibody staining revealed that Wg changes its localization depending on light conditions. Although Wg is secreted from glial cells, Wg appeared inside photoreceptor axons when flies were kept under light conditions, suggesting that an increase in neuronal activity causes Wg internalization into photoreceptors by endocytosis. Indeed, by blocking endocytosis in photoreceptors, the localization of Wg in photoreceptors disappeared. Interestingly, Wg accumulation was higher in axons with disassembled AZ structure than in axons whose AZ structure was stabilized at the single-cell level, indicating that Wg endocytosis may trigger AZ disassembly. Furthermore, when we genetically activated Wg signaling, Wg accumulation in photoreceptors decreased. Conversely, when we suppressed Wg signaling there was an increase in Wg accumulation. Through RNAi screening of Ca2+-binding proteins in photoreceptors, we found that Calcineurin is a key molecule that triggers Wg endocytosis. Overall, we propose that Wg signaling is regulated by a negative feedback loop driven by Wg endocytosis. The increase in neuronal activity is transmitted via calcium signaling, which leads to a decrease in Wg signaling and thereby promotes presynaptic remodeling.

ppGpp functions as an alarmone in metazoa.

Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.

Systematic identification of genes regulating synaptic remodeling in the Drosophila visual system.

In many animals, neural activity contributes to the adaptive refinement of synaptic properties, such as firing frequency and the number of synapses, for learning, memorizing and adapting for survival. However, the molecular mechanisms underlying such activity-dependent synaptic remodeling remain largely unknown. In the synapses of Drosophila melanogaster, the presynaptic active zone (AZ) forms a T-shaped presynaptic density comprising AZ proteins, including Bruchpilot (Brp). In a previous study, we found that the signal from a fusion protein molecular marker consisting of Brp and mCherry becomes diffuse under continuous light over three days (LL), reflecting disassembly of the AZ, while remaining punctate under continuous darkness. To identify the molecular players controlling this synaptic remodeling, we used the fusion protein molecular marker and performed RNAi screening against 208 neuron-related transmembrane genes that are highly expressed in the Drosophila visual system. Second analyses using the STaR (synaptic tagging with recombination) technique, which showed a decrease in synapse number under the LL condition, and subsequent mutant and overexpression analysis confirmed that five genes are involved in the activity-dependent AZ disassembly. This work demonstrates the feasibility of identifying genes involved in activity-dependent synaptic remodeling in Drosophila, and also provides unexpected insight into the molecular mechanisms involved in cholesterol metabolism and biosynthesis of the insect molting hormone ecdysone.

Cell surface molecule, Klingon, mediates the refinement of synaptic specificity in the Drosophila visual system.

In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, we found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and our genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain.

Two receptor tyrosine phosphatases dictate the depth of axonal stabilizing layer in the visual system.

Formation of a functional neuronal network requires not only precise target recognition, but also stabilization of axonal contacts within their appropriate synaptic layers. Little is known about the molecular mechanisms underlying the stabilization of axonal connections after reaching their specifically targeted layers. Here, we show that two receptor protein tyrosine phosphatases (RPTPs), LAR and Ptp69D, act redundantly in photoreceptor afferents to stabilize axonal connections to the specific layers of the Drosophila visual system. Surprisingly, by combining loss-of-function and genetic rescue experiments, we found that the depth of the final layer of stable termination relied primarily on the cumulative amount of LAR and Ptp69D cytoplasmic activity, while specific features of their ectodomains contribute to the choice between two synaptic layers, M3 and M6, in the medulla. These data demonstrate how the combination of overlapping downstream but diversified upstream properties of two RPTPs can shape layer-specific wiring.