We report here development of a novel gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based gene trap vector containing a splice acceptor and the GFP gene, performed a pilot screen for gene trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped gene. We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis.

Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival and differentiation during development and for synaptic function and plasticity in the mature brain. BDNF-containing vesicles are widely distributed and bidirectionally transported in neurons, and secreted BDNF can act on both presynaptic and postsynaptic cells. Activity-dependent BDNF secretion from neuronal cultures has been reported, but it remains unknown where the primary site of BDNF secretion is and whether neuronal activity can trigger BDNF secretion from axons and dendrites with equal efficacy. Using BDNF fused with pH-sensitive green fluorescent protein to visualize BDNF secretion, we found that BDNF-containing vesicles exhibited markedly different properties of activity-dependent exocytic fusion at the axon and dendrite of cultured hippocampal neurons. Brief spiking activity triggered a transient fusion pore opening, followed by immediate retrieval of vesicles without dilation of the fusion pore, resulting in very little BDNF secretion at the axon. On the contrary, the same brief spiking activity induced "full-collapse" vesicle fusion and substantial BDNF secretion at the dendrite. However, full vesicular fusion with BDNF secretion could occur at the axon when the neuron was stimulated by prolonged high-frequency activity, a condition neurons may encounter during epileptic discharge. Thus, activity-dependent axonal secretion of BDNF is highly restricted as a result of incomplete fusion of BDNF-containing vesicles, and normal neural activity induces BDNF secretion from dendrites, consistent with the BDNF function as a retrograde factor. Our study also revealed a novel mechanism by which differential exocytosis of BDNF-containing vesicles may regulate BDNF-TrkB signaling between connected neurons.

Ephrin-Eph signaling is involved in axon guidance during development, but it may also regulate synapse development after the axon has contacted the target cell. Here we report that the activation of ephrin-B reverse signaling in the developing Xenopus laevis optic tectum promotes morphological and functional maturation of retinotectal synapses. Elevation of ephrin-B signaling increased the number of retinotectal synapses and stabilized the axon arbors of retinal ganglion cells. It also enhanced basal synaptic transmission and activity-induced long-term potentiation (LTP) of retinotectal synapses. The functional effects were caused by a rapid enhancement of presynaptic glutamate release and a delayed increase in the postsynaptic glutamate responsiveness. The facilitated LTP induction occurred during the early phase of enhanced transmitter release and appeared to be causally related to the late-phase postsynaptic maturation via an NMDA receptor-dependent mechanism. This ephrin-B-dependent synapse maturation supports the notion that the ephrin/Eph protein families have multiple functions in neural development.

A phase 3, multicenter, open‐label, 52‐week study investigated the efficacy and safety of adalimumab 80 mg at week 0 followed by adalimumab 40 mg every other week (option to escalate to 80 mg when necessary) in Japanese patients with generalized pustular psoriasis (GPP). Adults (aged 15–75 years) with GPP, total skin score (overall erythema area, erythema area with pustules, and edema area) of 3 or more, and erythema with pustules (skin score, ≥1) based on the 2014 Japanese Dermatological Association severity index of GPP were enrolled. The primary efficacy end‐point was clinical response at week 16 (non‐responder imputation), defined as achieving remission (total skin score, 0) or improvement from baseline (reduction of ≥1 point from a baseline total skin score of 3 or ≥2 points from a baseline total skin score of ≥4). Of 10 enrolled patients (mean disease duration, 10.6 years), seven patients, including three with the dose escalated to 80 mg every other week before week 15, achieved clinical response at week 16, and five achieved clinical response at week 52. Mean change from baseline total GPP score was −4.6 at week 16 ( n = 8) and −6.0 at week 52 ( n = 5); change in total skin score was −3.1 ( n = 8) and −4.2 ( n = 5), respectively. Nine patients experienced one or more adverse events and three experienced serious adverse events. The most common adverse events were nasopharyngitis, pruritus and hypoalbuminemia. In conclusion, adalimumab was effective and well tolerated for up to 52 weeks in the treatment of Japanese patients with GPP.