Imperatorin ameliorates learning and memory deficits through BDNF/TrkB and ERK/CaMKIIα/CREB signaling in prenatally-stressed female offspring.

Prenatal stress (PS) can lead to impaired spatial learning and memory in offspring. Imperatorin (IMP) is a naturally occurring furanocoumarin with many pharmacological properties. However, the effects of IMP on cognitive impairment induced by PS and the underlying molecular mechanisms remain unclear. We investigated the protective effect of IMP treatment after PS on learning and memory deficits in female offspring at postnatal 60 days. After treating prenatally-stressed offspring with IMP (15 and 30 mg/kg) for 28 days, we found that IMP increased body weight and ameliorated spatial learning and memory and working memory deficits in female offspring rats. Meanwhile, hippocampal Glu and serum corticosterone levels in prenatally-stressed offspring were significantly decreased after IMP administration. Additionally, IMP treatment significantly increased BDNF, TrkB, CaMKII, and CREB mRNA expression in the hippocampus of offspring rats. Furthermore, PS-mediated induction of RKIP protein and mRNA expression and glucocorticoid receptor protein expression in the hippocampus of offspring rats were significantly decreased by IMP treatment, and the protein expression of BDNF and TrkB and relative levels of p-EKR/ERK, p-CaMKIIα/CaMKIIα, and p-CREB/CREB were remarkably increased after IMP treatment. Taken together, IMP can ameliorate PS-induced learning and memory deficits through BDNF/TrkB and ERK/CaMKIIα/CREB signaling pathway and hypothalamic-pituitary-adrenal axis.

Active nuclear import and passive nuclear export are the primary determinants of TDP-43 localization.

ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied "∆NES" mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.

Copine-6 Binds to SNAREs and Selectively Suppresses Spontaneous Neurotransmission.

Recent studies suggest that spontaneous and action potential-evoked neurotransmitter release processes are independently regulated. However, the mechanisms that uncouple the two forms of neurotransmission remain unclear. In cultured mouse and rat neurons, we show that the two C2 domain-containing protein copine-6 is localized to presynaptic terminals and binds to synaptobrevin2 as well as other SNARE proteins in a Ca2+-dependent manner. Ca2+-dependent interaction of copine-6 with synaptobrevin2 selectively suppresses spontaneous neurotransmission in a reaction that requires the tandem tryptophan residues at the C-terminal region of synaptobrevin2. Accordingly, copine-6 loss of function augmented presynaptic Ca2+ elevation-mediated neurotransmitter release. Intracellular Ca2+ chelation, on the other hand, occluded copine-6-mediated suppression of release. We also evaluated the molecular specificity of the copine-6-dependent regulation of spontaneous release and found that overexpression of copine-6 did not suppress spontaneous release in synaptobrevin2-deficient neurons. Together, these results suggest that copine-6 acts as a specific Ca2+-dependent suppressor of spontaneous neurotransmission.SIGNIFICANCE STATEMENT Synaptic transmission occurs both in response to presynaptic action potentials and spontaneously, in the absence of stimulation. Currently, much more is understood about the mechanisms underlying action potential-evoked neurotransmission compared with spontaneous release. However, recent studies have shown selective modulation of spontaneous neurotransmission process by several neuromodulators, suggesting specific molecular regulation of spontaneous release. In this study, we identify copine-6 as a specific regulator of spontaneous neurotransmission. By both gain-of-function and loss-of-function experiments, we show that copine-6 functions as a Ca2+-dependent suppressor of spontaneous release. These results further elucidate the mechanisms underlying differential regulation of evoked and spontaneous neurotransmitter release.

Synaptic Vesicle-Recycling Machinery Components as Potential Therapeutic Targets.

Presynaptic nerve terminals are highly specialized vesicle-trafficking machines. Neurotransmitter release from these terminals is sustained by constant local recycling of synaptic vesicles independent from the neuronal cell body. This independence places significant constraints on maintenance of synaptic protein complexes and scaffolds. Key events during the synaptic vesicle cycle-such as exocytosis and endocytosis-require formation and disassembly of protein complexes. This extremely dynamic environment poses unique challenges for proteostasis at synaptic terminals. Therefore, it is not surprising that subtle alterations in synaptic vesicle cycle-associated proteins directly or indirectly contribute to pathophysiology seen in several neurologic and psychiatric diseases. In contrast to the increasing number of examples in which presynaptic dysfunction causes neurologic symptoms or cognitive deficits associated with multiple brain disorders, synaptic vesicle-recycling machinery remains an underexplored drug target. In addition, irrespective of the involvement of presynaptic function in the disease process, presynaptic machinery may also prove to be a viable therapeutic target because subtle alterations in the neurotransmitter release may counter disease mechanisms, correct, or compensate for synaptic communication deficits without the need to interfere with postsynaptic receptor signaling. In this article, we will overview critical properties of presynaptic release machinery to help elucidate novel presynaptic avenues for the development of therapeutic strategies against neurologic and neuropsychiatric disorders.

Synaptotagmin-1- and Synaptotagmin-7-Dependent Fusion Mechanisms Target Synaptic Vesicles to Kinetically Distinct Endocytic Pathways.

Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca2+ dependent, but the exact role of Ca2+ and its key effector synaptotagmin-1 (syt1) in regulation of endocytosis is poorly understood. Here, we probed the role of syt1 in single- as well as multi-vesicle endocytic events using high-resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss of function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss of function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles toward slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin-7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca2+ levels.

How Do RIM-BPs Link Voltage-Gated Ca(2+) Channels to Evoked Neurotransmitter Release?

Coupling between voltage-gated Ca(2+) influx and synaptic vesicle exocytosis is essential for rapid evoked neurotransmission. Acuna et al. show that the knockout of RIM-BPs, which are key structural components of this coupling, decreases the reliability of evoked neurotransmitter release.

VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission.

Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca(2+)-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca(2+)-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle-associated SNAREs.

Laparoscopic splenectomy and periesophagogastric devascularization with endoligature for portal hypertension in children.

BACKGROUND: Bleeding from esophagogastric varices is an importment complication of portal hypertension. Recently, significant progress in laparoscopic technology has enabled the devascularization of the lower esophagus and upper stomach in a less invasive way. In this article, we report our preliminary experience with laparoscopic splenectomy and periesophagogastric devascularization by endoligature and its effectiveness for bleeding varices with hypersplenism in children. PATIENTS AND METHODS: Six children with bleeding portal hypertension and developed severe thrombocytopenia and/or leukopenia underwent laparoscopic splenectomy and selective pericardial devascularization by using silk endoligature combined with a Harmonic Scalpel (Ethicon Endosurgery, Cincinnati, OH). The patients included 5 males and 1 female, who ranged in age from 8 to 17 years. After a massive splenectomy was performed, we devascularized the periesophagogastric collateral vessels and perforating veins of the upper stomach to the level of the incisura angularis and the lower esophagus 5 or 6 cm away from the esophagocardia junction. The stem of the gastric coronary vein and paraesophageal collateral veins were not dissected in order to reserve portal blood flow toward the azygous shunt. RESULTS: All the procedures were completed successfully under a whole laparoscope. The operative time ranged from 180 to 270 minutes. Intraoperative blood loss was estimated to be from 80 to 200 mL. None of the patients required a blood transfusion. There were no significant complications either intra- operatively or postoperatively, and all patients had returned to usual activity by 5 days. Postoperative platelet count and white blood cell count increased in individual patients. The data were statistically significant (p = 0.006 and 0.002, respectively). During a postoperative follow-up period of 8-40 months, all children were asymptomatic, with improved growth and hematology and no rebleeding, sepsis, or encephalopathy. CONCLUSIONS: Laparoscopic massive splenectomy with selective periesophagogastric devascularization is a feasible, effective, and safe surgical procedure and has all the benefits of minimally invasive surgery. It offers a new alternative modality for children with bleeding portal hypertension and hypersplenism.