Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses.

After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37°C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.

The microtubule-dynamin binding inhibitor peptide PHDP5 rescues spatial learning and memory deficits in Alzheimer's disease model mice.

Dynamin is a microtubule (MT) binding protein playing a key role in vesicle endocytosis. In a brain slice model, tau loaded in presynaptic terminals assembles MTs, thereby impairing vesicle endocytosis via depletion of cytosolic dynamin. The peptide PHDP5, derived from the pleckstrin homology domain of dynamin 1, inhibits dynamin-MT interaction and rescues endocytosis and synaptic transmission impaired by tau when co-loaded in presynaptic terminals. We tested whether in vivo administration of PHDP5 could rescue the learning/memory deficits observed in Alzheimer's disease (AD) model mice. A modified PHDP5 incorporating a cell-penetrating peptide (CPP) and a FITC fluorescent marker was delivered intranasally to Tau609 transgenic (Tg) and 3xTg-AD mice. FITC-positive puncta were observed in the hippocampus of mice infused with PHDP5 or scrambled (SPHDP5) peptide, but not in saline-infused controls. In the Morris water maze (MWM) test for spatial learning/memory, AD model mice treated with FITC-PHDP5-CPP showed prominent improvements in learning and memory, performing close to the level of saline-infused WT mice control. In contrast, mice treated with a scrambled construct (FITC-SPHDP5-CPP) showed no significant improvement. We conclude that PHDP5 can be a candidate for human AD therapy.

Anoxia-induced hippocampal LTP is regeneratively produced by glutamate and nitric oxide from the neuro-glial-endothelial axis.

Transient anoxia causes amnesia and neuronal death. This is attributed to enhanced glutamate release and modeled as anoxia-induced long-term potentiation (aLTP). aLTP is mediated by glutamate receptors and nitric oxide (·NO) and occludes stimulation-induced LTP. We identified a signaling cascade downstream of ·NO leading to glutamate release and a glutamate-·NO loop regeneratively boosting aLTP. aLTP in entothelial ·NO synthase (eNOS)-knockout mice and blocking neuronal NOS (nNOS) activity suggested that both nNOS and eNOS contribute to aLTP. Immunostaining result showed that eNOS is predominantly expressed in vascular endothelia. Transient anoxia induced a long-lasting Ca2+ elevation in astrocytes that mirrored aLTP. Blocking astrocyte metabolism or depletion of the NMDA receptor ligand D-serine abolished eNOS-dependent aLTP, suggesting that astrocytic Ca2+ elevation stimulates D-serine release from endfeet to endothelia, thereby releasing ·NO synthesized by eNOS. Thus, the neuro-glial-endothelial axis is involved in long-term enhancement of glutamate release after transient anoxia.

Impact of Locoregional Therapies for Brain Lesions on Survival in Patients With Non-small Cell Lung Cancer With Multiple Extrathoracic Metastases.

BACKGROUND/AIM: The treatment of brain metastases in patients with non-small cell lung cancer (NSCLC) typically involves surgery, irradiation, and chemotherapy (single or combination therapy). However, the impact of these therapies on the survival of patients with NSCLC with multiple extrathoracic metastases has not yet been determined. Therefore, in the present study, we examined the prognostic effect of multimodal treatment for brain metastases in patients with NSCLC with multiple extrathoracic metastases in the absence of driver mutations. PATIENTS AND METHODS: Patients with NSCLC with multiple extrathoracic metastases (including at least one brain metastasis), who visited Saitama Medical Center, Saitama Medical University from January 1, 2010 to December 31, 2016, were enrolled in this study; follow-up was conducted until December 31, 2021. RESULTS: A total of 56 patients were enrolled, including 12 and 44 patients with single and multiple brain metastases, respectively. The median overall survival (OS) for all patients was 4.9 months, and did not differ significantly between patients with single and multiple brain metastases (3.0 vs. 4.9 months, respectively). The selection of locoregional treatment for brain metastases did not depend on Karnofsky performance status (p=0.0862). Among patients with multiple brain metastases, the OS for those who underwent craniotomy followed by whole brain radiation therapy (WBRT), those who received only WBRT, and those treated without locoregional therapy was 47.7, 3.9, and 15.9 months, respectively (p=0.00382). CONCLUSION: Surgical resection followed by radiation therapy is an effective treatment option for brain metastases in patients with multiple metastases. However, WBRT alone did not improve prognosis.

Development of a dose estimation system for external exposure assessment at the late phase for individuals living in areas affected by the Fukushima nuclear accident.

Although the elevated ambient dose equivalent (ADE) rates in areas radiologically affected by the 2011 Fukushima nuclear plant accident decreased as time passed, the assessment of Fukushima residents' external dose is still important. A dose estimation system for external exposure assessment at the late phase for individuals living in Fukushima Prefecture was developed for this purpose. The developed system enables the estimations of external doses over the future based on an individual behavioral pattern and ADE-rate distributions from aerial monitoring data and its predicted ADE rate. To validate the system, the estimated results were compared with the measured readings of personal dosemeters. The results indicate that the developed system properly reproduced the individual external doses of subjects living in areas where the outdoor ADE rates exceeded 0.2 µSv h-1.

Transfer of 137Cs and 90Sr from soil-to-potato: Interpretation of the association from global fallout in Aomori to accidental release in Fukushima and Chornobyl.

Ceasium-137 and 90Sr are major artificial radionuclides that have been released into the environment. Soil-to-plant transfer of radionuclides is an important route to food contamination. The radionuclide activity concentrations in crops must be quantitatively predicted for estimating the internal radiation doses from food ingestion. In this study, soil and potato samples were collected from three study sites contaminated with different sources of 137Cs and 90Sr: Aomori Prefecture (global fallout) and two accidental release areas (Fukushima Prefecture and the Chornobyl exclusion zone). The 137Cs activity concentrations in the soil and potato samples widely ranged from 1.0 to 250,000 and from 0.048 to 200,000 Bq kg-1 dry weight, respectively. The soil-to-potato transfer factor of 137Cs also ranged widely (0.0015-1.1) and decreased with increasing concentration of exchangeable K. Meanwhile, the activity concentrations of 90Sr in the soil and potato samples were 0.50-64,000 and 0.027-18,000 Bq kg-1 dry weight respectively, and the soil-to-potato transfer factor of 90Sr was 0.023-0.74, decreasing with increasing concentration of exchangeable Ca. The specific activity ratios of 137Cs/Cs and 90Sr/Sr in the exchangeable fraction were similar to those in potatoes, with a factor of 3 in the ±95 % confidence intervals over six orders of magnitude and a factor of 2 in the ±95 % confidence intervals over five orders of magnitude, respectively. According to the data, the accuracy of predicting the activity concentrations of 137Cs and 90Sr in potatoes can be improved by applying the specific activity ratios of 137Cs/Cs and 90Sr/Sr in the exchangeable fraction. This approach accounts for variable factors such as the effects of K and Ca fertilization and soil characteristics. It also emphasizes the benefit of determining the stable Cs and Sr concentrations in potatoes and other crops prior to possible future contamination.

Pneumocystis jirovecii Pneumonia Associated with COVID-19 in Patients with Interstitial Pneumonia.

Here, we report two cases of patients with interstitial pneumonia (IP) on steroids who developed Pneumocystis jirovecii pneumonia (PJP) following coronavirus disease 2019 (COVID-19) infection. Case 1: A 69-year-old man on 10 mg of prednisolone (PSL) daily for IP developed new pneumonia shortly after his COVID-19 infection improved and was diagnosed with PJP based on chest computed tomography (CT) findings and elevated serum ß-D-glucan levels. Trimethoprim-sulfamethoxazole (TMP-SMZ) was administered, and the pneumonia resolved. Case 2: A 70-year-old woman taking 4 mg/day of PSL for IP and rheumatoid arthritis developed COVID-19 pneumonia, which resolved mildly, but her pneumonia flared up and was diagnosed as PJP based on CT findings, elevated ß-D-glucan levels, and positive polymerase chain reaction for P. jirovecii DNA in the sputum. The autopsy revealed diffuse alveolar damage, increased collagen fiver and fibrotic foci, mucinous component accumulation, and the presence of a P. jirovecii cyst. In conclusion, steroids and immunosuppressive medications are well-known risk factors for PJP. Patients with IP who have been taking these drugs for a long time are frequently treated with additional steroids for COVID-19; thus, PJP complications should be avoided in such cases.

Moving towards a novel therapeutic strategy for hyperammonemia that targets glutamine metabolism.

Patients with urea cycle disorders intermittently develop episodes of decompensation with hyperammonemia. Although such an episode is often associated with starvation and catabolism, its molecular basis is not fully understood. First, we attempted to elucidate the mechanism of such starvation-associated hyperammonemia. Using a mouse embryonic fibroblast (MEF) culture system, we found that glucose starvation increases ammonia production, and that this increase is associated with enhanced glutaminolysis. These results led us to focus on α-ketoglutarate (AKG), a glutamate dehydrogenase inhibitor, and a major anaplerotic metabolite. Hence, we sought to determine the effect of dimethyl α-ketoglutarate (DKG), a cell-permeable AKG analog, on MEFs and found that DKG mitigates ammonia production primarily by reducing flux through glutamate dehydrogenase. We also verified that DKG reduces ammonia in an NH4 Cl-challenged hyperammonemia mouse model and observed that DKG administration reduces plasma ammonia concentration to 22.8% of the mean value for control mice that received only NH4 Cl. In addition, we detected increases in ornithine concentration and in the ratio of ornithine to arginine following DKG treatment. We subsequently administered DKG intravenously to a newborn pig with hyperammonemia due to ornithine transcarbamylase deficiency and found that blood ammonia concentration declined significantly over time. We determined that this effect is associated with facilitated reductive amination and glutamine synthesis. Our present data indicate that energy starvation triggers hyperammonemia through enhanced glutaminolysis and that DKG reduces ammonia accumulation via pleiotropic mechanisms both in vitro and in vivo. Thus, cell-permeable forms of AKG are feasible candidates for a novel hyperammonemia treatment.

Green fluorescent cAMP indicator of high speed and specificity suitable for neuronal live-cell imaging.

Cyclic adenosine monophosphate (cAMP) is a canonical intracellular messenger playing diverse roles in cell functions. In neurons, cAMP promotes axonal growth during early development, and mediates sensory transduction and synaptic plasticity after maturation. The molecular cascades of cAMP are well documented, but its spatiotemporal profiles associated with neuronal functions remain hidden. Hence, we developed a genetically encoded cAMP indicator based on a bacterial cAMP-binding protein. This indicator "gCarvi" monitors [cAMP]i at 0.2 to 20 µM with a subsecond time resolution and a high specificity over cyclic guanosine monophosphate (cGMP). gCarvi can be converted to a ratiometric probe for [cAMP]i quantification and its expression can be specifically targeted to various subcellular compartments. Monomeric gCarvi also enables simultaneous multisignal monitoring in combination with other indicators. As a proof of concept, simultaneous cAMP/Ca2+ imaging in hippocampal neurons revealed a tight linkage of cAMP to Ca2+ signals. In cerebellar presynaptic boutons, forskolin induced nonuniform cAMP elevations among boutons, which positively correlated with subsequent increases in the size of the recycling pool of synaptic vesicles assayed using FM dye. Thus, the cAMP domain in presynaptic boutons is an important determinant of the synaptic strength.

Perampanel markedly improved clinical seizures in a patient with a Rett-like phenotype and 960-kb deletion on chromosome 9q34.11 including the STXBP1.

Intractable epilepsy was successfully controlled using perampanel, an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor antagonist, in a 27-year-old woman who presented with a Rett syndrome-like phenotype and novel 960-kb deletion involving syntaxin-binding protein 1 on chromosome 9q34.11. Perampanel may be an effective antiepileptic drug for intractable epilepsy associated with STXBP1 mutations.

Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model.

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Thymic Adenocarcinoma with Positivity for Thyroid Transcription Factor-1 and a BRAF V600E Mutation.

Thymic adenocarcinomas are rare. We herein report for the first time a case of thymic adenocarcinoma with positivity for thyroid transcription factor-1 (TTF-1) and a BRAF V600E mutation. A 50-year-old woman had persistent suffocation and chest pain. Computed tomography revealed a 42×28-mm tumor in the anterior mediastinum. The patient underwent tumor resection using video-assisted thoracoscopic surgery. Histopathological findings showed thymic papillary adenocarcinoma, Masaoka stage II. Immunohistochemically, the tumor was positive for TTF-1. A sequencing analysis revealed a BRAF V600E mutation. The patient underwent postoperative radiotherapy and was in good health without recurrence at five months after resection.

Optimization of adenoviral gene transfer in human pluripotent stem cells.

Human pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have the potential to differentiate into a wide variety of cells in vitro and have applications in basic developmental biology research and regenerative medicine. To understand the process of differentiation from pluripotent stem cells to functional cells, it is necessary to efficiently and safely transfer and express exogenous genes. We attempted to optimize the efficient transfer of genes into pluripotent stem cells using adenoviral vectors. Comparative study of the activities of three representative ubiquitously active promoters revealed that only the CA promoter allowed robust transgene expression in human pluripotent stem cells. In addition, we established a protocol that allowed us to efficiently introduce target genes and ensure their expression even in small numbers of cells. Adenoviral vector infection of pluripotent stem cells in single-cell suspension culture yielded high gene transfer efficiency with low cytotoxicity, without losing the undifferentiated state of the pluripotent stem cells. This optimized system will facilitate developmental biology research and regenerative medicine using pluripotent stem cells.

Molecular phylogenetic position of Minamitalitrus zoltani elucidates a further troglobisation pattern in cave-dwelling terrestrial amphipods (Crustacea: Talitridae).

Talitrids are a highly diverse group of amphipod crustaceans that have colonized various terrestrial habitats. Three genera have successfully adapted to cave habitats on islands in the Pacific and Atlantic Oceans. However, the evolutionary origin of the Pacific troglobitic talitrids has remained unknown. We estimate the phylogenetic position of the troglobitic Minamitalitrus zoltani, which inhabits limestone caves on Minamidaito Island in the Northwestern Pacific, on the basis of the traditional multi-locus dataset. For the analyzed talitrids, we also reconstruct ancestral states of the maxilliped palp and male gnathopod 2. Our results indicate that Minamitalitrus zoltani is sister to the epigean Nipponorchestia curvatus with a deep divergence. Nipponorchestia curvatus inhabits coastal habitats in Japan, but is not indigenous to Minamidaito Island. A previous study estimated that the Atlantic troglobitic species had invaded subterranean habitats multiple times, but we provide new insight into the troglobisation history in talitrids. We also recover secondary shifts of character states of the maxilliped palp and male gnathopod 2 within the lineage composed of Minamitalitrus and its phylogenetically close genera. Our findings highlight the need for the genus-level reclassification of these genera; we split Nipponorchestia into two genera, establishing a new genus for Nipponorchestia nudiramus.

Hidden proteome of synaptic vesicles in the mammalian brain.

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an "ultra-definition" (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

Observation of morphological abnormalities in silkworm pupae after feeding 137CsCl-supplemented diet to evaluate the effects of low dose-rate exposure.

Since the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, morphological abnormalities in lepidopteran insects, such as shrinkage and/or aberration of wings, have been reported. Butterflies experimentally exposed to radiocesium also show such abnormalities. However, because of a lack of data on absorbed dose and dose-effect relationship, it is unclear whether these abnormalities are caused directly by radiation. We conducted a low dose-rate exposure experiment in silkworms reared from egg to fully developed larvae on a 137CsCl-supplemented artificial diet and estimated the absorbed dose to evaluate morphological abnormalities in pupal wings. We used 137CsCl at 1.3 × 103 Bq/g fresh weight to simulate 137Cs contamination around the FDNPP. Absorbed doses were estimated using a glass rod dosimeter and Monte Carlo particle transport simulation code PHITS. Average external absorbed doses were approximately 0.24 (on diet) and 0.016 mGy/day (near diet); the average internal absorbed dose was approximately 0.82 mGy/day. Pupal wing structure is sensitive to radiation exposure. However, no significant differences were observed in the wing-to-whole body ratio of pupae between the 137CsCl-exposure and control groups. These results suggest that silkworms are insensitive to low dose-rate exposure due to chronic ingestion of high 137Cs at a high concentration.

Meaningful word acquisition is associated with walking ability over 10 years in Rett syndrome.

PURPOSE: To investigate walking ability in Japanese patients with Rett syndrome (RTT). METHODS: Walking ability was assessed in 100 female Japanese patients with RTT using univariate and multivariate analysis in all age groups, and in patients over 10 years of age. We analyzed walking ability and confounding factors including prenatal-perinatal histories, developmental milestones, somatic and head growth, anthropometric data, body mass index, age of loss of purposeful hand use, age at onset of stereotypic hand movement, history of autistic behavior, age at regression, presence or absence of seizures, and the results of MECP2 genetic examination from the Japanese Rett syndrome database. RESULTS: Univariate analysis revealed that acquisition of walking in all age groups was significantly correlated with the acquisition of meaningful words, microcephaly, and crawling (P < 0.0001, P = 0.005, P < 0.0001, respectively). Univariate analysis revealed that walking ability over 10 years of age was significantly correlated with acquisition of meaningful words, microcephaly, and body mass index (P < 0,0001, P = 0.005, P = 0.0018, respectively). MECP2 mutations R306C, R133C, and R294X were significantly associated with different acquisition of crawling (P = 0.004) and walking (P = 0.01). Multivariate analysis revealed that only acquisition of meaningful words was significantly correlated with walking ability over 10 years of age. This trend excluded the genetic effects of R306C, R133C, and R294X. CONCLUSIONS: Meaningful word acquisition was robustly associated with walking ability over 10 years. Prognosis of walking ability may be predicted by the acquisition of meaningful words. This information is potentially useful for early intervention and the planning of comprehensive treatment for young children with RTT.

Estimation of dietary 14C dose coefficient using 13C-labelled compound administration analysis.

Carbon-14 released from nuclear facilities has been assessed to contribute significantly to the radiation dose that people are exposed to through the food chain. However, the current dose coefficient for members of public, which is the ratio of the 50-year committed effective dose to ingested 1 Bq 14C, recommended by the International Commission on Radiological Protection (ICRP) is not based on experimental human metabolic data for 14C in nutrients and diet. Therefore, to validate the coefficient, we administered 13C-labelled nutrients consisting of four amino acids, three fatty acids, and one monosaccharide to volunteers as substitutes for 14C labelled nutrients and measured the 13C concentration in various excreta samples. Although metabolic models were constructed from the excretion data, a significant fraction of administered 13C was not recovered from some nutrients. The dose coefficients of 14C in uniformly labelled Japanese diet, which were estimated under several assumptions about the unrecoverable fraction, varied from (6.2 ± 0.9) × 10-11 to (8.9 ± 4.4) × 10-10 Sv Bq-1 and were approximately comparable to the current value of 5.8 × 10-10 Sv Bq-1 recommended by the ICRP. Further studies are necessary to elucidate the metabolism of 14C in various nutrients in the unrecoverable fraction.

Frequency-Dependent Block of Excitatory Neurotransmission by Isoflurane via Dual Presynaptic Mechanisms.

Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses.SIGNIFICANCE STATEMENT Synaptic mechanisms of general anesthesia remain unidentified. In rat brainstem slices, isoflurane inhibits excitatory transmitter release by blocking presynaptic Ca2+ channels and exocytic machinery, with the latter mechanism predominating in its inhibitory effect on high-frequency transmission. Both in slice and in vivo, isoflurane preferentially inhibits spike transmission induced by high-frequency presynaptic inputs. This low-pass filtering action of isoflurane likely plays a significant role in general anesthesia.