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.

Disseminated intravascular coagulation induced by pazopanib following combination therapy of nivolumab plus ipilimumab in a patient with metastatic renal cell carcinoma.

Recently, combination therapy including immune checkpoint inhibition (ICI) has proven to be effective as first-line therapy for patients with metastatic renal cell carcinoma. Although the first-line combination therapies with ICI have shown clinical benefit, a number of patients require second-line treatment. We report a 60-year-old man with metastatic renal cell carcinoma who was treated with pazopanib soon after nivolumab plus ipilimumab combination therapy. He experienced Grade 3 disseminated intravascular coagulation (DIC). We suspect that this was caused by an interaction between pazopanib and nivolumab even though ICI therapy was discontinued. He was treated with thrombomodulin and platelet transfusion and recovered from DIC. Treatment with pazopanib was subsequently restarted. No evidence of DIC was observed thereafter. This severe adverse reaction may have been induced by an interaction between activated proinflammatory immune cells and cytokines from an exacerbated inflammatory state and pazopanib. This report highlights the need to perform careful monitoring of patients who receive molecular targeted therapy after ICI-based immunotherapy.

[Gas-Producing Splenic Abscess after Spleen Metastasis of Left Renal Cell Carcinoma Spleen Metastasis Successfully Treated with Sunitinib].

A 67-year-old man underwent open radical left nephrectomy for left renal cell carcinoma [pT4N0M1 (right lower lobe of lung)] and thoracoscopic partial right lung resection for lung metastasis. The patient subsequently developed a solitary lung metastasis at 10 months and then at 26 months postoperatively. He underwent partial lung resection on each occasion. During the 28 months postoperatively, he was found to have a 12 mm middle mediastinal lymph node metastasis and a 30 mm splenic metastasis, which gradually increased in size. Three months after discovery, sunitinib was initiated at 37.5 mg 2 weeks on/1 week off. Twelve days later, the patient presented with complaints of fever. A gas-producing splenic abscess was diagnosed and he was admitted on the same day. His condition improved with antibiotics and splenic drainage. On day 35 of hospitalization, he underwent laparoscopic splenectomy. The patient's postoperative clinical course was uneventful and he was discharged 7 days after the surgery.

The Change in Neutrophil Lymphocyte Ratio from the First to the Last Repeat Prostate Biopsy Proposed as a Marker of Carcinogenesis.

INTRODUCTION: We investigated whether the change in the neutrophil lymphocyte ratio (NLR) from the first to the last repeat prostate biopsy (ΔNLR) could be the diagnostic tool or not for prostate cancer (PCa) detection. MATERIALS AND METHODS: We retrospectively evaluated medical records of men who had undergone repeat prostate biopsy. The investigated parameters were white blood cell, neutrophil, lymphocyte counts, NLR at the last prostate biopsy, ΔNLR, prostate-specific antigen (PSA), PSA density (PSAD), and PSA velocity. Exclusion criteria were the presence of cancers other than prostate origin, medication, and diseases which induce the change of NLR. RESULTS: A total of 301 men who had undergone repeat prostate biopsy were selected for this study. After applying exclusion criteria, 223 patients were included. Of these patients, 94 were diagnosed with PCa (Group I) and 129 with no malignancy (Group II). Only a single patient had metastasis. On evaluating the area under the receiver operating characteristic curve of all study parameters, ΔNLR was the most accurate marker, followed by PSAD and then NLR measured at the last biopsy. CONCLUSIONS: ΔNLR was the most accurate marker to improve the total predictive value in repeat prostate biopsy for diagnosing PCa.

Small GTPase Rab17 regulates the surface expression of kainate receptors but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in hippocampal neurons via dendritic trafficking of Syntaxin-4 protein.

Glutamate receptors are fundamental for control synaptic transmission, synaptic plasticity, and neuronal excitability. However, many of the molecular mechanisms underlying their trafficking remain elusive. We previously demonstrated that the small GTPase Rab17 regulates dendritic trafficking in hippocampal neurons. Here, we investigated the role(s) of Rab17 in AMPA receptor (AMPAR) and kainate receptor (KAR) trafficking. Although Rab17 knockdown did not affect surface expression of the AMPAR subunit GluA1 under basal or chemically induced long term potentiation conditions, it significantly reduced surface expression of the KAR subunit GluK2. Rab17 co-localizes with Syntaxin-4 in the soma, dendritic shaft, the tips of developing hippocampal neurons, and in spines. Rab17 knockdown caused Syntaxin-4 redistribution away from dendrites and into axons in developing hippocampal neurons. Syntaxin-4 knockdown reduced GluK2 but had no effect on GluA1 surface expression. Moreover, overexpression of constitutively active Rab17 promoted dendritic surface expression of GluK2 by enhancing Syntaxin-4 translocation to dendrites. These data suggest that Rab17 mediates the dendritic trafficking of Syntaxin-4 to selectively regulate dendritic surface insertion of GluK2-containing KARs in rat hippocampal neurons.

Rabex-5 protein regulates dendritic localization of small GTPase Rab17 and neurite morphogenesis in hippocampal neurons.

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.

Differences in muscle power between the dominant and nondominant upper limbs of baseball players.

We examined the differences in muscle power between the dominant and nondominant upper limbs of 33 healthy, right-handed, university baseball players (mean age, 20.4 ± 1.1 years) with an average baseball experience >11 years. After measuring maximal voluntary contraction (MVC) of hand grip, elbow flexion, and shoulder internal rotation in both upper limbs, the muscle power of each joint was measured at 40%, 50%, and 60% MVC. No significant differences were observed in the main factors affecting MVC and elbow flexion power loads between dominant and nondominant upper limbs. For handgrip power, load factors at 40% MVC in the dominant hand were lower than those at 60% MVC in the same hand and those at 50% and 60% MVC in the nondominant hand. Significant differences were observed in shoulder internal rotation power between dominant and nondominant upper limbs, with the dominant limb having greater power at all loads. Correlations between muscle power of both upper limbs for handgrip and elbow flexion were significant and moderately high at all loads. For shoulder internal rotation power, the degree of correlation was significant and moderately high at 40% MVC but low to moderate at 50% and 60% MVC. Therefore, baseball players have marked lateral dominance in shoulder internal rotation power unlike handgrip and elbow flexion power, although the relationship between shoulder internal rotation muscle powers of both upper limbs becomes lower with increasing load. The dominance of muscle power of each joint varied even in the same upper limb. It is thus beneficial for baseball players to train with even loads on both arms or adopt simultaneous workout of both arms after adjusting for strength differences.

Gleason Pattern 5 May Be a Prognostic Factor in Radium-223 Treatment.

Background/Aim: Radium-223 treatment reduces the risk of death in patients with metastatic castration-resistant prostate cancer (CRPC). This study analyzed the prognostic factors in patients treated with radium-223 dichloride. Patients and Methods: Patients who received radium-223 dichloride were retrospectively analyzed. Prostate-specific antigen (PSA) response and alkaline phosphatase (ALP) decline rates were analyzed. Overall survival (OS) was evaluated using Kaplan-Meier curves, and prognostic factors for OS were assessed using Cox proportional hazards analysis. Results: Fifty-six patients were included in the study. The five-year OS rate in patients after diagnosis of CRPC was 62.2% [95% confidence interval (CI)=27.55-112.45], while the five-year OS rate in patients at the initiation of radium-223 treatment was 21.3% (95%CI=17.20-36.79). Six patients (11.1%) had a >50% PSA decline rate, and 10 (17.9%) had a >50% ALP decline rate. Cox proportional hazards analysis showed that PSA levels at the initiation of radium-223 treatment [hazard ratio (HR)=1.00; 95%CI=1.00-1.00; p=0.0054] and Gleason Pattern (GP) 5 (HR=5.42; 95%CI=1.08-27.27; p=0.0400) were associated with OS. Patients with GP 5 had a significantly poorer prognosis compared with patients with a GP ≤4. Early administration of radium-223 as a first- or second-line treatment was not associated with OS compared with late administration of radium-223 as a third-line or later treatment. Conclusion: GP 5 and high PSA levels at radium-223 initiation were associated with worse OS. Radium-223 as first- or second-line treatment was not associated with OS. Therefore, a treatment strategy for CRPC based on GP 5 is needed.

Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons.

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

Small GTPase Rab39A interacts with UACA and regulates the retinoic acid-induced neurite morphology of Neuro2A cells.

We screened for a Rab39-specific effector by performing a yeast two-hybrid assay with GTP-locked Rab39A/B as the bait and identified UACA (uveal autoantigen with coiled-coil domains and ankyrin repeats) as a specific Rab39A/B-binding protein. Deletion analysis revealed that a C-terminal coiled-coil domain of UACA functions as a GTP-dependent Rab39-binding domain. shRNA-mediated knockdown of endogenous Rab39A or UACA in mouse neuroblastoma Neuro2A cells resulted in a change in retinoic acid-induced neurite morphology from a multipolar morphology to a bipolar morphology. Taken together, these findings indicate that UACA functions as a Rab39A effector in the retinoic acid-induced differentiation of Neuro2A cells.

Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation.

Synaptic vesicle (SV) clusters, which reportedly result from synapsin's capacity to undergo liquid-liquid phase separation (LLPS), constitute the structural basis for neurotransmission. Although these clusters contain various endocytic accessory proteins, how endocytic proteins accumulate in SV clusters remains unknown. Here, we report that endophilin A1 (EndoA1), the endocytic scaffold protein, undergoes LLPS under physiologically relevant concentrations at presynaptic terminals. On heterologous expression, EndoA1 facilitates the formation of synapsin condensates and accumulates in SV-like vesicle clusters via synapsin. Moreover, EndoA1 condensates recruit endocytic proteins such as dynamin 1, amphiphysin, and intersectin 1, none of which are recruited in vesicle clusters by synapsin. In cultured neurons, like synapsin, EndoA1 is compartmentalized in SV clusters through LLPS, exhibiting activity-dependent dispersion/reassembly cycles. Thus, beyond its essential function in SV endocytosis, EndoA1 serves an additional structural function by undergoing LLPS, thereby accumulating various endocytic proteins in dynamic SV clusters in concert with synapsin.

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release.

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.

Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.

Prognostic factors in Japanese men with high-Gleason metastatic castration-resistant prostate cancer.

Background: Several therapeutic agents are available for metastatic castration-resistant prostate cancer (CRPC). However, prognosis is still not well developed. The Gleason score (GS) is a prognostic factor available for patients with metastatic CRPC. GSs ranging from 6 to 10 and GSs ≥8 are usually categorized as single prognostic factors. In this study, we evaluated the prognosis of high-GS metastatic CRPC in Japanese men. Methods: Overall, 105 patients with metastatic CRPC with a GS ≥8 were retrospectively analyzed. Multivariate analyses of patient age, GS, and Eastern Cooperative Oncology Group performance status (ECOG-PS) were performed using Cox proportional hazards analysis to predict overall survival (OS). Results: GS 8 had all Gleason patterns of 4+4. Thirty patients (28.6%) had GS of 8, and 75 (71.4%) had GS of 9 or 10. As a first-line treatment for metastatic CRPC, 42 patients (40%) received abiraterone, 35 (33.3%) received enzalutamide, and 26 (24.8%) received docetaxel. The 5-year OS in patients with GS of 8 was 65.0% [95% confidence interval (CI): 43.07-86.82%], while the 5-year OS in patients with GS of 9 or 10 was 37.0% (95% CI: 24.41-56.11%). There was a significant difference in OS between the GS 8 and GS 9-10 groups (log-rank test, P=0.038). Multivariate analysis showed that GS and ECOG-PS were significant prognostic factors for OS. Conclusions: Patients with metastatic CRPC with GS 9-10 had poor prognoses, suggesting the need for additional treatment options.

Synaptotagmin IV acts as a multi-functional regulator of Ca2+-dependent exocytosis.

In response to stimuli, secretary cells secrete a variety of signaling molecules packed in vesicles (e.g., neurotransmitters and peptide hormones) into the extracellular space by exocytosis. The vesicle secretion is often triggered by calcium ion (Ca(2+)) entered into secretary cells and achieved by the fusion of secretory vesicles with the plasma membrane. Recent accumulating evidence has indicated that members of the synaptotagmin (Syt) family play a major role in Ca(2+)-dependent exocytosis, and Syt I, in particular, is now widely accepted as the major Ca(2+)-sensor for synchronous neurotransmitter release. Involvement of other Syt isoforms in Ca(2+)-dependent exocytotic events other than neurotransmitter release has also been reported, and the Syt IV isoform is of particular interest, because Syt IV has several unique features not found in Syt I (e.g., immediate early gene product induced by deporalization and postsynaptic localization). In this article, we summarize the literature on the multi-functional role of Syt IV in Ca(2+)-dependent exocytosis.

The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons.

Upon the arrival of repetitive stimulation at the presynaptic terminals of neurons, replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained neurotransmitter release. Kinetics of replenishment and the available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability and speed is unknown. Here, based on comprehensive optical imaging of various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, all of which are implicated in organellar membrane fusion in non-neuronal cells, we show that part of the recycling pool bearing the endosomal Q-SNARE, syntaxin 7 (Stx7), is preferentially mobilized for release during high-frequency repetitive stimulation. Recruitment of the SV pool marked with an Stx7-reporter requires actin polymerization, as well as activation of the Ca2+/calmodulin signaling pathway, reminiscent of rapidly replenishing SVs characterized previously in calyx of Held synapses. Furthermore, disruption of Stx7 function by overexpressing its N-terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 forms rapidly replenishing vesicles essential for synaptic responses to high-frequency repetitive stimulation, and also highlight functional diversities of endosomal SNAREs in generating distinct exocytic vesicles in the presynaptic terminals.

Higher neutrophil-to-lymphocyte ratio after the first cycle of the first-line chemotherapy is associated with poor cancer specific survival of upper urinary tract carcinoma patients.

BACKGROUND: Inflammatory cytokines and immature myeloid derived suppressor cells (MDSCs), which increase during cancer progression, could lead to a neutrophil increase and lymphocyte reduction. Thus, the neutrophil-lymphocyte ratio (NLR) was used to predict survival of patients suffering from urological cancers including upper urinary tract carcinoma. We further determined whether the NLR during the first cycle of first-line chemotherapy could predict cancer specific survival. METHODS: We recruited patients with locally advanced or metastatic upper urinary tract urothelial carcinoma (UTUC) who received chemotherapy between January 2014 and July 2019. We investigated the impact of various clinical variables, including age, sex, performance status, and estimated creatinine clearance (CCr), and NLR before and after the first cycle of the first-line chemotherapy on prognosis. RESULTS: A total of 41 patients were included in our study. Cancer specific survival of the patients with lower NLR was significantly better than that of the patients with higher NLR measured after the first cycle of the first-line chemotherapy (log-rank test P=0.005, median 29.2 vs. 11.9 months, respectively). Cox proportional regression analysis showed that higher NLR after the first cycle of the first-line chemotherapy was a significant predictor of cancer specific survival. CONCLUSIONS: The NLR after the first cycle of the first-line chemotherapy could be an indication for patients with locally advanced or metastatic UTUC to maintain their first-line chemotherapy treatment.

Role of the polybasic sequence in the Doc2alpha C2B domain in dense-core vesicle exocytosis in PC12 cells.

The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2alpha, Doc2beta, and Doc2gamma, in humans and mice. Doc2alpha, the best-characterized, brain-specific isoform, exhibits Ca(2+)-dependent phospholipid-binding activity through its C2A domain, and the Ca(2+)-binding activity is thought to be important for the regulation of Ca(2+)-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2alpha (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2alpha(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of 'readily releasable' vesicles in neuroendocrine cells.

[A case of prostate cancer that developed following transurethral resection of the prostate: diagnostic efficiency of diffusion-weighted images on magnetic resonance imaging].

An 84-year-old man underwent transurethral resection of the prostate (TURP) with a diagnosis of prostatic hypertrophy at the age of 78. He had a moderately high prostate specific antigen (PSA) level of 4.5 ng/ml before TURP, but without pathological malignancy. Following surgery, his PSA level dropped to 1.7 ng/ml and the patient recovered almost completely. Four years later, however, he underwent a reexamination due to diminished urinary flow. His PSA at that time was 5.2 ng/ml, continuing to rise slowly thereafter. Six years following surgery, his PSA reached 13.7 ng/ml, and the diffusion-weighted image on magnetic resonance imaging showed a high intensity area in the prostatic portion of the urethra. A poorly differentiated adenocarcinoma was detected in TUR-derived tissue fragments of the tumor protruding from the prostate. Tissue obtained via a systematic needle biopsy showed no signs of malignancy. Hormonal therapy was introduced. Five months later, the PSA level fell to 0.130 ng/ml ; the patient is now in clinical follow-up.

[A case of infiltrating transitional cell carcinoma of kidney difficult to distinguish from xanthogranulomatous pyelonephritis].

The patient, a 67-year-old man, initially visited another institution with asymptomatic macroscopic hematuria as the major complaint. Since imaging revealed a tumor occupying the left kidney, he was referred to our hospital. Computed tomography (CT) was suggestive of a tumor involving the renal pelvis that had infiltrated the renal parenchyma. The patient was scheduled for surgery, which was postponed when pre-operative CT indicated a reduction in the tumor size, leading to a subsequent diagnosis of xanthogranulomatous pyelonephritis. Surgery was eventually conducted to address complaints such as persistent hematuria. The pathological diagnosis was infiltrating transitional cell carcinoma of the kidney. Urinary cytodiagnoses were class II-III throughout the observation period.