Anuria after kidney transplantation diagnosed as early recurrence of focal segmental glomerulosclerosis combined with acute calcineurin inhibitor nephrotoxicity: a case report and literature review.

BACKGROUND: Primary focal segmental glomerulosclerosis (FSGS) is a glomerular disease that sometimes recurs in patients after kidney transplantation (KT) and increases the risk of graft loss. Proteinuria is a common early sign of recurrent FSGS, but an abrupt decrease in urine volume is rare. Herein, we report a patient with early recurrence of FSGS with anuria following KT. CASE PRESENTATION: A 55-year-old man with end-stage kidney disease caused by primary FSGS experienced anuria on postoperative day 2 following deceased donor KT. Laboratory results revealed that serum tacrolimus trough levels were consistently elevated at the time of anuria. At first, we considered acute calcineurin inhibitor (CNI) nephrotoxicity based on graft biopsy on light microscopy, laboratory findings, and clinical courses. However, the allograft function did not recover even after discontinuation of CNI, and recurrent FSGS was diagnosed 2 weeks later on electron microscopy. A total of 13 sessions of plasmapheresis and two administrations of rituximab (375 mg/m2) were required to treat recurrent FSGS. The patient achieved a partial response, and the spot urine protein-to-creatinine ratio decreased from 15.5 g/g creatinine to 5.2 g/g creatinine. At 5 months following KT, the serum creatinine level was stable at 1.15 mg/dL. CONCLUSIONS: These findings highlight that anuria can occur in cases of early recurrence of FSGS combined with acute CNI nephrotoxicity.

Comparison of Light-Sheet Fluorescence Microscopy and Fast-Confocal Microscopy for Three-Dimensional Imaging of Cleared Mouse Brain.

Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images.

TAK-981, a SUMOylation inhibitor, suppresses AML growth immune-independently.

Acute myeloid leukemia (AML) generally has an unsatisfactory prognosis despite the recent introduction of new regimens, including targeted agents and antibodies. To find a new druggable pathway, we performed integrated bioinformatic pathway screening on large OHSU and MILE AML databases, discovered the SUMOylation pathway, and validated it independently with an external data set (totaling 2959 AML and 642 normal sample data). The clinical relevance of SUMOylation in AML was supported by its core gene expression which is correlated with patient survival, European LeukemiaNet 2017 risk classification, and AML-relevant mutations. TAK-981, a first-in-class SUMOylation inhibitor currently under clinical trials for solid tumors, showed antileukemic effects with apoptosis induction, cell-cycle arrest, and induction of differentiation marker expression in leukemic cells. It exhibited potent nanomolar activity, often stronger than that of cytarabine, which is part of the standard of care. TAK-981's utility was further demonstrated in in vivo mouse and human leukemia models as well as patient-derived primary AML cells. Our results also indicate direct and cancer cell-inherent anti-AML effects by TAK-981, different from the type 1 interferon and immune-dependent mechanism in a previous solid tumor study. Overall, we provide a proof-of-concept for SUMOylation as a new targetable pathway in AML and propose TAK-981 as a promising direct anti-AML agent. Our data should prompt studies on optimal combination strategies and transitions to clinical trials in AML.

Transcriptional Regulatory Role of NELL2 in Preproenkephalin Gene Expression.

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-likelike 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging.

Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.

Simultaneous Quantification of Four Marker Compounds in Bauhinia coccinea Extract and Their Potential Inhibitory Effects on Alzheimer's Disease Biomarkers.

Bauhinia coccinea is a tropical woody plant widely distributed in Vietnam and Unnan in southern China. Although many studies have shown the biological activities of extracts from various other species in the genus, no studies have investigated the effects of B. coccinea extracts on biological systems. In the present study, a quantitative analysis of four marker compounds of ethanol extracts of B. coccinea branches (EEBC) was performed using the high performance liquid chromatography (HPLC)-photodiode array (PDA) method. Among gallic acid, (+)-catechin, ellagic acid, and quercitrin contained in EEBC, the most abundant compound was (+)-catechin (18.736 mg/g). In addition, we investigated the EEBC on neuroprotection, antioxidation, and Alzheimer's disease (AD) marker molecules, acetylcholinesterase (AChE), and amyloid-ß (Aß). EEBC significantly inhibited hydrogen peroxide (H2O2)-induced cell death in a HT22 neuronal cell line and increased 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl scavenging activity markedly. EEBC also inhibited AChE and Aß aggregation. Among the four compounds, gallic acid exhibited strong inhibitory effects against AChE activation. In the Aß aggregation assay, the four marker compounds exhibited inhibitory effects lower than 30%. According to the results, EEBC could exert anti-AChE activation and Aß aggregation activities based on the interactive effects of the marker compounds. Our findings suggest that EEBC are sources of therapeutic candidates for application in the development of AD medication based on AChE and Aß dual targeting.

Symptom Profiles, Health-Related Quality of Life, and Clinical Blood Markers among Korean Community-Dwelling Older Adults Living with Chronic Conditions.

Older adults suffer from multiple symptoms, which negatively affects their health-related quality of life. The single-symptom management approach has been less than effective. The data of 2362 Korean community-dwelling older adults aged 70 and above were analyzed in the Korean Frailty and Aging Cohort Study (KFACS) study. A cluster analysis, correlation analysis, and logistic regression were used to analyze the data. We found three symptom clusters: high symptom burden (HSB, n = 1032); pain and fatigue group (PAF, n = 566); and the sleep deprivation group (SDP, n = 764). Participants in the HSB group are more likely to be of old age (OR = 1.1), be female (OR = 2.4), live in a rural area (OR = 1.4), have low physical activity (OR = 0.9), and have multiple chronic conditions (OR = 1.5). The clinical blood markers analysis showed a negative relationship among the physical health, free T4 (r = -0.083, p < 0.01) and insulin (r = -0.084, p < 0.01). The sex-specific blood markers analysis showed differences among three clusters. While free testosterone (male: r = 0.124, female: r = 0.110, p < 0.05) and dehydroepiandrosterone (DHEA) (male: r = 0.352 and female: r = 0.134, p < 0.05) were associated with physical health in the HSB group, only free testosterone was associated with mental health (male: r = -0.093, and female: r = -0.116, p < 0.05) in the SDP group. These findings suggest the potential role of the patient's sex and sex hormones in symptoms of Korean community-dwelling older adults. Understanding the symptom profiles and impact of biopsychosocial factors may enhance precision symptom management.

Accelerated Epigenetic Age in Normal Cognitive Aging of Korean Community-Dwelling Older Adults.

BACKGROUND: Epigenetic age acceleration has been studied as a promising biomarker of age-related conditions, including cognitive aging. This pilot study aims to explore potential cognitive aging-related biomarkers by investigating the relationship of epigenetic age acceleration and cognitive function and by examining the epigenetic age acceleration differences between successful cognitive aging (SCA) and normal cognitive aging (NCA) among Korean community-dwelling older adults (CDOAs). METHODS: We used data and blood samples of Korean CDOAs from the Korean Frailty and Aging Cohort Study. The participants were classified into two groups, SCA (above the 50th percentile in all domains of cognitive function) and NCA. The genome-wide DNA methylation profiling array using Illumina Infinium MethylationEPIC BeadChip was used to calculate the following: the DNA methylation age, universal epigenetic age acceleration, intrinsic epigenetic age acceleration (IEAA), and extrinsic epigenetic age acceleration (EEAA). We also used Pearson correlation analysis and independent t-tests to analyze the data. RESULTS: Universal age acceleration correlated with the Frontal Assessment Battery test results (r = -0.42, p = 0.025); the EEAA correlated with the Word List Recognition test results (r = -0.41, p = 0.027). There was a significant difference between SCA and NCA groups in IEAA (p = 0.041, Cohen's d = 0.82) and EEAA (p = 0.042, Cohen's d = 0.78). CONCLUSIONS: Epigenetic age acceleration can be used as a biomarker for early detection of cognitive decline in Korean community-dwelling older adults. Large longitudinal studies are warranted.

Effect of Obesity on Cognitive Impairment in Vascular Dementia Rat Model via BDNF-ERK-CREB Pathway.

The prevalence of vascular dementia continues to increase with no cure. Thus, it is important identify the aggravating factors of vascular dementia to delay disease progression in patients. Obesity is a well-known risk factor for vascular dementia and causes mild cognitive impairment. In the present study, we evaluated whether obesity exacerbates cognitive impairment in vascular dementia rats and how it affects synaptic plasticity through the BDNF pathway. We randomly assigned 30 Wistar male rats to three groups: sham surgery (Sham), vascular dementia (VaD), and vascular dementia with obesity (OB + VaD). We fed rats a 60% high-fat diet to establish obesity; we then induced vascular dementia using bilateral common carotid artery occlusion. After 6 weeks, we evaluated cognitive function using the Morris water maze and radial arm maze tests. We analyzed post-synaptic density-95 (PSD95) and growth-associated protein-43 (GAP43) to confirm synaptic plasticity. We also evaluated brain-derived neurotrophic factor (BDNF), phosphorylated extracellular signal-regulated kinase (pERK), and phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) in the hippocampus. The OB + VaD group showed the most impaired cognitive function on behavioral tests, with decreases in PSD95. The VaD group showed increased levels of BDNF, pERK, and pCREB, while the OB + VaD group displayed decreased levels. We suggest that obesity exacerbates cognitive impairment in vascular dementia by inhibiting the compensatory increases of BDNF-ERK-CREB pathway.

Microtubule-associated protein 2 mediates induction of long-term potentiation in hippocampal neurons.

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Evaluation of Animal Models by Comparison with Human Late-Onset Alzheimer's Disease.

Despite many efforts to alleviate the pathological conditions of Alzheimer's disease (AD), effective therapeutic drugs have not been developed, mainly because of the lack of molecular information about AD and animal models. We observed the reciprocal regulation of AD-associated genes (AD genes) and their related functions. Upregulated AD genes were positioned in central regions in the protein-protein interaction network and were involved in inflammation and DNA repair pathways. Downregulated AD genes positioned in the periphery of the network were associated with metabolic pathways. Using these features of AD genes, we found that 5×FAD, amyloid ß-injected mice, and rats in the initial phases after bilateral common carotid artery occlusion (BCCAO) exhibited patterns that were most similar to those of AD. In contrast, using differentially expressed genes from animal models, we observed that 3×Tg and animals in late phases of BCCAO were positioned close to AD genes.

Neuroprotective Effect of Corydalis ternata Extract and Its Phytochemical Quantitative Analysis.

The tubers of Corydalis ternata have been used to treat cardiovascular diseases such as hypertension and cardiac arrhythmia. Its active components have anticholinesterase, antiamnesic, and anti-inflammatory activities, and analgesic effects. In the present study, we performed quantitative analyses of the two components of C. ternata, coptisine and berberine, using HPLC. A 70% ethanol extract of C. ternata was prepared and the two components were separated using a C-18 analytical column on a gradient solvent system of acetonitrile and 0.1% (v/v) aqueous trifluoroacetic acid. Recordings were performed at a UV wavelength of 265 nm for two standard components. The established analytical method showed high linearity (correlation coefficient (r)=1.0000) and proper precision (0.49-3.88%), accuracy (97.88-102.7%), and recovery (95.12-103.79%) for two standard components. The amount of the coptisine and berberine was 4.968±0.089 mg/g and 3.73±0.075 mg/g, respectively. In addition, we investigated the effects of coptisine and berberine on acetylcholinesterase activity and amyloid-ß aggregation, which are major biomarkers of dementia. Coptisine and berberine decreased acetylcholinesterase activity in a dose-dependent manner (IC50=0.74 and 0.48 µM, respectively). The C. ternata extract exerted an antioxidant activity by stimulating the radical scavenging activity of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), but not 2,2-diphenyl-1-picrylhydrazyl (DPPH). Furthermore, the C. ternata extract reversed the hydrogen peroxide-induced death of HT22 hippocampal cells, indicating its neuroprotective effect. Our results suggest the potential of C. ternata as a therapeutic agent against dementia via the inhibition of acetylcholinesterase activity and neuronal cell death.

Phytochemical Quantification and the In Vitro Acetylcholinesterase Inhibitory Activity of Phellodendron chinense and Its Components.

The dried bark of Phellodendron chinense has been used as a traditional herbal medicine to remove damp heat, relieve consumptive fever, and cure dysentery and diarrhea. In the present study, we performed quantitative analyses of the two components of P. chinense, phellodendrine and berberine, using high-performance liquid chromatography. A 70% ethanol extract of P. chinense was prepared and the two components were separated on a C-18 analytical column using a gradient solvent system of acetonitrile and 0.1% (v/v) aqueous trifluoroacetic acid. The ultraviolet wavelength used for detection was 200 nm for phellodendrine and 226 nm for berberine. The analytical method established here showed high linearity (correlation coefficient, ≥0.9991). The amount of phellodendrine and berberine used was 22.255 ± 0.123 mg/g and 269.651 ± 1.257 mg/g, respectively. Moreover, we performed an in vitro acetylcholinesterase (AChE) activity assay and an amyloid-ß aggregation test to examine the biological properties of phellodendrine and berberine as therapeutic drugs for Alzheimer's disease. Phellodendrine and berberine inhibited AChE activity in a dose-dependent manner (IC50 = 36.51 and 0.44 µM, respectively). In contrast, neither phellodendrine nor berberine had an effect on amyloid-ß aggregation. The P. chinense extract and phellodendrine, but not berberine, exhibited antioxidant activity by increasing radical scavenging activity. Moreover, P. chinense demonstrated a neuroprotective effect in hydrogen peroxide-treated HT22 hippocampal cells. Overall, our findings suggest that P. chinense has potential as an anti-Alzheimer's agent via the suppression of the enzymatic activity of acetylcholinesterase and the stimulation of antioxidant activity.

nArgBP2 regulates excitatory synapse formation by controlling dendritic spine morphology.

Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown. Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine-synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.

Acute exposure to silica nanoparticles aggravate airway inflammation: different effects according to surface characteristics.

Silica nanoparticles (SNPs) are widely used in many scientific and industrial fields despite the lack of proper evaluation of their potential toxicity. This study examined the effects of acute exposure to SNPs, either alone or in conjunction with ovalbumin (OVA), by studying the respiratory systems in exposed mouse models. Three types of SNPs were used: spherical SNPs (S-SNPs), mesoporous SNPs (M-SNPs), and PEGylated SNPs (P-SNPs). In the acute SNP exposure model performed, 6-week-old BALB/c female mice were intranasally inoculated with SNPs for 3 consecutive days. In the OVA/SNPs asthma model, the mice were sensitized two times via the peritoneal route with OVA. Additionally, the mice endured OVA with or without SNP challenges intranasally. Acute SNP exposure induced significant airway inflammation and airway hyper-responsiveness, particularly in the S-SNP group. In OVA/SNPs asthma models, OVA with SNP-treated group showed significant airway inflammation, more than those treated with only OVA and without SNPs. In these models, the P-SNP group induced lower levels of inflammation on airways than both the S-SNP or M-SNP groups. Interleukin (IL)-5, IL-13, IL-1ß and interferon-γ levels correlated with airway inflammation in the tested models, without statistical significance. In the mouse models studied, increased airway inflammation was associated with acute SNPs exposure, whether exposed solely to SNPs or SNPs in conjunction with OVA. P-SNPs appear to be relatively safer for clinical use than S-SNPs and M-SNPs, as determined by lower observed toxicity and airway system inflammation.

SNX14 is a bifunctional negative regulator for neuronal 5-HT6 receptor signaling.

The 5-hydroxytryptamine (5-HT, also known as serotonin) subtype 6 receptor (5-HT6R, also known as HTR6) plays roles in cognition, anxiety and learning and memory disorders, yet new details concerning its regulation remain poorly understood. In this study, we found that 5-HT6R directly interacted with SNX14 and that this interaction dramatically increased internalization and degradation of 5-HT6R. Knockdown of endogenous SNX14 had the opposite effect. SNX14 is highly expressed in the brain and contains a putative regulator of G-protein signaling (RGS) domain. Although its RGS domain was found to be non-functional as a GTPase activator for Gαs, we found that it specifically bound to and sequestered Gαs, thus inhibiting downstream cAMP production. We further found that protein kinase A (PKA)-mediated phosphorylation of SNX14 inhibited its binding to Gαs and diverted SNX14 from Gαs binding to 5-HT6R binding, thus facilitating the endocytic degradation of the receptor. Therefore, our results suggest that SNX14 is a dual endogenous negative regulator in 5-HT6R-mediated signaling pathway, modulating both signaling and trafficking of 5-HT6R.

ADP-ribosylation factor 6 (ARF6) bidirectionally regulates dendritic spine formation depending on neuronal maturation and activity.

Recent studies have reported conflicting results regarding the role of ARF6 in dendritic spine development, but no clear answer for the controversy has been suggested. We found that ADP-ribosylation factor 6 (ARF6) either positively or negatively regulates dendritic spine formation depending on neuronal maturation and activity. ARF6 activation increased the spine formation in developing neurons, whereas it decreased spine density in mature neurons. Genome-wide microarray analysis revealed that ARF6 activation in each stage leads to opposite patterns of expression of a subset of genes that are involved in neuronal morphology. ARF6-mediated Rac1 activation via the phospholipase D pathway is the coincident factor in both stages, but the antagonistic RhoA pathway becomes involved in the mature stage. Furthermore, blocking neuronal activity in developing neurons using tetrodotoxin or enhancing the activity in mature neurons using picrotoxin or chemical long term potentiation reversed the effect of ARF6 on each stage. Thus, activity-dependent dynamic changes in ARF6-mediated spine structures may play a role in structural plasticity of mature neurons.

SCAMP5 plays a critical role in synaptic vesicle endocytosis during high neuronal activity.

Secretory carrier membrane protein 5 (SCAMP5), a recently identified candidate gene for autism, is brain specific and highly abundant in synaptic vesicles (SVs), but its function is currently unknown. Here, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs in cultured rat hippocampal neurons resulted in a reduction in total vesicle pool size as well as in recycling pool size, but the recycling/resting pool ratio was significantly increased. SCAMP5 KD slowed endocytosis after stimulation, but impaired it severely during strong stimulation. We also found that KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during a stimulus. Reintroducing shRNA-resistant SCAMP5 reversed these endocytic defects. Therefore, our results suggest that SCAMP5 functions during high neuronal activity when a heavy load is imposed on endocytosis. Our data also raise the possibility that the reduction in expression of SCAMP5 in autistic patients may be related to the synaptic dysfunction observed in autism.

Hypoxia-mediated mechanism of MUC5AC production in human nasal epithelia and its implication in rhinosinusitis.

BACKGROUND: Excessive mucus production is typical in various upper airway diseases. In sinusitis, the expression of MUC5AC, a major respiratory mucin gene, increases. However, the mechanisms leading to mucus hypersecretion in sinusitis have not been characterized. Hypoxia due to occlusion of the sinus ostium is one of the major pathologic mechanisms of sinusitis, but there have been no reports regarding the mechanism of hypoxia-induced mucus hypersecretion. METHODS AND FINDINGS: This study aims to identify whether hypoxia may induce mucus hypersecretion and elucidate its mechanism. Normal human nasal epithelial (NHNE) cells and human lung mucoepidermoid carcinoma cell line (NCI-H292) were used. Sinus mucosa from patients was also tested. Anoxic condition was in an anaerobic chamber with a 95% N2/5% CO2 atmosphere. The regulatory mechanism of MUC5AC by anoxia was investigated using RT-PCR, real-time PCR, western blot, ChIP, electrophoretic mobility shift, and luciferase assay. We show that levels of MUC5AC mRNA and the corresponding secreted protein increase in anoxic cultured NHNE cells. The major transcription factor for hypoxia-related signaling, HIF-1α, is induced during hypoxia, and transfection of a mammalian expression vector encoding HIF-1α results in increased MUC5AC mRNA levels under normoxic conditions. Moreover, hypoxia-induced expression of MUC5AC mRNA is down-regulated by transfected HIF-1α siRNA. We found increased MUC5AC promoter activity under anoxic conditions, as indicated by a luciferase reporter assay, and mutation of the putative hypoxia-response element in MUC5AC promoter attenuated this activity. Binding of over-expressed HIF-1α to the hypoxia-response element in the MUC5AC promoter was confirmed. In human sinusitis mucosa, which is supposed to be hypoxic, expression of MUC5AC and HIF-1α is higher than in control mucosa. CONCLUSION: The results indicate that anoxia up-regulates MUC5AC by the HIF-1α signaling pathway in human nasal epithelia and suggest that hypoxia might be a pathogenic mechanism of mucus hypersecretion in sinusitis.

Human salivary gland stem cells ameliorate hyposalivation of radiation-damaged rat salivary glands.

Salivary function in mammals may be defective for various reasons, such as aging, Sjogren's syndrome or radiation therapy in head and neck cancer patients. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. In the present study, we isolated tissue-specific stem cells from the human submandibular salivary gland (hSGSCs). To efficiently isolate and amplify hSGSCs in large amounts, we developed a culture system (lasting 4-5 weeks) without any selection. After five passages, we obtained adherent cells that expressed mesenchymal stem cell surface antigen markers, such as CD44, CD49f, CD90 and CD105, but not the hematopoietic stem cell markers, CD34 and CD45, and that were able to undergo adipogenic, osteogenic and chondrogenic differentiation. In addition, hSGSCs were differentiated into amylase-expressing cells by using a two-step differentiation method. Transplantation of hSGSCs to radiation-damaged rat salivary glands rescued hyposalivation and body weight loss, restored acinar and duct cell structure, and decreased the amount of apoptotic cells. These data suggest that the isolated hSGSCs, which may have characteristics of mesenchymal-like stem cells, could be used as a cell therapy agent for the damaged salivary gland.