Reduced chondroitin sulfate content prevents diabetic neuropathy through transforming growth factor-ß signaling suppression.

Diabetic neuropathy (DN) is a major complication of diabetes mellitus. Chondroitin sulfate (CS) is one of the most important extracellular matrix components and is known to interact with various diffusible factors; however, its role in DN pathology has not been examined. Therefore, we generated CSGalNAc-T1 knockout (T1KO) mice, in which CS levels were reduced. We demonstrated that diabetic T1KO mice were much more resistant to DN than diabetic wild-type (WT) mice. We also found that interactions between pericytes and vascular endothelial cells were more stable in T1KO mice. Among the RNA-seq results, we focused on the transforming growth factor ß signaling pathway and found that the phosphorylation of Smad2/3 was less upregulated in T1KO mice than in WT mice under hyperglycemic conditions. Taken together, a reduction in CS level attenuates DN progression, indicating that CS is an important factor in DN pathogenesis.

Very-long-chain fatty acids are crucial to neuronal polarity by providing sphingolipids to lipid rafts.

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

Are HPV-negative lesions concerned for the introduction of primary HPV testing for cervical cancer screening in Japan?

AIM: In Japan, primary human papilloma virus (HPV) testing has not been introduced for cervical cancer screening due to concerns that HPV-negative lesions may be missed and a lack of Japanese data. The purpose of this study was to evaluate the validity of primary HPV testing in Japan by analyzing cervical intraepithelial neoplasia 2 (CIN2) or more/high-risk HPV- (CIN2+/hrHPV-) cases in cervical cancer screening. METHODS: Data from 35 525 cervical cancer screenings with HPV testing and cervical cytology from 2011 to 2019 in Saga City, Japan, were reviewed. The cases with low-grade squamous intraepithelial lesion (LSIL+)/hrHPV- were analyzed in detail. RESULTS: The results of the 35 525 examinees were as follows: 31 123 were negative for intraepithelial lesion or malignancy (NILM)/hrHPV-, 2612 were NILM/hrHPV+, 262 were atypical squamous cells of undetermined significance (ASC-US)/hrHPV-, 213 were ASC-US/hrHPV+, 291 were LSIL+/hrHPV-, and 1024 were LSIL+/hrHPV+. Of the 256 LSIL+/hrHPV- examinees for whom histology was available, CIN2+ were CIN2 9.4% (24/256), CIN3 3.9% (10/256), cervical adenocarcinoma 0.4% (1/256), uterine corpus cancer 1.2% (3/256), and uterine sarcoma 0.4% (1/256). Overall, the rate of LSIL+/hrHPV- was 0.82% (291/35.525), 0.1% (36/35525) of which were cervical lesions with CIN2+. Only one cervical adenocarcinoma was detected, but gastric-type adenocarcinoma was not included. CONCLUSION: HPV-negative CIN2+ or cervical adenocarcinoma is not a concern for the introduction of primary HPV screening in Japan. Primary HPV testing in cervical cancer screening is considered a feasible method that can be used in Japan, although an algorithm suitable for Japan and a national-level management system need to be established.

The difference in the effectiveness of human papillomavirus vaccine based on smoking status.

AIM: This study aimed to clarify (1) the effectiveness of human papillomavirus (HPV) vaccine against precancerous lesions of uterine cervical cancer and (2) the difference in these effectiveness based on smoking status. METHODS: This retrospective cross-sectional study considered women aged 20-24 who underwent cervical cancer screening in Saga City from April 2014 to March 2020. Cervical cytology and histological diagnosis were compared with or without HPV vaccination and smoking. RESULTS: The study included 7253 women (2467 vaccinated and 4786 unvaccinated). Among the vaccinated women, 462 were smokers, 2003 were nonsmokers: among the nonvaccinated women, the numbers were 1217 and 3554, respectively. 0.28% (7/2467) of participants with vaccination had HSIL+ compared to 0.77% (37/4786) without vaccination (odds ratio [OR] 0.36, 95% confidence interval [CI], 0.16-0.81). About 0.32% (8/2467) with vaccination had cervical intraepithelial neoplasia (CIN) 2+ compared to 0.69% (33/4786) without vaccination (OR 0.46, 95% CI, 0.21-1.00). Four women without vaccination had CIN3+. In nonsmokers, HPV vaccination significantly suppressed the incidence of HSIL+ from 0.42% (15/3554) to 0.1% (2/2003) (OR 0.21, 95% CI, 0.05-0.95), but the suppressive effect was not significant in smokers (OR 0.59, 95% CI, 0.22-1.56). In vaccinated women, the incidence of CIN2+ was 0.20% (4/2003) in nonsmokers and 0.87% (4/462) in smokers (OR 0.22, 95% CI, 0.05-0.89, p = 0.02). CONCLUSIONS: HPV bivalent/quadrivalent vaccination is effective in protecting against CIN but insufficient in smokers. The nine-valent vaccine should be introduced into routine vaccination as soon as possible to prevent high-risk HPV infection other than 6/11/16/18.

Neuronal Signaling Involved in Neuronal Polarization and Growth: Lipid Rafts and Phosphorylation.

Neuronal polarization and growth are developmental processes that occur during neuronal cell differentiation. The molecular signaling mechanisms involved in these events in in vivo mammalian brain remain unclear. Also, cellular events of the neuronal polarization process within a given neuron are thought to be constituted of many independent intracellular signal transduction pathways (the "tug-of-war" model). However, in vivo results suggest that such pathways should be cooperative with one another among a given group of neurons in a region of the brain. Lipid rafts, specific membrane domains with low fluidity, are candidates for the hotspots of such intracellular signaling. Among the signals reported to be involved in polarization, a number are thought to be present or translocated to the lipid rafts in response to extracellular signals. As part of our analysis, we discuss how such novel molecular mechanisms are combined for effective regulation of neuronal polarization and growth, focusing on the significance of the lipid rafts, including results based on recently introduced methods.

Phosphoproteomic and bioinformatic methods for analyzing signaling in vertebrate axon growth and regeneration.

Phosphorylation is the most important post-translational modification of proteins in many cells, including neurons. Phosphoproteomics is a relatively new technique for comprehensively identifying phosphorylation sites in the whole proteome of a given system. We applied this method to developmental neurobiology research to understand the signaling pathways that regulate the mammalian growth cone, which is formed at the tips of developing neurites to ensure accurate neuronal network formation. Using this powerful technique, we identified at least four phosphorylation sites tightly associated with axon growth. Because phosphoproteomic results include relatively large numbers of phosphopeptides, the data are typically analyzed using bioinformatics. We utilized three bioinformatics tools to identify the responsible protein kinases, the putative functions of the phosphorylated protein groups, and the evolutional aspects of the phosphorylated proteins. Collectively, these data indicate phosphoproteomics is a cutting-edge tool for neuroscience research.

Phosphorylation sites of microtubule-associated protein 1B (MAP 1B) are involved in axon growth and regeneration.

The growth cone is a specialized structure that forms at the tip of extending axons in developing and regenerating neurons. This structure is essential for accurate synaptogenesis at developmental stages, and is also involved in plasticity-dependent synaptogenesis and axon regeneration in the mature brain. Thus, understanding the molecular mechanisms utilized by growth cones is indispensable to understanding neuronal network formation and rearrangement. Phosphorylation is the most important and commonly utilized protein modification in signal transduction. We previously identified microtubule-associated protein 1B (MAP 1B) as the most frequently phosphorylated protein among ~ 1200 phosphorylated proteins. MAP 1B has more than 10 phosphorylation sites that were present more than 50 times among these 1200 proteins. Here, we produced phospho-specific antibodies against phosphorylated serines at positions 25 and 1201 of MAP 1B that specifically recognize growing axons both in cultured neurons and in vivo in various regions of the embryonic brain. Following sciatic nerve injury, immunoreactivity with each antibody increased compared to the sham operated group. Experiments with transected and sutured nerves revealed that regenerating axons were specifically recognized by these antibodies. These results suggest that these MAP 1B phosphorylation sites are specifically involved in axon growth and that phospho-specific antibodies against MAP 1B are useful markers of growing/regenerating axons.

What Has Changed Since the Introduction of Human Papillomavirus Testing with the Cytology-Based Cervical Cancer Screening System in Japan A Social Experiment.

BACKGROUND: Uterine cervical cancer is the fourth most common female cancer in the world. In Japan, we have an apparently low rate of joining cervical cancer screening programs compared with Western countries. Furthermore, the incidence and mortality rate of cervical cancer among the younger generation has been increasing. OBJECT: The aim of this study was to assess the effectiveness of cervical cancer screening with human papillomavirus (HPV) testing and cytology in Japan. METHODS: Collaborating with Saga City government, we initiated a cervical cancer screening system consisting of HPV testing and baseline cervical cytology from April 2011 as a social experiment. A total of 17,284 participants have been screened with this new combination system. RESULTS: After HPV testing with cytology-based cervical cancer screening, the number of screenings done in women aged under 40 years has significantly increased. In addition, the number of women diagnosed with cervical intraepithelial neoplasia grade 3 has increased (25 of 14,025 vs. 146 of 23,049 under 50 years: p < 0.001). CONCLUSION: These data suggested that the introduction of HPV testing with cytology-based cervical cancer screening as an adjunct to conventional cytology resulted in better efficiency and more accurate screening among the Japanese population.

Glycoprotein M6a as a signaling transducer in neuronal lipid rafts.

Neuronal development is composed of the complex steps which involve many signaling proteins. On the other hand, there are many proteins highly expressed in the differentiated neurons at developmental stages, but of which physiological functions are not precisely known so far. Glycoprotein 6a (GPM6a) currently belongs to such proteins. This protein has four-transmembrane domains and is a member of proteolipid protein family. Recently, we demonstrated that GPM6a is highly concentrated in lipid rafts of the developing neuron with its palmitoylation, and that this molecule is involved in rapid determination of the neuronal polarity, in response to laminin, an extracellular matrix protein (Honda et al., J Neurosci 37: 4046-64 [2017]). Since lipid rafts are membrane domains enriched in sphingolipids and cholesterol, have lower fluidity than other membrane areas and are believed to be signaling "hot-spots", and here, we discuss the functions of this protein in neuronal lipid raft signaling for neuronal development.

Rufy3 is an adapter protein for small GTPases that activates a Rac guanine nucleotide exchange factor to control neuronal polarity.

RUN and FYVE domain-containing 3 (Rufy3) is an adapter protein for small GTPase proteins and is bound to activated Rap2, a Ras family protein in the developing neuron. Previously, we reported the presence of a rapid cell polarity determination mechanism involving Rufy3, which is likely required for in vivo neuronal development. However, the molecular details of this mechanism are unclear. To this end, here we produced Rufy3 knock-out (Rufy3-KO) mice to study the role of Rufy3 in more detail. Examining Rufy3-KO neurons, we found that Rufy3 is recruited via glycoprotein M6A to detergent-resistant membrane domains, which are biochemically similar to lipid rafts. We also clarified that Rufy3, as a component of a ternary complex, induces the assembly of Rap2 in the axonal growth cone, whereas in the absence of Rufy3, the accumulation of a Rac guanine nucleotide exchange factor, T-cell lymphoma invasion and metastasis 2 (Tiam2/STEF), is inhibited downstream of Rap2. We also found that Rufy3 regulates the cellular localization of Rap2 and Tiam2/STEF. Taken together, we conclude that Rufy3 is a physiological adapter for Rap2 and activates Tiam2/STEF in glycoprotein M6A-regulated neuronal polarity and axon growth.

Extracellular Signals Induce Glycoprotein M6a Clustering of Lipid Rafts and Associated Signaling Molecules.

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

Identification of functional marker proteins in the mammalian growth cone.

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins.

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369-375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.

Ca2+ influx through distinct routes controls exocytosis and endocytosis at drosophila presynaptic terminals.

Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca(2+). However, it is not known whether Ca(2+) influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, we separately measured exocytosis and endocytosis using FM1-43. In a temperature-sensitive Ca(2+) channel mutant, cacophony(TS2), exocytosis induced by high K(+) decreased at nonpermissive temperatures, while endocytosis remained unchanged. In wild-type larvae, a spider toxin, PLTXII, preferentially inhibited exocytosis, whereas the Ca(2+) channel blockers flunarizine and La(3+) selectively depressed endocytosis. None of these blockers affected exocytosis or endocytosis induced by a Ca(2+) ionophore. Evoked synaptic potentials were depressed regardless of stimulus frequency in cacophony(TS2) at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La(3+) gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibire(ts1), a dynamin mutant, flunarizine or La(3+) inhibited assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function.

Impaired vascular endothelium-dependent relaxation in Henoch-Schönlein purpura.

Few reports have focused on vascular endothelial function in children with Henoch-Schönlein purpura (HSP). The purpose of the present study was to assess endothelial function and to follow serial changes from the acute to convalescent phases in children with HSP. Forearm flow-mediated vasodilation was evaluated in 21 patients with HSP, aged 4.0-10.3 years (median 6.2 years), and in 14 control subjects. Vascular dimension, mean velocity, and flow volume were measured by ultrasonography in brachial artery before and after hyperemia, and during incremental infusions of nitroglycerin (0.5, 1.0 microg/kg per min). In the controls, significant increases in dimension, mean velocity, and flow volume were observed in reactive hyperemia ( P<0.01). In contrast, patients in the acute phase of HSP showed a flow velocity profile indicating a highly resistant forearm circulation, and significantly attenuated responses after hyperemia ( P<0.01 vs. control), whereas the responses to nitroglycerin were well preserved. In addition, the impaired hyperemic responses recovered in the convalescent phase, with no significant differences compared with controls. These results clearly suggest that forearm vascular endothelium-dependent relaxation was attenuated in patients with acute HSP.

Serum KL-6 level in newborns with meconium aspiration syndrome.

BACKGROUND: It has been reported that serum KL-6 increases in babies with progressing chronic lung disease (CLD). However, there have been few reports assessing KL-6 in meconium aspiration syndrome (MAS). KL-6 was measured in neonates with respiratory diseases including MAS. METHODS: Thirty-eight neonates with respiratory disease were enrolled in the study. These patients were classified into three groups, 14 patients with respiratory distress syndrome (RDS), 14 with MAS, and 10 with transient tachypnea of the newborn (TTN). The control group consisted of 12 healthy neonates. KL-6 levels were measured 1 day (median) after the birth. In the RDS group, measurement was repeated just prior to 36 weeks' postmenstrual age. RESULTS: The levels of KL-6 were 116 +/- 40 U/mL in the RDS, 281 +/- 138 U/mL in the MAS, and 106 +/- 41 U/mL in the TTN groups. KL-6 levels were significantly higher in the MAS group than in the control group (134 +/- 71 U/mL; P < 0.01). In addition, the levels were significantly higher in those with severe MAS than those with mild MAS (P < 0.05). In patients with RDS, KL-6 increased in patients who developed CLD (P < 0.05), while KL-6 levels did not change in those who did not develop CLD. CONCLUSION: These data confirm the high level of KL-6 in CLD, and suggest that KL-6 is increased in MAS.

Direct, Ca2+-dependent interaction between tubulin and synaptotagmin I: a possible mechanism for attaching synaptic vesicles to microtubules.

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.