Rab6-Mediated Polarized Transport of Synaptic Vesicle Precursors Is Essential for the Establishment of Neuronal Polarity and Brain Formation.

Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.

Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

EHBP1L1, an apicobasal polarity regulator, is critical for nuclear polarization during enucleation of erythroblasts.

Cell polarity, the asymmetric distribution of proteins and organelles, is permanently or transiently established in various cell types and plays an important role in many physiological events. epidermal growth factor receptor substrate 15 homology domain-binding protein 1-like 1 (EHBP1L1) is an adapter protein that is localized on recycling endosomes and regulates apical-directed transport in polarized epithelial cells. However, the role of EHBP1L1 in nonepithelial cells, remains unknown. Here, Ehbp1l1-/- mice showed impaired erythroblast enucleation. Further analyses showed that nuclear polarization before enucleation was impaired in Ehbp1l1-/- erythroblasts. It was also revealed that EHBP1L1 interactors Rab10, Bin1, and dynamin were involved in erythroblast enucleation. In addition, Ehbp1l1-/- erythrocytes exhibited stomatocytic morphology and dehydration. These defects in erythroid cells culminated in early postnatal anemic lethality in Ehbp1l1-/- mice. Moreover, we found the mislocalization of nuclei and mitochondria in the skeletal muscle cells of Ehbp1l1-/- mice, as observed in patients with centronuclear myopathy with genetic mutations in Bin1 or dynamin 2. Taken together, our findings indicate that the Rab8/10-EHBP1L1-Bin1-dynamin axis plays an important role in multiple cell polarity systems in epithelial and nonepithelial cells.

The Rab GTPase-binding protein EHBP1L1 and its interactors CD2AP/CIN85 negatively regulate the length of primary cilia via actin remodeling.

Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of WT EHBP1L1, although this was not observed in the CD2AP/CIN85-binding-deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.

The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6.

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.

Autophagy is an indispensable process that degrades cytoplasmic materials to maintain cellular homeostasis. During autophagy, double-membrane autophagosomes surround cytoplasmic materials and either fuse with endosomes (called amphisomes) and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents by lysosomal hydrolases. However, it remains unclear if there are specific mechanisms and/or conditions which distinguish these alternate routes. Here, we identified PACSIN1 as a novel autophagy regulator. PACSIN1 deletion markedly decreased autophagic activity under basal nutrient-rich conditions but not starvation conditions, and led to amphisome accumulation as demonstrated by electron microscopic and co-localization analysis, indicating inhibition of lysosome fusion. PACSIN1 interacted with SNAP29, an autophagic SNARE, and was required for proper assembly of the STX17 and YKT6 complexes. Moreover, PACSIN1 was required for lysophagy, aggrephagy but not mitophagy, suggesting cargo-specific fusion mechanisms. In C. elegans, deletion of sdpn-1, a homolog of PACSINs, inhibited basal autophagy and impaired clearance of aggregated protein, implying a conserved role of PACSIN1. Taken together, our results demonstrate the amphisome-lysosome fusion process is preferentially regulated in response to nutrient state and stress, and PACSIN1 is a key to specificity during autophagy.

Minimally Invasive "Crab-Shaped Fixation" for Treating Patients with Fragility Fractures of the Pelvis.

INTRODUCTION: In the aging society, fragility fracture of the pelvis (FFP) has become a problem. Although strong and minimally invasive fixation is required in FFP surgery, reports on relevant surgical results are scarce. Crab-shaped fixation (CSF) is a spinopelvic fixation that involves reconstruction of the posterior pelvic ring using spinal instrumentation. This study aimed to evaluate the walking ability and perioperative complications of patients who underwent CSF for FFP. METHODS: We included patients diagnosed with unstable FFPs, including sacral fracture, who required hospitalization. Demographic data, such as age, sex, bone mineral density, and Rommens classification, were evaluated. Surgical findings included estimated blood loss, operation time, and perioperative complications. The improvement in postoperative walking ability was evaluated based on the length of the postoperative period before the initiation of walker training and whether walking aids were required at 6 months post-surgery, compared with those who received conservative treatment for FFP. RESULTS: Our study included 6 cases that received CSF and 16 cases that received conservative treatment, in which all were females and presented with sacral fractures. The average age of the surgical patients, the operation time, and the estimated blood loss were 79.5±14.7 years, 180.7±28.7 min, and 124.2±29.4 mL, respectively. The average length of the postoperative period prior to the initiation of walker training was 10.8±12.3 days, which was significantly shorter than that of conservative treatment. With regard to perioperative complications, poor reduction of fracture dislocation was observed in one case as well as surgical site infection in another case, but the walking ability improved in all cases. CONCLUSIONS: We evaluated the results of the posterior pelvic ring reconstruction with CSF for patients with vertically unstable FFPs, including sacral fractures. CSF is minimally invasive and would be useful as it allowed walker training during the early postoperative period, which improved patients' walking ability even during the in situ pelvic ring reconstruction.

SNAP23 deficiency causes severe brain dysplasia through the loss of radial glial cell polarity.

In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)-specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.

Loss of Rab6a in the small intestine causes lipid accumulation and epithelial cell death from lactation.

Intestinal epithelial cells (IECs) are not only responsible for the digestion and absorption of dietary substrates but also function as a first line of host defense against commensal and pathogenic luminal bacteria. Disruption of the epithelial layer causes malnutrition and enteritis. Rab6 is a small GTPase localized to the Golgi, where it regulates anterograde and retrograde transport by interacting with various effector proteins. Here, we generated mice with IEC-specific deletion of Rab6a (Rab6a∆IEC mice). While Rab6aΔIEC mice were born at the Mendelian ratio, they started to show IEC death, inflammation, and bleeding in the small intestine shortly after birth, and these changes culminated in early postnatal death. We further found massive lipid accumulation in the IECs of Rab6a∆IEC neonates. In contrast to Rab6a∆IEC neonates, knockout embryos did not show any of these abnormalities. Lipid accumulation and IEC death became evident when Rab6a∆IEC embryos were nursed by a foster mother, suggesting that dietary milk-derived lipids accumulated in Rab6a-deficient IECs and triggered IEC death. These results indicate that Rab6a plays a crucial role in regulating the lipid transport and maintaining tissue integrity.

Extremely short setting of optimal sensed atrioventricular interval in patients after Fontan procedure with implanted dual-chamber pacemaker.

BACKGROUND: Atrioventricular interval optimisation is important in patients with dual-chamber pacing, especially with heart failure. In patients with CHD, especially in those with Fontan circulation, the systemic atrial contraction is supposed to be more important than in patients without structural heart disease. METHODS: We retrospectively evaluated two patients after Fontan procedure with dual-chamber pacemaker with a unique setting of optimal sensed atrioventricular interval. RESULTS: The optimal sensed atrioventricular interval determined by echocardiogram was extremely short sensed atrioventricular interval at 25 and 30 ms in both cases; however, the actual P wave and ventricular pacing interval showed 180 and 140 ms, respectively. In both cases, the atrial epicardial leads were implanted on the opposite site of the origin of their own atrial rhythm. The time differences between sensed atrioventricular interval and actual P wave and ventricular pacing interval occurred because of the site of the epicardial atrial pacing leads and the intra-atrial conduction delay. CONCLUSION: We need to consider the origin of the atrial rhythm, the site of the epicardial atrial lead, and the atrial conduction delay by using electrocardiogram and X-ray when we set the optimal sensed atrioventricular interval in complicated CHD.

Standards of Practice in Acute Ischemic Stroke Intervention International Recommendations.

After five positive randomized controlled trials showed benefit of mechanical thrombectomy in the management of acute ischemic stroke with emergent large-vessel occlusion, a multi-society meeting was organized during the 17th Congress of the World Federation of Interventional and Therapeutic Neuroradiology in October 2017 in Budapest, Hungary. This multi-society meeting was dedicated to establish standards of practice in acute ischemic stroke intervention aiming for a consensus on the minimum requirements for centers providing such treatment. In an ideal situation, all patients would be treated at a center offering a full spectrum of neuroendovascular care (a level 1 center). However, for geographical reasons, some patients are unable to reach such a center in a reasonable period of time. With this in mind, the group paid special attention to define recommendations on the prerequisites of organizing stroke centers providing medical thrombectomy for acute ischemic stroke, but not for other neurovascular diseases (level 2 centers). Finally, some centers will have a stroke unit and offer intravenous thrombolysis, but not any endovascular stroke therapy (level 3 centers). Together, these level 1, 2, and 3 centers form a complete stroke system of care. The multi-society group provides recommendations and a framework for the development of medical thrombectomy services worldwide.

Standards of practice in acute ischemic stroke intervention: International recommendations.

This article was first published in JNIS. Cite this article as: Pierot L, Jayaraman MV, Szikora I, et al. Standards of practice in acute ischemic stroke intervention: international recommendations. Journal of NeuroInterventional Surgery. Published Online First: 28 August 2018. doi: 10.1136/neurintsurg-2018-014287.

RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat.

Common wheat originated from interspecific hybridization between cultivated tetraploid wheat and its wild diploid relative Aegilops tauschii followed by amphidiploidization. This evolutionary process can be reproduced artificially, resulting in synthetic hexaploid wheat lines. Here we performed RNA sequencing (RNA-seq)-based bulked segregant analysis (BSA) using a bi-parental mapping population of two synthetic hexaploid wheat lines that shared identical A and B genomes but included with D-genomes of distinct origins. This analysis permitted identification of D-genome-specific polymorphisms around the Net2 gene, a causative locus to hybrid necrosis. The resulting single nucleotide polymorphisms (SNPs) were classified into homoeologous polymorphisms and D-genome allelic variations, based on the RNA-seq results of a parental tetraploid and two Ae. tauschii accessions. The difference in allele frequency at the D-genome-specific SNP sites between the contrasting bulks (ΔSNP-index) was higher on the target chromosome than on the other chromosomes. Several SNPs with the highest ΔSNP-indices were converted into molecular markers and assigned to the Net2 chromosomal region. These results indicated that RNA-seq-based BSA can be applied efficiently to a synthetic hexaploid wheat population to permit molecular marker development in a specific chromosomal region of the D genome.

LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis.

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson's disease and Crohn's disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

Continuous infusion of lipo-prostaglandin E1 for Takayasu's arteritis with heart failure in an 11-month-old baby: a case report.

BACKGROUND: Takayasu's arteritis is extremely rare in children aged below 6 years. At the onset of Takayasu's arteritis in children, symptoms are varied but differ from those in adults. Corticosteroids are the mainstay of treatment for preventing irreversible vascular damage but there is no standard treatment for progressive vascular stenosis. CASE PRESENTATION: A Japanese 11-month-old baby boy presented with Takayasu's arteritis and heart failure, possibly due to afterload mismatch caused by high blood pressure. Computed tomography was performed and revealed thoracic and abdominal aortic aneurysms. It also revealed severe celiac artery stenosis and bilateral renal artery stenosis. Prednisolone was initiated as first-line therapy. The fever resolved, and C-reactive protein levels returned to normal. Although his general condition improved, deterioration of vascular lesions was evident. Celiac artery occlusion, severe right renal artery stenosis, and new superior mesenteric artery stenosis were observed. We decided to use a continuous infusion of lipo-prostaglandin E1 for prevention of branch stenosis of his abdominal aorta. The progression of vascular stenosis was stopped and our patient's cardiac function gradually improved. CONCLUSIONS: A differential diagnosis of heart failure with high blood pressure should be considered in babies. The progression of vascular stenosis may be suppressed by lipo-prostaglandin E1.

The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution.

Cholesterol, which is endocytosed to the late endosome (LE)/lysosome, is delivered to other organelles through vesicular and nonvesicular transport mechanisms. In this study, we discuss a novel mechanism of cholesterol transport from recycling endosomes (REs) to the trans-Golgi network (TGN) through RELCH/KIAA1468, which is newly identified in this study as a Rab11-GTP- and OSBP-binding protein. After treating cells with 25-hydroxycholesterol to induce OSBP relocation from the cytoplasm to the TGN, REs accumulated around the TGN area, but this accumulation was diminished in RELCH- or OSBP-depleted cells. Cholesterol content in the TGN was decreased in Rab11-, RELCH-, and OSBP-depleted cells and increased in the LE/lysosome. According to in vitro reconstitution experiments, RELCH tethers Rab11-bound RE-like and OSBP-bound TGN-like liposomes and promotes OSBP-dependent cholesterol transfer from RE-like to TGN-like liposomes. These data suggest that RELCH promotes nonvesicular cholesterol transport from REs to the TGN through membrane tethering.

BIG1 is required for the survival of deep layer neurons, neuronal polarity, and the formation of axonal tracts between the thalamus and neocortex in developing brain.

BIG1, an activator protein of the small GTPase, Arf, and encoded by the Arfgef1 gene, is one of candidate genes for epileptic encephalopathy. To know the involvement of BIG1 in epileptic encephalopathy, we analyzed BIG1-deficient mice and found that BIG1 regulates neurite outgrowth and brain development in vitro and in vivo. The loss of BIG1 decreased the size of the neocortex and hippocampus. In BIG1-deficient mice, the neuronal progenitor cells (NPCs) and the interneurons were unaffected. However, Tbr1+ and Ctip2+ deep layer (DL) neurons showed spatial-temporal dependent apoptosis. This apoptosis gradually progressed from the piriform cortex (PIR), peaked in the neocortex, and then progressed into the hippocampus from embryonic day 13.5 (E13.5) to E17.5. The upper layer (UL) and DL order in the neocortex was maintained in BIG1-deficient mice, but the excitatory neurons tended to accumulate before their destination layers. Further pulse-chase migration assay showed that the migration defect was non-cell autonomous and secondary to the progression of apoptosis into the BIG1-deficient neocortex after E15.5. In BIG1-deficient mice, we observed an ectopic projection of corticothalamic axons from the primary somatosensory cortex (S1) into the dorsal lateral geniculate nucleus (dLGN). The thalamocortical axons were unable to cross the diencephalon-telencephalon boundary (DTB). In vitro, BIG1-deficient neurons showed a delay in neuronal polarization. BIG1-deficient neurons were also hypersensitive to low dose glutamate (5 µM), and died via apoptosis. This study showed the role of BIG1 in the survival of DL neurons in developing embryonic brain and in the generation of neuronal polarity.