Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

Infrequent RAS mutation is not associated with specific histological phenotype in gliomas.

BACKGROUND: Mutations in driver genes such as IDH and BRAF have been identified in gliomas. Meanwhile, dysregulations in the p53, RB1, and MAPK and/or PI3K pathways are involved in the molecular pathogenesis of glioblastoma. RAS family genes activate MAPK through activation of RAF and PI3K to promote cell proliferation. RAS mutations are a well-known driver of mutation in many types of cancers, but knowledge of their significance for glioma is insufficient. The purpose of this study was to reveal the frequency and the clinical phenotype of RAS mutant in gliomas. METHODS: This study analysed RAS mutations and their clinical significance in 242 gliomas that were stored as unfixed or cryopreserved specimens removed at Kyoto University and Osaka National Hospital between May 2006 and October 2017. The hot spots mutation of IDH1/2, H3F3A, HIST1H3B, and TERT promoter and exon 2 and exon 3 of KRAS, HRAS, and NRAS were analysed with Sanger sequencing method, and 1p/19q codeletion was analysed with multiplex ligation-dependent probe amplification. DNA methylation array was performed in some RAS mutant tumours to improve accuracy of diagnosis. RESULTS: RAS mutations were identified in four gliomas with three KRAS mutations and one NRAS mutation in one anaplastic oligodendroglioma, two anaplastic astrocytomas (IDH wild-type in each), and one ganglioglioma. RAS-mutant gliomas were identified with various types of glioma histology. CONCLUSION: RAS mutation appears infrequent, and it is not associated with any specific histological phenotype of glioma.

[Risk Factors for Early Progression of Carotid Stenosis after Cervical Radiation Therapy].

OBJECTIVE: Carotid stenosis may occur as a late complication following cervical radiation therapy(RT);however, it may also progress in the early post-RT period. This study aimed to characterize the clinical features associated with the early progression of post-RT carotid stenosis. METHODS: We retrospectively reviewed clinical records of 30 patients who had undergone unilateral or bilateral cervical RT between January 2010 and November 2014. We analyzed the pre- and post-RT stenosis of their carotid arteries using contrast-enhanced computed tomography images. The arteries were classified as progressive or non-progressive according to the presence or absence of stenosis progression within five years after RT. Using univariate and multivariate analyses, we evaluated the following potential clinical risk factors:age;gender;history of hypertension, hyperlipidemia, diabetes mellitus, coronary artery disease, or smoking habit;antiplatelet or statin use;radiation dose;and prior presence of carotid stenosis before RT. RESULTS: In total, we reviewed 57 irradiated carotid arteries and identified 9 with early post-RT progression. Carotid stenosis before RT was observed in 88.9% of arteries in the progressive group but only 2% of arteries in the non-progressive group and it predicted progression(univariate and multiple logistic regression analyses, p<0.0001). No other clinical characteristics had a significant association with the progression of carotid stenosis. CONCLUSION: Prior presence of carotid stenosis may be a risk factor for its early progression after RT. Pre-RT screening of cervical arteries may be useful, and strict management of carotid stenosis is critical in patients with cervical radiation therapy.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

[Therapeutic Effects of Low-Dose Bevacizumab for the Treatment of Recurrent Brain Metastases].

OBJECTIVE: Molecularly targeted therapy has been adopted to treat a number of cancers. Bevacizumab, a recombinant humanized monoclonal antibody against vascular endothelial growth factor, is a representative agent used in molecularly targeted therapeutic regimens. However, the therapeutic effect of bevacizumab for the treatment of brain metastases remains unknown. We report the clinical effects of low dose bevacizumab(≤2.5mg/kg/week)to treat recurrent brain metastases. METHODS: We retrospectively analyzed patients with brain metastases who had been treated with bevacizumab between 2012 and 2016 at our institution. We identified clinical characteristics, including age, gender, primary tumor site, dose of bevacizumab, therapeutic and adverse effects, and magnetic resonance imaging results. The lesions were assessed with the RECIST criteria based on gadolinium-enhanced T1-weighted, T2-weighted, and FLAIR images. Statistical analysis was performed using t-test and Fisher's exact test. RESULTS: The cohort comprised 26 patients(8 men, 18 women)with a median age of 61 years(range 39-82 years). There were no significant clinical differences between the low dose and non-low dose groups. Patients in the low dose group did not report any adverse effects from bevacizumab. Three patients with brain metastases from colon cancer are illustrated to report the clinical course of low dose bevacizumab. CONCLUSION: Low dose bevacizumab may be a safe and effective therapeutic option to treat recurrent brain metastases from bevacizumab-sensitive cancers.

Cdk5 and its substrates, Dcx and p27kip1, regulate cytoplasmic dilation formation and nuclear elongation in migrating neurons.

Neuronal migration is crucial for development of the mammalian-specific six-layered cerebral cortex. Migrating neurons are known to exhibit distinct features; they form a cytoplasmic dilation, a structure specific to migrating neurons, at the proximal region of the leading process, followed by nuclear elongation and forward movement. However, the molecular mechanisms of dilation formation and nuclear elongation remain unclear. Using ex vivo chemical inhibitor experiments, we show here that rottlerin, which is widely used as a specific inhibitor for PKCδ, suppresses the formation of a cytoplasmic dilation and nuclear elongation in cortical migrating neurons. Although our previous study showed that cortical neuronal migration depends on Jnk, another downstream target of rottlerin, Jnk inhibition disturbs only the nuclear elongation and forward movement, but not the dilation formation. We found that an unconventional cyclin-dependent kinase, Cdk5, is a novel downstream target of rottlerin, and that pharmacological or knockdown-mediated inhibition of Cdk5 suppresses both the dilation formation and nuclear elongation. We also show that Cdk5 inhibition perturbs endocytic trafficking as well as microtubule organization, both of which have been shown to be required for dilation formation. Furthermore, knockdown of Dcx, a Cdk5 substrate involved in microtubule organization and membrane trafficking, or p27(kip1), another Cdk5 substrate involved in actin and microtubule organization, disturbs the dilation formation and nuclear elongation. These data suggest that Cdk5 and its substrates, Dcx and p27(kip1), characterize migrating neuron-specific features, cytoplasmic dilation formation and nuclear elongation in the mouse cerebral cortex, possibly through the regulation of microtubule organization and an endocytic pathway.

Extra-cell cycle regulatory functions of cyclin-dependent kinases (CDK) and CDK inhibitor proteins contribute to brain development and neurological disorders.

In developing brains, neural progenitors exhibit cell cycle-dependent nuclear movement within the ventricular zone [interkinetic nuclear migration (INM)] and actively proliferate to produce daughter progenitors and/or neurons, whereas newly generated neurons exit from the cell cycle and begin pial surface-directed migration and maturation. Dysregulation of the balance between the proliferation and the cell cycle exit in neural progenitors is one of the major causes of microcephaly (small brain). Recent studies indicate that cell cycle machinery influences not only the proliferation but also INM in neural progenitors. Furthermore, several cell cycle-related proteins, including p27(kip1) , p57(kip2) , Cdk5, and Rb, regulate the migration of neurons in the postmitotic state, suggesting that the growth arrest confers dual functions on cell cycle regulators. Consistently, several types of microcephaly occur in conjunction with neuronal migration disorders, such as periventricular heterotopia and lissencephaly. However, cell cycle re-entry by disturbance of growth arrest in mature neurons is thought to trigger neuronal cell death in Alzheimer's disease. In this review, we introduce the cell cycle protein-mediated regulation of two types of nuclear movement, INM and neuronal migration, during cerebral cortical development, and discuss the roles of growth arrest in cortical development and neurological disorders.

Cdk5 regulates multiple cellular events in neural development, function and disease.

Cyclin-dependent kinases (CDKs) generally regulate cell proliferation in dividing cells, including neural progenitors. In contrast, an unconventional CDK, Cdk5, is predominantly activated in post-mitotic cells, and involved in various cellular events, such as microtubule and actin cytoskeletal organization, cell-cell and cell-extracellular matrix adhesions, and membrane trafficking. Interestingly, recent studies have indicated that Cdk5 is associated with several cell cycle-related proteins, Cyclin-E and p27(kip1) . Taking advantage of multiple functionality, Cdk5 plays important roles in neuronal migration, layer formation, axon elongation and dendrite arborization in many regions of the developing brain, including cerebral cortex and cerebellum. Cdk5 is also required for neurogenesis at least in the cerebral cortex. Furthermore, Cdk5 is reported to control neurotransmitter release at presynaptic sites, endocytosis of the NMDA receptor at postsynaptic sites and dendritic spine remodeling, and thereby regulate synaptic plasticity and memory formation and extinction. In addition to these physiological roles in brain development and function, Cdk5 is associated with many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. In this review, I will introduce the physiological and pathological roles of Cdk5 in mammalian brains from the viewpoint of not only in vivo phenotypes but also its molecular and cellular functions.

Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons.

Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.

Exchangeable leaders of collectively migrating glioma abuse N-cadherin trafficking.

Collectively migrating cells consist of leaders and followers with different features. In this issue, Kim et al. (https://doi.org/10.1083/jcb.202401057) characterize the leader and follower cells in collective glioma migration and uncover important roles of YAP1/TAZ-mediated regulation of N-cadherin in the leader cells.

Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex.

Primary neuronal culture is a valuable in vitro model for analyzing the molecular mechanisms underlying the development and function of neural circuits. In contrast to neurons in vivo, primary cultured neurons can easily be transfected with genes of interest or treated with chemicals such as agonists and inhibitors of a specific target molecule. Furthermore, time-dependent morphological changes, such as the acquisition of neuronal polarity, axon elongation, and dendrite branch formation, can be analyzed by using primary neuronal cultures. Here, we describe a method for preparing a primary culture of neurons from the developing cerebral cortex, together with a method for gene transfer to primary cultured cortical neurons.

Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration.

p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.

Feto-maternal cholesterol transport regulated by ß-Klotho-FGF15 axis is essential for fetal growth.

ß-Klotho (ß-KL) is indispensable to regulate lipid, glucose, and energy metabolism in adult animals. ß-KL is highly expressed in the yolk sac, but its role in the developmental stages has not been established. We hypothesized that ß-KL is required for metabolic regulation in the embryo and aimed to clarify the role of ß-KL during development. Here, we show that ß-KL regulates feto-maternal cholesterol transport through the yolk sac by mediating FGF 15 signaling, and also that impairment of the ß-KL-FGF15 axis causes fetal growth restriction (FGR). Embryos of ß- kl knockout (ß-kl-/-) mice were morphologically normal but exhibited FGR before placental maturation. The body weight of ß-kl-/- mice remained lower after birth. ß-KL deletion reduced cholesterol supply from the maternal blood and led to lipid shortage in the embryos. These phenotypes were similar to those of embryos lacking FGF15, indicating that ß-KL-FGF15 axis is essential for growth and lipid regulation in the embryonic stages. Our findings suggest that lipid abnormalities in early gestation provoke FGR, leading to reduced body size in later life.

RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.

RAB35 is a multifunctional small GTPase that regulates endocytic recycling, cytoskeletal rearrangement, and cytokinesis. However, its physiological functions in mammalian development remain unclear. Here, we generated Rab35-knockout mice and found that RAB35 is essential for early embryogenesis. Interestingly, brain-specific Rab35-knockout mice displayed severe defects in hippocampal lamination owing to impaired distribution of pyramidal neurons, although defects in cerebral cortex formation were not evident. In addition, Rab35-knockout mice exhibited defects in spatial memory and anxiety-related behaviors. Quantitative proteomics indicated that the loss of RAB35 significantly affected the levels of other RAB proteins associated with endocytic trafficking, as well as some neural cell adhesion molecules, such as contactin-2. Collectively, our findings revealed that RAB35 is required for precise neuronal distribution in the developing hippocampus by regulating the expression of cell adhesion molecules, thereby influencing spatial memory.

The outermost region of the developing cortical plate is crucial for both the switch of the radial migration mode and the Dab1-dependent "inside-out" lamination in the neocortex.

Mammalian neocortex has a laminated structure that develops in a birth-date-dependent "inside-out" pattern. This layered structure is established by neuronal migration with sequential changes of the migratory mode regulated by several signaling cascades, including the Reelin-Disabled homolog 1 (Dab1) pathway. Although the importance of "locomotion," the major migratory mode, has been well established, the physiological significance of the mode change from locomotion to "terminal translocation," the final migratory mode, is unknown. In this study, we found that the outermost region of the mouse cortical plate has several histologically distinct features and named this region the primitive cortical zone (PCZ). Time-lapse analyses revealed that "locomoting" neurons paused transiently just beneath the PCZ before migrating into it by "terminal translocation." Furthermore, whereas Dab1-knockdown (KD) neurons could reach beneath the PCZ, they failed to enter the PCZ, suggesting that the Dab1-dependent terminal translocation is necessary for entry of the neurons into the PCZ. Importantly, sequential in utero electroporation experiments directly revealed that failure of the Dab1-dependent terminal translocation resulted in disruption of the inside-out alignment within the PCZ and that this disrupted pattern was still preserved in the mature cortex. Conversely, Dab1-KD locomoting neurons could pass by both wild-type and Dab1-KD predecessors beneath the PCZ. Our data indicate that the PCZ is a unique environment, passage of neurons through which involves molecularly and behaviorally different migratory mechanisms, and that the migratory mode change from locomotion to terminal translocation just beneath the PCZ is critical for the Dab1-dependent inside-out lamination in the mature cortex.

Cell adhesion and its endocytic regulation in cell migration during neural development and cancer metastasis.

Cell migration is a crucial event for tissue organization during development, and its dysregulation leads to several diseases, including cancer. Cells exhibit various types of migration, such as single mesenchymal or amoeboid migration, collective migration and scaffold cell-dependent migration. The migration properties are partly dictated by cell adhesion and its endocytic regulation. While an epithelial-mesenchymal transition (EMT)-mediated mesenchymal cell migration requires the endocytic recycling of integrin-mediated adhesions after the disruption of cell-cell adhesions, an amoeboid migration is not dependent on any adhesions to extracellular matrix (ECM) or neighboring cells. In contrast, a collective migration is mediated by both cell-cell and cell-ECM adhesions, and a scaffold cell-dependent migration is regulated by the endocytosis and recycling of cell-cell adhesion molecules. Although some invasive carcinoma cells exhibit an EMT-mediated mesenchymal or amoeboid migration, other cancer cells are known to maintain cadherin-based cell-cell adhesions and epithelial morphology during metastasis. On the other hand, a scaffold cell-dependent migration is mainly utilized by migrating neurons in normal developing brains. This review will summarize the structures of cell adhesions, including adherens junctions and focal adhesions, and discuss the regulatory mechanisms for the dynamic behavior of cell adhesions by endocytic pathways in cell migration in physiological and pathological conditions, focusing particularly on neural development and cancer metastasis.

NRF2 pathway activation attenuates ageing-related renal phenotypes due to α-klotho deficiency.

Oxidative stress is one of the major causes of the age-related functional decline in cells and tissues. The KEAP1-NRF2 system plays a central role in the regulation of redox balance, and NRF2 activation exerts antiageing effects by controlling oxidative stress in aged tissues. α-Klotho was identified as an ageing suppressor protein based on the premature ageing phenotypes of its mutant mice, and its expression is known to gradually decrease during ageing. Because α-klotho has been shown to possess antioxidant function, ageing-related phenotypes of α-klotho mutant mice seem to be attributable to increased oxidative stress at least in part. To examine whether NRF2 activation antagonizes ageing-related phenotypes caused by α-klotho deficiency, we crossed α-klotho-deficient (Kl-/-) mice with a Keap1-knockdown background, in which the NRF2 pathway is constitutively activated in the whole body. NRF2 pathway activation in Kl-/- mice extended the lifespan and dramatically improved ageing-related renal phenotypes. With elevated expression of antioxidant genes accompanied by an oxidative stress decrease, the antioxidant effects of NRF2 seem to make a major contribution to the attenuation of ageing-related renal phenotypes of Kl-/- mice. Thus, NRF2 is expected to exert an antiageing function by partly compensating for the functional decline of α-Klotho during physiological ageing.

Diagnosis of spinal dural defect using three-dimensional fast steady-state MR in patient with superficial siderosis: A case report.

Background: Spinal dural defects can result in superficial siderosis (SS) of the central nervous system. Closure of the defect can stop or slow the progression of the disease. Here, we evaluated, whether preoperative three-dimensional fast steady-state acquisition MR could adequately detect these defects and, thus, facilitate their closure and resolution. Case Description: A 65-year-old right-handed male presented with a 33-year history of the left C8 root avulsion and a 3-year history of slowly progressive gait difficulties and hearing loss. The T2*-weighted imaging revealed symmetrical hemosiderin deposition throughout his central nervous system. A left C6-C7 dural defect involving only inner layer was identified using a three-dimensional MR (3D-FIESTA). It was treated through a left C6-7 hemilaminectomy and successfully sealed with adipose tissue and fibrin glue. Subsequently, the progression of cerebellar ataxia was halted, nevertheless the sensorineural hearing loss worsened even over the next 2 years. Conclusion: 3D-FIESTA reconstruction was approved to be useful tool for identifying the tiny hole of the inner dural layer responsible for SS.

Dissecting the factors involved in the locomotion mode of neuronal migration in the developing cerebral cortex.

Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cdelta (PKCdelta), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCdelta hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCdelta in the early and final phases of migration, respectively.