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.

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.

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.

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.

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.

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.

Alternative Functions of Cell Cycle-Related and DNA Repair Proteins in Post-mitotic Neurons.

Proper regulation of neuronal morphological changes is essential for neuronal migration, maturation, synapse formation, and high-order function. Many cytoplasmic proteins involved in the regulation of neuronal microtubules and the actin cytoskeleton have been identified. In addition, some nuclear proteins have alternative functions in neurons. While cell cycle-related proteins basically control the progression of the cell cycle in the nucleus, some of them have an extra-cell cycle-regulatory function (EXCERF), such as regulating cytoskeletal organization, after exit from the cell cycle. Our expression analyses showed that not only cell cycle regulators, including cyclin A1, cyclin D2, Cdk4/6, p21cip1, p27kip1, Ink4 family, and RAD21, but also DNA repair proteins, including BRCA2, p53, ATM, ATR, RAD17, MRE11, RAD9, and Hus1, were expressed after neurogenesis, suggesting that these proteins have alternative functions in post-mitotic neurons. In this perspective paper, we discuss the alternative functions of the nuclear proteins in neuronal development, focusing on possible cytoplasmic roles.

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.

Characterization and Stage-Dependent Lineage Analysis of Intermediate Progenitors of Cortical GABAergic Interneurons.

Intermediate progenitors of both excitatory and inhibitory neurons, which can replenish neurons in the adult brain, were recently identified. However, the generation of intermediate progenitors of GABAergic inhibitory neurons (IPGNs) has not been studied in detail. Here, we characterized the spatiotemporal distribution of IPGNs in mouse cerebral cortex. IPGNs generated neurons during both embryonic and postnatal stages, but the embryonic IPGNs were more proliferative. Our lineage tracing analyses showed that the embryonically proliferating IPGNs tended to localize to the superficial layers rather than the deep cortical layers at 3 weeks after birth. We also found that embryonic IPGNs derived from the medial and caudal ganglionic eminence (CGE) but more than half of the embryonic IPGNs were derived from the CGE and broadly distributed in the cerebral cortex. Taken together, our data indicate that the broadly located IPGNs during embryonic and postnatal stages exhibit a different proliferative property and layer distribution.

Prognostic stratification for IDH-wild-type lower-grade astrocytoma by Sanger sequencing and copy-number alteration analysis with MLPA.

The characteristics of IDH-wild-type lower-grade astrocytoma remain unclear. According to cIMPACT-NOW update 3, IDH-wild-type astrocytomas with any of the following factors show poor prognosis: combination of chromosome 7 gain and 10 loss (+ 7/- 10), and/or EGFR amplification, and/or TERT promoter (TERTp) mutation. Multiplex ligation-dependent probe amplification (MLPA) can detect copy number alterations at reasonable cost. The purpose of this study was to identify a precise, cost-effective method for stratifying the prognosis of IDH-wild-type astrocytoma. Sanger sequencing, MLPA, and quantitative methylation-specific PCR were performed for 42 IDH-wild-type lower-grade astrocytomas surgically treated at Kyoto University Hospital, and overall survival was analysed for 40 patients who underwent first surgery. Of the 42 IDH-wild-type astrocytomas, 21 were classified as grade 4 using cIMPACT-NOW update 3 criteria and all had either TERTp mutation or EGFR amplification. Kaplan-Meier analysis confirmed the prognostic significance of cIMPACT-NOW criteria, and World Health Organization grade was also prognostic. Cox regression hazard model identified independent significant prognostic indicators of PTEN loss (risk ratio, 9.75; p < 0.001) and PDGFRA amplification (risk ratio, 13.9; p = 0.002). The classification recommended by cIMPACT-NOW update 3 could be completed using Sanger sequencing and MLPA. Survival analysis revealed PTEN and PDGFRA were significant prognostic factors for IDH-wild-type lower-grade astrocytoma.

Simple method for large-scale production of macrophage activating factor GcMAF.

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

Two Patients Who Underwent Emergency Stenting for Iatrogenic Cervical Internal Carotid Artery Dissection during Thrombectomy.

Objective: Iatrogenic artery dissection during reperfusion therapy is one of the complications causing a poor prognosis. We report two cases of emergent stent placement for iatrogenic cervical carotid artery dissection during reperfusion therapy for acute ischemic stroke. Case Presentation: Two patients, a 77-year-old woman and a 77-year-old man, were diagnosed with acute major cerebral artery occlusion, and underwent reperfusion therapy. The iatrogenic internal carotid artery dissection was caused by derivation of a 6-Fr catheter and 0.014-inch wire in the tortuous cervical internal carotid artery, and emergent stent placement was performed. Recanalization was confirmed and no deterioration caused by the iatrogenic dissection was found. Conclusion: In patients in whom cerebral infarction is localized on MRI, additional stent placement may be effective for preventing adverse events caused by iatrogenic cervical internal carotid artery dissection during reperfusion therapy for intracranial cerebral artery occlusion related to atherosclerotic change.

A Survived Case of Acute Bilateral Internal Carotid Artery Occlusion Treated by Mechanical Thrombectomy.

Objective: We report a survived case of acute bilateral internal carotid artery occlusion successfully treated by mechanical thrombectomy. Case Presentation: The patient was an 82-year-old right-handed man. Sudden consciousness disturbance and aphasia appeared, and cranial magnetic resonance angiography (MRA) revealed bilateral internal carotid artery occlusion. Cerebral angiography demonstrated occlusion between the cervical and cavernous portions of the bilateral internal carotid artery, and the growth of collateral circulation to the areas where the bilateral internal carotid artery perfused. We performed mechanical thrombectomy first on the left side, which was the main cause of his symptoms and relatively wide ischemic penumbra, and achieved recanalization of the bilateral internal carotid artery and suppressed extensive enlargement of the infarction. Conclusion: Acute bilateral internal carotid artery occlusion requires an optimal treatment strategy based on the clinical symptoms and preoperative imaging.

Occipital Sinus Dural Arteriovenous Fistula Presenting with Cerebellar Hemorrhage.

BACKGROUND: Occipital sinus (OS) dural arteriovenous fistula (DAVF) is extremely rare, and we are aware of no case accompanied by cerebral hemorrhage. We present a case of OS DAVF presenting with cerebellar hemorrhage, treated successfully by transvenous embolization. CASE DESCRIPTION: A 62-year-old female presented with headache and nausea of recent onset. Computed tomography revealed left cerebellar hemorrhage with perihematomal edema. Angiography showed OS DAVF fed by bilateral occipital and posterior meningeal arteries, with drainage into the left inferior hemispheric vein and right transverse sinus receiving the shunt flow from OS. The caudal side of the OS was occluded. The inferior hemispheric vein was dilated with 2 varices, and the junction between the OS and right transverse sinus was narrowed. Because the OS was not involved in normal cerebellar drainage, transvenous embolization of the OS was performed. The microcatheter was advanced to the OS from the transverse sinus during balloon occlusion at the confluence of sinuses. Coils were placed in the OS from the caudal to cranial side, and complete occlusion of the shunt was obtained. CONCLUSIONS: This is the first report of OS DAVF presenting with cerebellar hemorrhage. Transvenous embolization of the affected OS appears ideal when transvenous access is feasible, and the OS is not involved in normal venous drainage of the cerebellum.

Breakage and Retrieval of an Aspiration Catheter Coil with a Stent Retriever During Mechanical Thrombectomy.

BACKGROUND: Some mechanical thrombectomy techniques for acute ischemic stroke use a combination of an aspiration catheter and stent retriever. We experienced a rare case of aspiration catheter coil breakage and subsequent retrieval using a stent retriever. CASE DESCRIPTION: A 79-year-old man suddenly developed somnolence, global aphasia, and right hemiplegia. Magnetic resonance imaging revealed acute infarction of the left frontal lobe and occlusion of the left common carotid artery. Thus, using an aspiration catheter and a stent retriever, mechanical thrombectomy was performed. The stent retriever was deployed from the middle cerebral artery to the internal carotid artery and retracted into the aspiration catheter placed in the internal carotid artery proximal to the thrombus. The catheter was bent during retraction of the stent retriever. After thrombus aspiration, the internal carotid and anterior and middle cerebral arteries were successfully reperfused; however, the stent retriever captured a broken section of the winding coil of the aspiration catheter. We suspected that an X-ray marker on the stent retriever broke the winding coil at the bent segment of the aspiration catheter and the stent captured the broken coil. CONCLUSIONS: The combined use of an aspiration catheter and a stent retriever may cause unexpected device breakage, especially when the catheter is bent.

Growth Arrest Triggers Extra-Cell Cycle Regulatory Function in Neurons: Possible Involvement of p27kip1 in Membrane Trafficking as Well as Cytoskeletal Regulation.

Cell cycle regulation is essential for the development of multicellular organisms, but many cells in adulthood, including neurons, exit from cell cycle. Although cell cycle-related proteins are suppressed after cell cycle exit in general, recent studies have revealed that growth arrest triggers extra-cell cycle regulatory function (EXCERF) in some cell cycle proteins, such as p27(kip1), p57(kip2), anaphase-promoting complex/cyclosome (APC/C), and cyclin E. While p27 is known to control G1 length and cell cycle exit via inhibition of cyclin-dependent kinase (CDK) activities, p27 acquires additional cytoplasmic functions in growth-arrested neurons. Here, we introduce the EXCERFs of p27 in post-mitotic neurons, mainly focusing on its actin and microtubule regulatory functions. We also show that a small amount of p27 is associated with the Golgi apparatus positive for Rab6, p115, and GM130, but not endosomes positive for Rab5, Rab7, Rab8, Rab11, SNX6, or LAMTOR1. p27 is also colocalized with Dcx, a microtubule-associated protein. Based on these results, we discuss here the possible role of p27 in membrane trafficking and microtubule-dependent transport in post-mitotic cortical neurons. Collectively, we propose that growth arrest leads to two different fates in cell cycle proteins; either suppressing their expression or activating their EXCERFs. The latter group of proteins, including p27, play various roles in neuronal migration, morphological changes and axonal transport, whereas the re-activation of the former group of proteins in post-mitotic neurons primes for cell death.

[Pharmacological approach to cerebral cortical development].

In the developing mammalian cerebral cortex, newly generated neurons migrate toward the pial surface to form a mammalian-specific six-layered cerebral cortex. Genetic studies of human neurological diseases have suggested the involvement of several molecules in cortical neuronal migration. In vivo electroporation is another powerful tool for understanding the molecular mechanisms of neuronal migration. By using these techniques, however, it is difficult to understand molecular basis of time-dependent changes of neuronal morphologies. Here, we introduce a pharmacological approach to cerebral cortical development. Major advantages of the pharmacological approach include the transient suppression of molecules of interest and analyzing time-dependent changes of neuronal morphologies. It also allows us to search molecules regulating neuronal migration with comparative ease. We propose the complementarity between the pharmacological approach and genetics or in vivo electroporation experiments.