Impacts of methotrexate on survival, dendrite development, and synapse formation of cortical neurons.

Methotrexate (MTX) is an anti-metabolite that has been used for the treatment of patients of acute lymphocytic leukemia or non-Hodgikin lymphoma for decades. In some cases, MTX-treated patients suffer from neurological side effects, including seizures and cognitive dysfunctions. While most patients are at developmental stages, information of the mechanisms of the side effects of MTX treatment on the developing neurons has been limited. Neurons develop in five steps in the human brain: neurogenesis, polarity formation, dendrite and axon development, synapse formation, and neuronal death. Except for neurogenesis, these processes can be recapitulated in the primary culture system of cortical neurons. Using primary cultured cortical neurons, we studied the impact of MTX treatment on dendrite development, synapse formation, and neuronal death in the present report. MTX treatment impaired neuronal survival, dendrite development, and synapse formation. Interestingly, half maximal effective concentrations (EC50 s) of MTX for all three processes are at the similar range and lower than the MTX concentration in the cerebrospinal fluid in treated patients. Our results provide possible mechanisms of neurological side effects in treated patients.

Correction: Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

[This corrects the article DOI: 10.1371/journal.pbio.2002457.].

Super-resolved 3D-STED microscopy identifies a layer-specific increase in excitatory synapses in the hippocampal CA1 region of Neuroligin-3 KO mice.

The chemical synapse is one type of cell-adhesion system that transmits information from a neuron to another neuron in the complex neuronal network in the brain. Synaptic transmission is the rate-limiting step during the information processing in the neuronal network and its plasticity is involved in cognitive functions. Thus, morphological and electrophysiological analyses of synapses are of particular importance in neuroscience research. In the current study, we applied super-resolved three-dimensional stimulated emission depletion (3D-STED) microscopy for the morphological analyses of synapses. This approach allowed us to estimate the precise number of excitatory and inhibitory synapses in the mouse hippocampal tissue. We discovered a region-specific increase in excitatory synapses in a model mouse of autism spectrum disorder, Neuroligin-3 KO, with this method. This type of analysis will open a new field in developmental neuroscience in the future.

Drebrin expression patterns in patients with refractory temporal lobe epilepsy and hippocampal sclerosis.

OBJECTIVE: Drebrins are crucial for synaptic function and dendritic spine development, remodeling, and maintenance. In temporal lobe epilepsy (TLE) patients, a significant hippocampal synaptic reorganization occurs, and synaptic reorganization has been associated with hippocampal hyperexcitability. This study aimed to evaluate, in TLE patients, the hippocampal expression of drebrin using immunohistochemistry with DAS2 or M2F6 antibodies that recognize adult (drebrin A) or adult and embryonic (pan-drebrin) isoforms, respectively. METHODS: Hippocampal sections from drug-resistant TLE patients with hippocampal sclerosis (HS; TLE, n = 33), of whom 31 presented with type 1 HS and two with type 2 HS, and autopsy control cases (n = 20) were assayed by immunohistochemistry and evaluated for neuron density, and drebrin A and pan-drebrin expression. Double-labeling immunofluorescences were performed to localize drebrin A-positive spines in dendrites (MAP2), and to evaluate whether drebrin colocalizes with inhibitory (GAD65) and excitatory (VGlut1) presynaptic markers. RESULTS: Compared to controls, TLE patients had increased pan-drebrin in all hippocampal subfields and increased drebrin A-immunopositive area in all hippocampal subfields but CA1. Drebrin-positive spine density followed the same pattern as total drebrin quantification. Confocal microscopy indicated juxtaposition of drebrin-positive spines with VGlut1-positive puncta, but not with GAD65-positive puncta. Drebrin expression in the dentate gyrus of TLE cases was associated negatively with seizure frequency and positively with verbal memory. TLE patients with lower drebrin-immunopositive area in inner molecular layer (IML) than in outer molecular layer (OML) had a lower seizure frequency than those with higher or comparable drebrin-immunopositive area in IML compared with OML. SIGNIFICANCE: Our results suggest that changes in drebrin-positive spines and drebrin expression in the dentate gyrus of TLE patients are associated with lower seizure frequency, more preserved verbal memory, and a better postsurgical outcome.

Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain.

Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function.

The role of drebrin in dendritic spines.

Dendritic spines form typical excitatory synapses in the brain and their shapes vary depending on synaptic inputs. It has been suggested that the morphological changes of dendritic spines play an important role in synaptic plasticity. Dendritic spines contain a high concentration of actin, which has a central role in supporting cell motility, and polymerization of actin filaments (F-actin) is most likely involved in spine shape changes. Drebrin is an actin-binding protein that forms stable F-actin and is highly accumulated within dendritic spines. Drebrin has two isoforms, embryonic-type drebrin E and adult-type drebrin A, that change during development from E to A. Inhibition of drebrin A expression results in a delay of synapse formation and inhibition of postsynaptic protein accumulation, suggesting that drebrin A has an important role in spine maturation. In mature synapses, glutamate stimulation induces rapid spine-head enlargement during long-term potentiation (LTP) formation. LTP stimulation induces Ca2+ entry through N-methyl-d-aspartate (NMDA) receptors, which causes drebrin exodus from dendritic spines. Once drebrin exits from dendritic spine heads, the dynamic actin pool increases in spine heads to facilitate F-actin polymerization. To maintain enlarged spine heads, drebrin-decorated F-actin is thought to reform within the spine heads. Thus, drebrin plays a pivotal role in spine plasticity through regulation of F-actin.

The role of drebrin in neurons.

Drebrin is an actin-binding protein that changes the helical pitch of actin filaments (F-actin), and drebrin-decorated F-actin shows slow treadmilling and decreased rate of depolymerization. Moreover, the characteristic morphology of drebrin-decorated F-actin enables it to respond differently to the same signals from other actin cytoskeletons. Drebrin consists of two major isoforms, drebrin E and drebrin A. In the developing brain, drebrin E appears in migrating neurons and accumulates in the growth cones of axons and dendrites. Drebrin E-decorated F-actin links lamellipodium F-actin to microtubules in the growth cones. Then drebrin A appears at nascent synapses and drebrin A-decorated F-actin facilitates postsynaptic molecular assembly. In the adult brain, drebrin A-decorated F-actin is concentrated in the central region of dendritic spines. During long-term potentiation initiation, NMDA receptor-mediated Ca2+ influx induces the transient exodus of drebrin A-decorated F-actin via myosin II ATPase activation. Because of the unique physical characteristics of drebrin A-decorated F-actin, this exodus likely contributes to the facilitation of F-actin polymerization and spine enlargement. Additionally, drebrin reaccumulation in dendritic spines is observed after the exodus. In our drebrin exodus model of structure-based synaptic plasticity, reestablishment of drebrin A-decorated F-actin is necessary to keep the enlarged spine size during long-term potentiation maintenance. In this review, we introduce the genetic and biochemical properties of drebrin and the roles of drebrin in early stage of brain development, synaptic formation and synaptic plasticity. Further, we discuss the pathological relevance of drebrin loss in Alzheimer's disease. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

Drebrin in Neuronal Migration and Axonal Growth.

During development, production of neurons from neural stem cells, migration of neurons from their birthplace to their final location, and extension of neurites, axons, and dendrites are important for the formation of functional neuronal circuits. The actin cytoskeleton has major roles in the morphological development of neurons. In this chapter, we focused on the distribution and function of the actin-binding protein, drebrin, to elucidate the importance of drebrin-bound F-actin in neurons during early developmental stages of neurons in embryonic, postnatal, and adult brains. There are three major isoforms of drebrin in the chicken brain (E1, E2, and A) and two major isoforms in the mammalian brain (E and A). Among these drebrin isoforms, drebrin E1 and E2 in chicken and drebrin E in the mammalian brain are involved in these neuronal stages. In migrating neurons of the developing and adult brain, drebrin is localized at the base of filopodia of leading processes, to regulate neuronal migration. In axonal growth cones, drebrin is localized in the transitional zone to regulate axonal growth by inhibiting actomyosin interactions and mediating the interactions between F-actin and microtubules. For axonal collateral branching, drebrin is localized at axonal actin patches and the base of filopodia, to accelerate the transition from actin patches to filopodia and stabilize the filopodia.

Drebrin in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder accompanied by severe progressive memory and cognitive impairment. The brain of AD patients has an abundance of two abnormal structures, amyloid plaques (senile plaques) and neurofibrillary tangles. In addition, drebrin loss is another hallmark of AD brains, which is a common feature in the brain of both AD patients and AD mouse models. Strong evidence from human genetics and transgenic mouse models has indicated that amyloid ß (Aß) is part of the etiology and pathogenesis of AD. Recently, it has become clear that synaptic dysfunction, including reduced synaptic transmission and loss of dendritic spines, occurs prior to the formation of amyloid plaques and neuronal cell loss. Furthermore, immunohistochemistry using postmortem human brains and AD mouse models has shown that drebrin loss in postsynaptic sites occurs earlier than the presynaptic change in AD brains. In addition, dysregulation of glutamate receptor trafficking and the p21-activated kinase/LIM kinase pathway has been observed in AD brains. It is now believed that soluble Aß oligomers, namely, Aß-derived diffusible ligands (ADDLs), but not insoluble Aß aggregation mediates Aß toxicity. ADDLs bind to the postsynaptic site and induce the aberrant morphology and density of dendritic spines. Consistent with the AD mouse models, the surface expression of glutamate receptors decreases after ADDL exposure. Importantly, the ADDL-induced drebrin loss in dendritic spines occurs prior to aberrations in dendritic spine morphology and density. These observations indicate that drebrin loss in dendritic spines occurs at the prodromal stage of AD, before the density and morphology of dendritic spines change. Quantitation of drebrin may be a possible tool for diagnosing the prodromal stage of AD, before dementia development in AD.

Localization of Drebrin: Light Microscopy Study.

Developmental changes in the expression and localization of drebrin has been mainly analyzed in chick embryo and young rat by various anti-drebrin polyclonal and monoclonal antibodies. Immunoblot analysis demonstrated that the adult drebrin isoform (drebrin A) is restricted to neural tissues, while the embryonic drebrin isoforms (drebrin E1 and E2 in chicken and drebrin E in mammals) are found in a wide variety of tissues. In the developing brain, drebrin E (including chicken drebrin E2) is expressed in newly generated neurons. During neuronal migration, drebrin E is distributed ubiquitously within the neurons. Once drebrin A is expressed in the developing neuron, drebrin E is no longer present within the cell soma and accumulates in the growth cone of growing processes, resulting in the cessation of neuronal migration. The limited subcellular localization of drebrin A, which is possibly regulated by a drebrin A-specific mechanism, is likely to affect the localization of drebrin E. In the adult brain, drebrin is mainly localized in dendritic spines, but in some nuclei, drebrin can be detected in neuronal somata as well as dendritic spines. The fact that the developmental changes in drebrin expression highly correlate in time with the sensitive period of visual cortical plasticity in kittens suggests that synaptic plasticity depends on drebrin.

Spikar, a novel drebrin-binding protein, regulates the formation and stabilization of dendritic spines.

Dendritic spines are small, actin-rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin-binding protein that is thought to initiate spine formation through a unique drebrin-actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin-binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co-activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA-interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin-mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.

Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

The murine ortholog of Kaufman oculocerebrofacial syndrome gene Ube3b is crucial for the maintenance of the excitatory synapses in the young adult stage.

Kaufman oculocerebrofacial syndrome (KOS) is an autosomal recessive developmental disorder. Inactivating mutations in UBE3B, an E3 ubiquitin ligase gene are causative for KOS. We have reported that towards postnatal week three, its murine ortholog, Ube3b, acts as a negative regulator of the number of dendritic spines. In this study, we investigated the role of Ube3b at the synapse in the young adult mice. With an improved estimation method, images from the hippocampal CA1 and CA2 regions acquired with 3D Stimulated Emission Depletion (3D-STED) microscopy were used to quantify the excitatory synapse numbers. In the young adult mice, the excitatory synapse density was decreased in brain-specific Ube3b conditional knockout mice as compared to the control. Our results indicate the novel role of Ube3b in the maintenance of synapse numbers in the young adult period.

Phldb2 is essential for regulating hippocampal dendritic spine morphology through drebrin in an adult-type isoform-specific manner.

Morphologically dynamic dendritic spines are the major sites of neuronal plasticity in the brain; however, the molecular mechanisms underlying their morphological dynamics have not been fully elucidated. Phldb2 is a protein that contains two predicted coiled-coil domains and the pleckstrin homology domain, whose binding is highly sensitive to PIP3. We have previously demonstrated that Phldb2 regulates synaptic plasticity, glutamate receptor trafficking, and PSD-95 turnover. Drebrin is one of the most abundant neuron-specific F-actin-binding proteins that are pivotal for synaptic morphology and plasticity. We observed that Phldb2 bound to drebrin A (adult-type drebrin), but not to drebrin E (embryonic-type drebrin). In the absence of Phldb2, the subcellular localization of drebrin A in the hippocampal spines and its distribution in the hippocampus were altered. Immature spines, such as the filopodium type, increased relatively in the CA1 regions of the hippocampus, whereas mushroom spines, a typical mature type, decreased in Phldb2-/- mice. Phldb2 suppressed the formation of an abnormal filopodium structure induced by drebrin A overexpression. Taken together, these findings demonstrate that Phldb2 is pivotal for dendritic spine morphology and possibly for synaptic plasticity in mature animals by regulating drebrin A localization.

Synthetic torpor protects rats from exposure to accelerated heavy ions.

Hibernation or torpor is considered a possible tool to protect astronauts from the deleterious effects of space radiation that contains high-energy heavy ions. We induced synthetic torpor in rats by injecting adenosine 5'-monophosphate monohydrate (5'-AMP) i.p. and maintaining in low ambient temperature room (+ 16 °C) for 6 h immediately after total body irradiation (TBI) with accelerated carbon ions (C-ions). The 5'-AMP treatment in combination with low ambient temperature reduced skin temperature and increased survival following 8 Gy C-ion irradiation compared to saline-injected animals. Analysis of the histology of the brain, liver and lungs showed that 5'-AMP treatment following 2 Gy TBI reduced activated microglia, Iba1 positive cells in the brain, apoptotic cells in the liver, and damage to the lungs, suggesting that synthetic torpor spares tissues from energetic ion radiation. The application of 5'-AMP in combination with either hypoxia or low temperature environment for six hours following irradiation of rat retinal pigment epithelial cells delays DNA repair and suppresses the radiation-induced mitotic catastrophe compared to control cells. We conclude that synthetic torpor protects animals from cosmic ray-simulated radiation and the mechanism involves both hypothermia and hypoxia.

Evidence for cell density affecting C2C12 myogenesis: possible regulation of myogenesis by cell-cell communication.

INTRODUCTION: Community effect is a phenomenon caused by cell-cell communication during myogenesis. In myogenic C2C12 cells in vitro, the confluent phase is needed for myogenesis induction. METHODS: To examine the cell-density effect, growth kinetics and myogenic differentiation were investigated in cells plated at four different cell densities. RESULTS: We found that expression of a myogenic differentiation marker was high in a density-dependent manner. At high density, where cell-cell contact was obvious, contact inhibition after the proliferation stage was accompanied by microarray findings demonstrating upregulation of negative regulating cell-cycle markers, including CDKI p21 and the muscle differentiation markers MyoD and myogenin. Interestingly, developmentally regulated protein expression (drebrin) protein expression was also upregulated in a density-dependent manner. CONCLUSIONS: These results suggest that contact inhibition after the proliferation stage may induce growth arrest via cell-cell communication through the expression of CDKI p21 and may be responsible for progressing cell fusion.

Hibernation as a Tool for Radiation Protection in Space Exploration.

With new and advanced technology, human exploration has reached outside of the Earth's boundaries. There are plans for reaching Mars and the satellites of Jupiter and Saturn, and even to build a permanent base on the Moon. However, human beings have evolved on Earth with levels of gravity and radiation that are very different from those that we have to face in space. These issues seem to pose a significant limitation on exploration. Although there are plausible solutions for problems related to the lack of gravity, it is still unclear how to address the radiation problem. Several solutions have been proposed, such as passive or active shielding or the use of specific drugs that could reduce the effects of radiation. Recently, a method that reproduces a mechanism similar to hibernation or torpor, known as synthetic torpor, has started to become possible. Several studies show that hibernators are resistant to acute high-dose-rate radiation exposure. However, the underlying mechanism of how this occurs remains unclear, and further investigation is needed. Whether synthetic hibernation will also protect from the deleterious effects of chronic low-dose-rate radiation exposure is currently unknown. Hibernators can modulate their neuronal firing, adjust their cardiovascular function, regulate their body temperature, preserve their muscles during prolonged inactivity, regulate their immune system, and most importantly, increase their radioresistance during the inactive period. According to recent studies, synthetic hibernation, just like natural hibernation, could mitigate radiation-induced toxicity. In this review, we see what artificial hibernation is and how it could help the next generation of astronauts in future interplanetary missions.

Soy Isoflavones Accelerate Glial Cell Migration via GPER-Mediated Signal Transduction Pathway.

Soybean isoflavones, such as genistein, daidzein, and its metabolite, S-equol, are widely known as phytoestrogens. Their biological actions are thought to be exerted via the estrogen signal transduction pathway. Estrogens, such as 17ß-estradiol (E2), play a crucial role in the development and functional maintenance of the central nervous system. E2 bind to the nuclear estrogen receptor (ER) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. In addition to binding to nuclear ER, E2 also binds to the G-protein-coupled estrogen receptor (GPER) and activates the nongenomic estrogen signaling pathway. Soybean isoflavones also bind to the ER and GPER. However, the effect of soybean isoflavone on brain development, particularly glial cell function, remains unclear. We examined the effects of soybean isoflavones using an astrocyte-enriched culture and astrocyte-derived C6 clonal cells. Isoflavones increased glial cell migration. This augmentation was suppressed by co-exposure with G15, a selective GPER antagonist, or knockdown of GPER expression using RNA interference. Isoflavones also activated actin cytoskeleton arrangement via increased actin polymerization and cortical actin, resulting in an increased number and length of filopodia. Isoflavones exposure increased the phosphorylation levels of FAK (Tyr397 and Tyr576/577), ERK1/2 (Thr202/Tyr204), Akt (Ser473), and Rac1/cdc42 (Ser71), and the expression levels of cortactin, paxillin and ERα. These effects were suppressed by knockdown of the GPER. Co-exposure of isoflavones to the selective RhoA inhibitor, rhosin, selective Cdc42 inhibitor, casin, or Rac1/Cdc42 inhibitor, ML-141, decreased the effects of isoflavones on cell migration. These findings indicate that soybean isoflavones exert their action via the GPER to activate the PI3K/FAK/Akt/RhoA/Rac1/Cdc42 signaling pathway, resulting in increased glial cell migration. Furthermore, in silico molecular docking studies to examine the binding mode of isoflavones to the GPER revealed the possibility that isoflavones bind directly to the GPER at the same position as E2, further confirming that the effects of the isoflavones are at least in part exerted via the GPER signal transduction pathway. The findings of the present study indicate that isoflavones may be an effective supplement to promote astrocyte migration in developing and/or injured adult brains.

PKN1 promotes synapse maturation by inhibiting mGluR-dependent silencing through neuronal glutamate transporter activation.

Abnormal metabotropic glutamate receptor (mGluR) activity could cause brain disorders; however, its regulation has not yet been fully understood. Here, we report that protein kinase N1 (PKN1), a protein kinase expressed predominantly in neurons in the brain, normalizes group 1 mGluR function by upregulating a neuronal glutamate transporter, excitatory amino acid transporter 3 (EAAT3), and supports silent synapse activation. Knocking out PKN1a, the dominant PKN1 subtype in the brain, unmasked abnormal input-nonspecific mGluR-dependent long-term depression (mGluR-LTD) and AMPA receptor (AMPAR) silencing in the developing hippocampus. mGluR-LTD was mimicked by inhibiting glutamate transporters in wild-type mice. Knocking out PKN1a decreased hippocampal EAAT3 expression and PKN1 inhibition reduced glutamate uptake through EAAT3. Also, synaptic transmission was immature; there were more silent synapses and fewer spines with shorter postsynaptic densities in PKN1a knockout mice than in wild-type mice. Thus, PKN1 plays a critical role in regulation of synaptic maturation by upregulating EAAT3 expression.

Drebrin E is involved in the regulation of axonal growth through actin-myosin interactions.

Drebrin is a well-known side-binding protein of F-actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F-actins and microtubules overlapped. Over-expression of drebrin E caused drebrin E and F-actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F-actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over-expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin-myosin interactions in the transitional zone of axonal growth cones.