Neurite Outgrowth In Hippocampal Neurons Research Articles

Hericerin derivatives activates a pan-neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory.

The traditional medicinal mushroom Hericium erinaceus is known for enhancing peripheral nerve regeneration through targeting nerve growth factor (NGF) neurotrophic activity. Here, we purified and identified biologically new active compounds from H. erinaceus, based on their ability to promote neurite outgrowth in hippocampal neurons. N-de phenylethyl isohericerin (NDPIH), an isoindoline compound from this mushroom, together with its hydrophobic derivative hericene A, were highly potent in promoting extensive axon outgrowth and neurite branching in cultured hippocampal neurons even in the absence of serum, demonstrating potent neurotrophic activity. Pharmacological inhibition of tropomyosin receptor kinase B (TrkB) by ANA-12 only partly prevented the NDPIH-induced neurotrophic activity, suggesting a potential link with BDNF signaling. However, we found that NDPIH activated ERK1/2 signaling in the absence of TrkB in HEK-293T cells, an effect that was not sensitive to ANA-12 in the presence of TrkB. Our results demonstrate that NDPIH acts via a complementary neurotrophic pathway independent of TrkB with converging downstream ERK1/2 activation. Mice fed with H. erinaceus crude extract and hericene A also exhibited increased neurotrophin expression and downstream signaling, resulting in significantly enhanced hippocampal memory. Hericene A therefore acts through a novel pan-neurotrophic signaling pathway, leading to improved cognitive performance.

Rab3A interacts with spastin to regulate neurite outgrowth in hippocampal neurons

Investigating novel mechanisms of neurite outgrowth via cytoskeleton is critical for developing therapeutic strategies against neural disorders. Rab3A is a vesicle-related protein distributed throughout the nervous system, but the detailed mechanism related to cytoskeleton remains largely unknown. Our previous reports show that spastin serves microtubule to regulate neurite outgrowth. Here, we asked whether Rab3A could function via modulating spastin during neuronal development. The results revealed that Rab3A colocalized with spastin in cultured hippocampal neurons. Immunoprecipitation assays showed that Rab3A physically interacted with spastin in rat brain lysates. Rab3A overexpression significantly induced spastin degradation; this effect was reversed by leupeptin- or MG-132- administration, suggesting the lysosomal and ubiquitin-mediated degradation system. Immunofluorescence staining further confirmed that Rab3A and spastin immune-colocalized with the lysosome marker lysotracker. In COS7 cells, Rab3A overexpression significantly downregulated spastin expression and abolished the spastin-mediated microtubule severing. Furthermore, overexpression inhibited while genetic knockdown of Rab3A promoted neurite outgrowth. However, this inhibitory effect on neurite outgrowth in hippocampal neurons could be reversed via co-transfection of spastin, indicating that Rab3A functions via its interaction protein spastin. In general, our data identify an interaction between Rab3A and spastin, and this interaction affects the protein stability of spastin and eliminates its microtubule severing function, thereby modulating neurite outgrowth.

MiR-182-5p Regulates Nogo-A Expression and Promotes Neurite Outgrowth of Hippocampal Neurons In Vitro.

Nogo-A protein is a key myelin-associated inhibitor of axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in the CNS, such as neuronal differentiation, neuritogenesis, and plasticity. Our results show that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3′UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data, we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.

Effects of ethanol-and choline-treated astrocytes on hippocampal neuron neurite outgrowth in vitro.

Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for β-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

Dexmedetomidine Alleviates LPS-Induced Neuronal Dysfunction by Modulating the AKT/GSK-3β/CRMP-2 Pathway in Hippocampal Neurons.

ObjectiveDexmedetomidine, an α2-adrenergic receptor agonist, mitigates cognitive dysfunction in elderly patients after surgery with general anesthesia. However, the underlying mechanism by which dexmedetomidine reduces cognitive dysfunction remains to be fully elucidated. The aim of this study was to investigate the effects of dexmedetomidine on lipopolysaccharide (LPS)-induced neuronal dysfunction in cultured hippocampal neurons.MethodsLPS, in the presence and absence of dexmedetomidine, was applied to cultured hippocampal neurons to mimic post-surgical inflammation. Neuronal morphology, including neurite outgrowth and synaptic transmission, was observed, and miniature excitatory postsynaptic currents were recorded by electrophysiological patch-clamp.ResultsLPS significantly impaired neurite outgrowth in hippocampal neurons in a concentration- and time-dependent manner, which was reversed by dexmedetomidine treatment. Electrophysiological patch-clamp results showed that LPS induced synaptic transmission dysfunction, which was restored after dexmedetomidine addition. Furthermore, Western blotting assays showed that LPS suppressed the AKT/GSK-3β/CRMP-2 signaling pathway and dexmedetomidine countered the inhibitory effect of LPS by re-activating this pathway.ConclusionIn general, dexmedetomidine protected against the effects of LPS-induced hippocampal neuron damage, including neurite outgrowth and synaptic transmission. Overall, dexmedetomidine modulated the AKT/GSK-3β/CRMP-2 signaling pathway to alleviate LPS-induced neurological dysfunction.

The lncRNA MALAT1/miR-30/Spastin Axis Regulates Hippocampal Neurite Outgrowth.

Spastin, a microtubule-severing enzyme, is important for neurite outgrowth. However, the mechanisms underlying the post-transcriptional regulation of spastin during microtubule-related processes are largely unknown. We demonstrated that the spastin expression level is controlled by a long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-30 (miR-30) axis during neurite outgrowth. The miR-30 expression level decreased in hippocampal neurons with increasing days in culture, and miR-30 overexpression suppressed while miR-30 inhibition promoted neurite outgrowth in hippocampal neurons. Spastin was validated as a target gene of miR-30 using the luciferase reporter assay. The protein expression, microtubule severing activity, and neurite promoting effect of spastin were suppressed by the overexpression of miR-30 mimics and increased by miR-30 inhibitors. MALAT1 expression increased during neurite outgrowth and MALAT1 silencing impaired neurite outgrowth. miR-30 was a sponge target of MALAT1 and MALAT1/miR-30 altered neurite outgrowth in hippocampal neurons. MALAT1 overexpression reversed the inhibitory effect of miR-30 on the activity of a luciferase reporter construct containing spastin, as well as spastin mRNA and protein expression, indicating that spastin was a downstream effector of MALAT1/miR-30. The MALAT1/miR-30 cascade also modulated spastin-induced microtubule severing, and the MALAT1/miR-30/spastin axis regulated neurite outgrowth in hippocampal neurons. This study suggests a new mechanism governing neurite outgrowth in hippocampal neurons involving MALAT1/miR-30-regulated spastin expression.

The Vps52 subunit of the GARP and EARP complexes is a novel Arf6-interacting protein that negatively regulates neurite outgrowth of hippocampal neurons

ADP ribosylation factor 6 (Arf6) is a small GTP-binding protein implicated in neuronal morphogenesis through endosomal trafficking and actin remodeling. In this study, we identified Vps52, a core subunit of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes, as a novel Arf6-binding protein by yeast two-hybrid screening. Vps52 interacted specifically with GTP-bound Arf6 among the Arf family. Immunohistochemical analyses of hippocampal pyramidal cells revealed that fine punctate immunolabeling for Vps52 was distributed throughout neuronal compartments, most densely in the cell body and dendritic shafts, and was largely associated with trans-Golgi network and vesicular endomembranes. In cultured hippocampal neurons, knockdown of Vps52 increased total length of axons and dendrites; these phenotypes were completely restored by co-expression of shRNA-resistant full-length Vps52. However, co-expression of a Vps52 mutant lacking the ability to interact with Arf6 restored only the Vps52-knockdown phenotype of the dendritic length. The present findings suggest that Vps52 is a novel Arf6-interacting protein that regulates neurite outgrowth in hippocampal neurons.

Site-specific HNK-1 epitope on alternatively spliced fibronectin type-III repeats in tenascin-C promotes neurite outgrowth of hippocampal neurons through contactin-1.

The human natural killer-1 (HNK-1) carbohydrate epitope, composed of a unique sulfated trisaccharide (HSO3–3GlcAβ1–3Galβ1–4GlcNAc-R), is highly expressed during brain development and regulates higher brain function. However, it remains unclear which glycoprotein carries the HNK-1 epitope in the embryonic brain and the functional role it plays. Here, we showed that one of the major HNK-1 carrier proteins in the embryonic brain is tenascin-C (TNC), an extracellular matrix protein that regulates neurite outgrowth by interacting with the GPI-anchored protein contactin-1 (CNTN). Because the alternatively spliced fibronectin type-III (FNIII) repeats in TNC give rise to many isoforms and affect neuronal function, we evaluated neurite outgrowth of primary hippocampal neurons on purified recombinant FNIII repeats with or without the HNK-1 epitope as a substrate. We found that the presence of the HNK-1 epitope on the C domain of TNC promoted neurite outgrowth, and that this signal was mediated by CNTN, which is an HNK-1-expressing neuronal receptor. The neurite-promoting activity of the HNK-1 epitope on TNC required neuronal HNK-1 expression, which was defective in neurons lacking the glucuronyltransferases GlcAT-P and GlcAT-S. These results suggest that the HNK-1 epitope is a key modifier of TNC and CNTN in the regulation of embryonic brain development.

Spastin Interacts with CRMP5 to Promote Neurite Outgrowth by Controlling the Microtubule Dynamics.

Changing the microtubule dynamics is sufficient to alter the axon and dendrite specification and development. Spastin participates in the growth and regeneration of neurites by severing microtubules into small segments, and collapsin response mediator protein 5 (CRMP5) provides structural support and serves as a track for cargo transport by promoting microtubule polymerization. Nevertheless, how spastin and CRMP5 cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. In our present study, spastin interacted with CRMP5 in vitro and in vivo. The binding domains for the spastin and CRMP5 interaction were the N-terminal fragment of spastin (residues 270-328) and the C-terminal fragment of CRMP5 (residues 472-564). Spastin and its truncation mutants, including the microtubule-binding domain (MTBD) and ATPases associated with diverse cellular activities (AAA) domain, were necessary for the severing of microtubules. Furthermore, we demonstrated that microtubule polymerization of CRMP5 interfered with the microtubule-severing function of spastin. Knocking down either spastin or CRMP5 inhibited neurite outgrowth in hippocampal neurons. However, co-transfected spastin and CRMP5 promoted the outgrowth of neurites including dendrites and axons. Taken together, our data support a model in which the spastin interaction with CRMP5 promotes neurite outgrowth by controlling the microtubule dynamics.

Identification of keratan sulfate disaccharide at C-3 position of glucuronate of chondroitin sulfate from Mactra chinensis.

Glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate, heparin, heparan sulfate and keratan sulfate (KS) are linear sulfated repeating disaccharide sequences containing hexosamine and uronic acid [or galactose (Gal) in the case of KS]. Among the GAGs, CS shows structural variations, such as sulfation patterns and fucosylation, which are responsible for their physiological functions through CS interaction with CS-binding proteins. Here, we solved the structure of KS-branched CS-E derived from a clam, Mactra chinensis. KS disaccharide [d-GlcNAc6S-(1→3)-β-d-Gal-(1→] was attached to the C-3 position of GlcA, and consecutive KS-branched disaccharide sequences were found in a CS chain. KS-branched polysaccharides clearly exhibited resistance to degradation by chondroitinase ABC or ACII (at low concentrations) compared with typical CS structures. Furthermore, KS-branched polysaccharides stimulated neurite outgrowth of hippocampal neurons. These results strongly suggest that M. chinensis is a rich source of KS-branched CS, and it has important biological activities.

ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6in mouse hippocampal neurons.

ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3in hippocampal neurons. In addition, knockdown and knockout of Arf6in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.

Synergic interaction between amyloid precursor protein and neural cell adhesion molecule promotes neurite outgrowth.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide. The main features of AD are the pathological changes of density and distribution of intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. The processing of amyloid beta precursor protein (APP) to β-amyloid peptide (Aβ) is one of the critical events in the pathogenesis of AD. In this study, we evaluated the role of the interaction of neural cell adhesion molecule (NCAM) and APP in neurite outgrowth using two different experimental systems: PC12E2 cells and hippocampal neurons that were isolated from wild type, APP knock-in and APP knock-out mice. PC12E2 cells or hippocampal neurons were co-cultured with NCAM-negative or NCAM-positive fibroblasts L929 cells. We found that APP promoted neurite outgrowth of PC12E2 cells and hippocampal neurons in either the presence or absence of NCAM. Secreted APP can rescue the neurite outgrowth in hippocampal neurons from APP knock-out mice. The interaction of APP and NCAM had synergic effect in promoting neurite outgrowth in both PC12E2 cells and hippocampal neurons. Our results suggested that the interaction of APP with NCAM played an important role in AD development and therefore could be a potential therapeutic target for AD treatment.

The ethanol extract of the rhodophyte Kappaphycus alvarezii promotes neurite outgrowth in hippocampal neurons

Neurodegenerative disorders, which affect memory, cognition, and social functions, can be treated using neurotrophic agents to support neuronal development and protect mature neurons from atrophy. We screened 34 tropical seaweed species collected from Indonesian coastal areas for their neurite-outgrowth-promoting activity (NOPA) in fetal rat hippocampal neurons in vitro. Based on the number and total length of primary neurites, red seaweeds had greater NOPA than green and brown seaweeds. The red seaweed Kappaphycus alvarezii showed the highest NOPA. Addition of the ethanol extract to the culture (1 μg mL−1) significantly accelerated initial neuronal maturation from stage I to stage II (70 %; P < 0.05) within 24 h and increased the number of neurites that developed multipolar characteristics (48 %; P < 0.05). These results indicate that the aquaculturable carrageenan producer K. alvarezii might be a promising source of neurotrophic compounds to enhance memory and learning.

Neurite outgrowth stimulation by n-3 and n-6 PUFAs of phospholipids in apoE-containing lipoproteins secreted from glial cells

PUFAs, which account for 25-30% of the total fatty acids in the human brain, are important for normal brain development and cognitive function. However, it remains unclear how PUFAs are delivered to neurons and exert their effects. In this study, we demonstrated that n-3 and n-6 PUFAs added to the medium are incorporated into membrane phospholipids of primary glial cells from rat cortices, and then secreted as the fatty acid moiety of phospholipids in apoE-containing lipoproteins (LpEs). Tandem mass spectrometry analysis further showed that LpEs secreted from glial cells contain a variety of metabolites of PUFAs produced in glial cells by elongation and unsaturation. LpEs are absorbed by endocytosis into neurons via LDL receptor-related protein 1. LpE-containing n-3 and n-6 PUFAs exhibit a strong effect on neurite outgrowth of hippocampal neurons by increasing the number of branches. This study sheds light on the novel role of LpEs in the central nervous system and also a novel pathway in which PUFAs act on neurons.

Functional chondroitin sulfate from Enteroctopus dofleini containing a 3-O-sulfo glucuronic acid residue

There are several reports that chondroitin sulfate containing K-type units [GlcA (3S)-GalNAc (4S)] exhibiting similar levels of neurite outgrowth promoting activities as CS having high amounts of B-, D- and E-type disulfated disaccharides. Although CS containing K-type units possess important biological activities, there are only few sources, such as king crab cartilage, squid cartilage or sea cucumber. In this study, CS containing 13.9% of K-type units was found in octopus (Enteroctopus dofleini) cartilage using different substrate specificities of chondroitinases. The 2D NMR spectra showed cross-peaks assigned to protons on sugar ring of GlcA (3S), demonstrating the presence of K-type units in octopus CS. Furthermore, proportion of fucosylated disaccharide units in octopus CS was very low. Octopus CS showed high affinity for growth factors and stimulated neurite outgrowth of hippocampal neurons, similar to the activity of squid CS-E. These results strongly suggest that octopus cartilage is a rich source of CS-K and has important biological activities.

Composition of glycosaminoglycans in elasmobranchs including several deep-sea sharks: identification of chondroitin/dermatan sulfate from the dried fins of Isurus oxyrinchus and Prionace glauca.

Shark fin, used as a food, is a rich source of glycosaminoglyans (GAGs), acidic polysaccharides having important biological activities, suggesting their nutraceutical and pharmaceutical application. A comprehensive survey of GAGs derived from the fin was performed on 11 elasmobranchs, including several deep sea sharks. Chondroitin sulfate (CS) and hyaluronic acid (HA) were found in Isurus oxyrinchus, Prionace glauca, Scyliorhinus torazame, Deania calcea, Chlamydoselachus anguineus, Mitsukurina owatoni, Mustelus griseus and Dasyatis akajei, respectively. CS was only found from Chimaera phantasma, Dalatias licha, and Odontaspis ferox, respectively. Characteristic disaccharide units of most of the CS were comprised of C- and D-type units. Interestingly, substantial amount of CS/dermatan sulfate (DS) was found in the dried fin (without skin and cartilage) of Isurus oxyrinchus and Prionace glauca. 1H-NMR analysis showed that the composition of glucuronic acid (GlcA) and iduronic acid (IdoA) in shark CS/DS was 41.2% and 58.8% (Isurus oxyrinchus), 36.1% and 63.9% (Prionace glauca), respectively. Furthermore, a substantial proportion of this CS/DS consisted of E-, B- and D-type units. Shark CS/DS stimulated neurite outgrowth of hippocampal neurons at a similar level as DS derived from invertebrate species. Midkine and pleiotrophin interact strongly with CS/DS from Isurus oxyrinchus and Prionace glauca, affording Kd values of 1.07 nM, 6.25 nM and 1.70 nM, 1.88 nM, respectively. These results strongly suggest that the IdoA-rich domain of CS/DS is required for neurite outgrowth activity. A detailed examination of oligosaccharide residues, produced by chondroitinase ACII digestion, suggested that the IdoA and B-type units as well as A- and C-type units were found in clusters in shark CS/DS. In addition, it was discovered that the contents of B-type units in these IdoA-rich domain increased in a length dependent manner, while C- and D-type units were located particularly in the immediate vicinity of the IdoA-rich domain.

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2

Numerous studies have indicated that nano-titanium dioxide (TiO2) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO2 affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO2 for 24 h, and nano-TiO2 internalization, dendritic growth, glutamate metabolism, expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (INa) and potassium current (IK) were examined. Our findings demonstrated that nano-TiO2 crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO2 induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca2+]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the INa and IK. In addition, nano-TiO2 increased nitric oxide and nitrice synthase, attenuated the activities of Ca2+-ATPase and Na+/K+-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO2 inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function.

Bcl-xL is necessary for neurite outgrowth in hippocampal neurons.

B-cell lymphoma-extra large (Bcl-xL) protects survival in dividing cells and developing neurons, but was not known to regulate growth. Growth and synapse formation are indispensable for neuronal survival in development, inextricably linking these processes. We have previously shown that, during synaptic plasticity, Bcl-xL produces changes in synapse number, size, activity, and mitochondrial metabolism. In this study, we determine whether Bcl-xL is required for healthy neurite outgrowth and whether neurite outgrowth is necessary for survival in developing neurons in the presence or absence of stress. Depletion of endogenous Bcl-xL impairs neurite outgrowth in hippocampal neurons followed by delayed cell death which is dependent on upregulation of death receptor 6 (DR6), a molecule that regulates axonal pruning. Under hypoxic conditions, Bcl-xL-depleted neurons demonstrate increased vulnerability to neuronal process loss and to death compared with hypoxic controls. Endogenous DR6 expression and upregulation during hypoxia are associated with worsened neurite damage; depletion of DR6 partially rescues neuronal process loss, placing DR6 downstream of the effects of Bcl-xL on neuronal process outgrowth and protection. In vivo ischemia produces early increases in DR6, suggesting a role for DR6 in brain injury. We suggest that DR6 levels are usually suppressed by Bcl-xL; Bcl-xL depletion leads to upregulation of DR6, failure of neuronal outgrowth in nonstressed cells, and exacerbation of hypoxia-induced neuronal injury. Bcl-xL regulates neuronal outgrowth during development and protects neurites from hypoxic insult, as opposed by DR6. Factors that enhance neurite formation may protect neurons against hypoxic injury or neurodegenerative stimuli.

SH2B1 increases the numbers of IRSp53-induced filopodia

BackgroundFilopodia are actin-rich membrane protrusions that play instrumental roles in development, cell migration, pathogen detection, and wound healing. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53kDa (IRSp53) has been implicated in regulating the formation of filopodia. Our previous results suggest that a signaling adaptor protein SH2B1β is required for neurite outgrowth of hippocampal neurons and neurite initiation of PC12 cells. Thus, we hypothesize that IRSp53 and SH2B1β may act together to regulate filopodium formation. MethodsTo determine the contribution of IRSp53 and SH2B1β in the formation of filopodia, we transiently transfect IRSp53 and/or SH2B1β to 293T cells. Cell morphology and protein distribution are assessed via confocal microscopy and subcellular fractionation. Total numbers of filopodia and filopodium numbers per perimeter are calculated to show the relative contribution of IRSp53 and SH2B1β. ResultsIn this study, we show that SH2B1β interacts with IRSp53 and increases the number of IRSp53-induced filopodia. One mechanism for this enhancement is that IRSp53 recruits SH2B1β to the plasma membrane to actively promote membrane protrusion. The increased numbers of filopodia likely result from SH2B1-mediated cytoplasmic extension and thus increased cell perimeter as well as IRSp53-mediated filopodium formation. ConclusionsTaken together, this study provides a novel finding that SH2B1β interacts with IRSp53-containing complexes to increase the number of filopodia. General significanceA better understanding of how SH2B1β and IRSp53 promote filopodium formation may have clinical implication in neurogenesis and regeneration.

Astrocytes protect against diazinon- and diazoxon-induced inhibition of neurite outgrowth by regulating neuronal glutathione

Evidence demonstrating that human exposure to various organophosphorus insecticides (OPs) is associated with neurobehavioral deficits in children continues to emerge. The present study focused on diazinon (DZ) and its active oxygen metabolite, diazoxon (DZO), and explored their ability to impair neurite outgrowth in rat primary hippocampal neurons as a mechanism of developmental neurotoxicity. Both DZ and DZO (0.5–10μM) significantly inhibited neurite outgrowth in hippocampal neurons, at concentrations devoid of any cyototoxicity. These effects appeared to be mediated by oxidative stress, as they were prevented by antioxidants (melatonin, N-t-butyl-alpha-phenylnitrone, and glutathione ethyl ester). Inhibition of neurite outgrowth was observed at concentrations below those required to inhibit the catalytic activity of acetylcholinesterase. The presence of astrocytes in the culture was able to provide protection against inhibition of neurite outgrowth by DZ and DZO. Astrocytes increased neuronal glutathione (GSH) in neurons, to levels comparable to those of GSH ethyl ester. Astrocytes depleted of GSH by l-buthionine-(S,R)-sulfoximine no longer conferred protection against DZ- and DZO-induced inhibition of neurite outgrowth. The findings indicate that DZ and DZO inhibit neurite outgrowth in hippocampal neurons by mechanisms involving oxidative stress, and that these effects can be modulated by astrocytes and astrocyte-derived GSH. Oxidative stress from other chemical exposures, as well as genetic abnormalities that result in deficiencies in GSH synthesis and regulation, may render individuals more susceptible to these developmental neurotoxic effects of OPs.