Neuroblastic Cells Research Articles

Antidepressant-like effect of Ganoderma lucidum spore polysaccharide-peptide mediated by upregulation of prefrontal cortex brain-derived neurotrophic factor.

A 28-kDa polysaccharide-peptide (PGL) with antidepressant-like activities was isolated from spores of the mushroom Ganoderma lucidum. It was unadsorbed on DEAE-cellulose. Its internal amino acid sequences manifested pronounced similarity with proteins from the mushrooms Lentinula edodes and Agaricus bisporus. The monosaccharides present in 28-kDa PGL comprised predominantly of glucose (over 90%) and much fewer galactose, mannose residues, and other residues. PGL manifested antidepressant-like activities as follows. It enhanced viability and DNA content in corticosterone-injured PC12 cells(a cell line derived from a pheochromocytoma of the rat adrenal medulla with an embryonic origin from the neural crest containing a mixture of neuroblastic cells and eosinophilic cells) and reduced LDH release. A single acute PGL treatment shortened the duration of immobility of mice in both tail suspension and forced swimming tests. PGL treatment enhanced sucrose preference and shortened the duration of immobility in mice exposed to chronic unpredictable mild stress (CUMS). Chronic PGL treatment reversed the decline in mouse brain serotonin and norepinephrine levels but did not affect dopamine levels. PGL decreased serum corticosterone levels and increased BDNF mRNA and protein levels and increased synapsin I and PSD95 levels in the prefrontal cortex. This effect was completely blocked by pretreatment with the BDNF antagonist K252a, indicating that PGL increased synaptic proteins in a BDNF-dependent manner.Key points• An antidepressive polysaccharide-peptide PGL was isolated from G. lucidum spores.• PGL protected PC12 nerve cells from the toxicity of corticosterone.• PGL upregulated BDNF expression and influenced key factors in the prefrontal cortex.

Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus.

The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.

Tumour‐derived substrate‐adherent cells promote neuroblastoma survival through secreted trophic factors

Neuroblastoma (NB) is the most common extracranial solid tumour in children. NB is highly heterogeneous and is comprised of a mixture of neuroblastic cancer cells and stromal cells. We previously reported that N‐type cells (neuroblastic cells) and S‐type cells (substrate‐adherent cells) in the SK‐N‐SH cell line shared almost identical genetic backgrounds. Sublines of N‐ and S‐type cells were isolated from an early passage (P35) of SK‐N‐SH. Sequencing analysis revealed that all sublines harboured the anaplastic lymphoma kinase (ALK) F1174L mutation, indicating that they were tumour derived. Surprisingly, over 74% resembled S‐type cells. In coculture experiments, S‐type cells protected N‐type cells from apoptosis induced by the oncogenic ALK inhibitor TAE684. Western blotting analyses showed that ALK, protein kinase A (AKT) and STAT3 signalling were stimulated in the cocultures. Furthermore, the conditioned medium from S‐type cells activated these downstream signalling molecules in the N‐type cells. The activation of STAT3 in the N‐type cells was ALK‐independent, while AKT was regulated by the ALK activation status. To identify the responsible soluble factors, we used a combination of transcriptomic and proteomic analysis and found that plasminogen activator inhibitor 1, secreted protein acidic and cysteine rich, periostin and galectin‐1 were potential mediators of STAT3 signalling. The addition of recombinant proteins to the tumour cells treated with the ALK inhibitor partially enhanced cell viability. Overall, the tumour‐derived S‐type cells prevented apoptosis in the N‐type cells via ALK‐independent STAT3 activation triggered by secreted factors. The inhibition of these factors in combination with ALK inhibition could provide a new direction for targeted therapies to treat high‐risk NB.

TNF signal, ROS, and caspase-11 play important role in HIV gp120 with morphine-Induced Neuropathic Pain.

Recent clinical evidence suggests that repeated use of opioid medications increases chronic pain in individuals living with HIV. It is important to elucidate the exact molecular mechanisms of neuropathic pain (NP) in opioid users living with HIV. HIV-related neuron damage is induced by activating glial cells releasing neurotoxic factors (such as, TNFα). Chronic morphine use also induces TNFα product. Spinal mitochondrial superoxide (mtO2●-) plays important role in neuropathic pain. Non-canonical inflammasome, caspase-11 signal pathway is involved in methamphetamine-induced neuron apoptosis. Increased ROS enhances caspase-11 expression during enteric pathogen infection. In the present study, we investigated the role of TNFRI, mtO2●- and caspase-11 in a HIV coat glycoprotein gp120 with morphine (gp120/M)-induced neuropathic pain state. HIV-gp120 with morphine induced mechanical allodynia lasting for 3 weeks. HIV-gp120/M upregulated the expression of TNFRI, mitochondrial superoxide, with caspase-11, and down-regulated Sirt3 in the spinal dorsal horn. TNFRI-/- knockout mice were higher mechanical threshold and longer thermal latency than that in wildtype mice. Intrathecal administration of antisense oligonucleotides against TNFRI, Mito-Temple (mitochondrial scavenger of mtO2●-), or siRNA agonist caspase-11 mRNA reduced mechanical allodynia in the gp120/M neuropathic pain model. Antisense oligonucleotides against TNFRI reduced the number of MitoSox (a marker of mtO2●-) positive cells in the SCDH in gp120/M group. The rat neuronal neuroblast (B35) cell line (ATCC) were treated with rTNFα (10 ng/mL, Peprotech) increased expression of mtO2●- and caspase-11, and caspase-11 related inflammatory factors such as, caspase-1, and IL-1β. In conclusions, spinal TNFRI signal plays a significant role in the HIV gp120-related neuropathic pain state, and provide evidence for a novel approach to treating chronic pain in opioid users living with HIV. NIH R01DA34749, R01DA047089, and R01DA047157, and R01DA047157-03S1.

The involvement of Nile tilapia (Oreochromis niloticus) Neu4 sialidase in neural differentiation during early ontogenesis

Neurogenesis is an important process for the formation of the central nervous system during ontogenesis. Mammalian sialidases are involved in neurogenesis through desialylation of sialo-glycoconjugates. However, the significance of fish sialidases, unlike that of mammals, in neurogenesis has not been investigated. The present study focuses on Nile tilapia (Oreochromis niloticus) because of its unique profiles of sialidases related to enzymatic properties, subcellular localization, and tissue-specific gene expression. First, the fish were cultured under aphotic condition, which is known to cause the delayed development of the retina and brain in various fish. Next, we investigate the effect of aphotic condition on the levels of tilapia sialidases. Our results revealed that the tilapia showed a decrease in the number of ganglion cell in the retina. The expression level of neu4 mRNA is up-regulated in the eyes from tilapia reared in Dark accompanied by the increase of retinal differentiation markers. These results indicated that tilapia Neu4 is involved in retinal development in Nile tilapia. Furthermore, we tried to clarify the function of tilapia Neu4 in the neuronal cells using two neuroblast cell lines (SH-SY5Y and Neuro2a cell lines). Tilapia Neu4 decreased sialic acid level of both nuclear glycoproteins as well as glycolipids. Moreover, tilapia Neu4 accelerated neurite formation in both two neural cell lines and, increased the acetylcholinesterase activity, but it did not affect cell proliferation. Collectively, these results suggest that Neu4 accelerates neurite differentiation during ontogenesis in tilapia.

Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.

Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila.

The final size and function of the adult central nervous system (CNS) are determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. Here, we explore non-autonomous glial regulation of NB proliferation. We show that lipid droplets (LDs) which reside within the glial niche are closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation. Upon FGF-mediated cortex glial overgrowth, glial Hh non-autonomously activates Hh signalling in the NBs, which in turn disrupts NB cell cycle progression and its ability to produce neurons. Glial Hh's ability to signal to NB is further modulated by lipid storage regulator lipid storage droplet-2 (Lsd-2) and de novo lipogenesis gene fatty acid synthase 1 (Fasn1). Together, our data suggest that glial-derived Hh modified by lipid metabolism mechanisms can affect the neighbouring NB's ability to proliferate and produce neurons.

Semi-Automated Cell and Tissue Analyses Reveal Regionally Specific Morphological Alterations of Immune and Neural Cells in a Porcine Middle Cerebral Artery Occlusion Model of Stroke.

Histopathological analysis of cellular changes in the stroked brain provides critical information pertaining to inflammation, cell death, glial scarring, and other dynamic injury and recovery responses. However, commonly used manual approaches are hindered by limitations in speed, accuracy, bias, and the breadth of morphological information that can be obtained. Here, a semi-automated high-content imaging (HCI) and CellProfiler histological analysis method was developed and used in a Yucatan miniature pig permanent middle cerebral artery occlusion (pMCAO) model of ischemic stroke to overcome these limitations. Evaluation of 19 morphological parameters in IBA1+ microglia/macrophages, GFAP+ astrocytes, NeuN+ neuronal, FactorVIII+ vascular endothelial, and DCX+ neuroblast cell areas was conducted on porcine brain tissue 4 weeks post pMCAO. Out of 19 morphological parameters assessed in the stroke perilesional and ipsilateral hemisphere regions (38 parameters), a significant change in measured IBA1+ parameters, GFAP+ parameters, NeuN+ parameters, FactorVIII+ parameters, and DCX+ parameters were observed in stroked vs. non-stroked animals. Principal component analysis (PCA) and correlation analyses demonstrated that stroke-induced significant and predictable morphological changes that demonstrated strong relationships between IBA1+, GFAP+, and NeuN+ areas. Ultimately, this unbiased, semi-automated HCI and CellProfiler histopathological analysis approach revealed regional and cell specific morphological signatures of immune and neural cells after stroke in a highly translational porcine model. These identified features can provide information of disease pathogenesis and evolution with high resolution, as well as be used in therapeutic screening applications.

N-Acetyl-D-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration.

Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK–NudC–Lis1–dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein–NudC–Lis1 complex at the nuclear envelope is required for the regulation of cell migration.

Insulin‐like growth factor 1 (IGF1) gene therapy modulates behavior, neurogenesis, and synaptic proteins in a rat model of sporadic Alzheimer's disease

AbstractBackgroundSporadic Alzheimer’s disease (SAD) is the most prevalent neurodegenerative disorder worldwide and it is characterized by progressive memory loss and cognitive impairment. Brain activation of insulin signaling preserves memory in animal models and appears beneficial for patients. In contrast, the role of insulin‐like growth factor 1 (IGF1) remains controversial. Our aim was to determine the possible protective actions of IGF1 in a rat SAD model induced by intracerebroventricular injection of streptozotocin (icv‐STZ). To this end, we drove the expression of IGF1 using a recombinant adenoviral vector, RAd‐IGF1.MethodWe evaluated 3 animal groups: Sham, STZ and STZ+IGF1. STZ and STZ+IGF1 groups were injected with 3 mg/kg icv‐STZ and, 7 days later, the STZ+IGF1 group received the icv‐RAd‐IGF1. During the last two weeks until the end of the study (day 24 post icv‐STZ), we performed several behavioral tests. Additionally, we analyzed neuroblast cells, synaptic proteins and IGF1‐signaling pathway proteins in the hippocampus.ResultIGF1 improved marble‐burying behavior, hippocampus‐dependent spatial memory, object recognition memory and decreased depression‐like behavior, all features affected by STZ. Also, brain IGF1 overexpression restored neurogenesis, enhanced SYT2, PSD95 and GAD65/67 levels, and modulated the IGF1 receptor signaling pathway in STZ animals.ConclusionResults indicate a protective role of IGF1 in our SAD model, as it was effective against behavior deficits and ameliorated hippocampal protein alterations induced by STZ. We conclude that IGF1 therapy has an interesting potential for SAD treatment.

Glial granules contain germline proteins in the Drosophila brain, which regulate brain transcriptome

Membraneless RNA-protein granules play important roles in many different cell types and organisms. In particular, granules found in germ cells have been used as a paradigm to study large and dynamic granules. These germ granules contain RNA and proteins required for germline development. Here, we unexpectedly identify large granules in specific subtypes of glial cells (“glial granules”) of the adult Drosophila brain which contain polypeptides with previously characterized roles in germ cells including scaffold Tudor, Vasa, Polar granule component and Piwi family proteins. Interestingly, our super-resolution microscopy analysis shows that in the glial granules, these proteins form distinct partially overlapping clusters. Furthermore, we show that glial granule scaffold protein Tudor functions in silencing of transposable elements and in small regulatory piRNA biogenesis. Remarkably, our data indicate that the adult brain contains a small population of cells, which express both neuroblast and germ cell proteins. These distinct cells are evolutionarily conserved and expand during aging suggesting the existence of age-dependent signaling. Our work uncovers previously unknown glial granules and indicates the involvement of their components in the regulation of brain transcriptome.

Host sex and transplanted human induced pluripotent stem cell phenotype interact to influence sensorimotor recovery in a mouse model of cortical contusion injury

Traumatic brain injury (TBI) is a substantial cause of disability and death worldwide. Primary head trauma triggers chronic secondary injury mechanisms in the brain that are a focus of therapeutic efforts to treat TBI. Currently, there is no successful clinical strategy to repair brain injury. Cell transplantation therapies have demonstrated promise in attenuating secondary injury mechanisms of neuronal death and dysfunction in animal models of brain injury. In this study, we used a unilateral cortical contusion injury (CCI) model of sensorimotor brain injury to examine the effects of human induced pluripotent stem cell (hiPSC) transplantation on pathology in male and female adult mice. We determined transplanted hiPSC-derived neural stem cells (NSCs) and neuroblasts but not astrocytes best tolerate the injured host environment. Surviving NSC and neuroblast cells were clustered at the site of injection within the deep layers of the cortex and underlying corpus callosum. Cell grafts extended neuritic processes that crossed the midline into the contralateral corpus callosum or continued laterally within the external capsule to enter the ipsilateral entorhinal cortex. To determine the effect of transplantation on neuropathology, we performed sensorimotor behavior testing and stereological estimation of host neurons, astrocytes, and microglia within the contused cortex. These measures did not reveal a consistent effect of transplantation on recovery post-injury. Rather the positive and negative effects of cell transplantation were dependent on the host sex, highlighting the importance of developing patient-specific approaches to treat TBI. Our study underscores the complex interactions of sex, neuroimmune responses and cell therapy in a common experimental model of TBI.

Protection of neuronal cell lines, antimicrobial and photocatalytic behaviours of eco-friendly TiO2 nanoparticles

Eco-friendly synthesis of nanoparticles is promising as a futuristic alternative for chemical and physical protocols. In the present investigation, TiO2 nanoparticles were synthesised using the peel extract of Citrus aurantium fruit for the first time. The biosynthesised TiO2 nanoparticles were characterized by various techniques such as X- ray diffraction (XRD), UV–vis spectrophotometer, Fourier Transform Infra-Red Spectroscopy (FT-IR), Field Emission Scanning Electron Microscope (FE-SEM), Energy dispersive X-ray (EDAX), Transmission Electron Microscope (TEM) and Atomic Force Microscope (AFM). The crystal structure and phase purity of TiO2 nanoparticles were analysed using XRD. The optical properties and functional groups of TiO2 nanoparticles were investigated with the help of UV visible absorption and FT-IR analysis. The morphology and topography of TiO2 nanoparticles were examined through FE-SEM, TEM and AFM measurements. Biosynthesised TiO2 nanoparticles were evaluated for their antimicrobial activities against Staphylococcus aureus and Staphylococcus epidermidis (Gram-positive), Pseudomonas aeruginosa and Klebsiella pneumonia (Gram- negative), Candida albicans and Aspergillus niger (Fungi) using agar well method. In addition, cytotoxicity study was performed through MTT assay, the cell cycle analysis, and apoptosis assays of cells by flow cytometry and DNA fragmentation analysis. The photocatalytic behaviour against methylene blue dye was studied. Results of the present study revealed that biosynthesised TiO2 nanoparticles with an average size of 34 nm showed excellent antimicrobial, cell viability in PC12 cell lines (a combination of neuroblastic and eosinophilic cell lines) with IC50 ranged between 31.2–62.5 μg/ mL and excellent photo degradation property. Therefore, the present study concluded that biosynthesised TiO2 nanoparticles have potential antimicrobial, anti-apoptotic and photocatalytic behaviours towards neuronal cell lines.

Down-Regulation of AHNAK2 Inhibits Cell Proliferation, Migration and Invasion Through Inactivating the MAPK Pathway in Lung Adenocarcinoma.

AHNAK nucleoprotein 2 (AHNAK2) has been emerged as a crucial protein for neuroblast differentiation and cell migration, thereby involving in the development of various cancers. However, the specific molecular mechanism of AHNAK2 in lung adenocarcinoma is inconclusive. By accessing to the Oncomine dataset and GEPIA website, a higher expression level of AHNAK2 was observed in lung adenocarcinoma tissue samples. Overall survival (OS) curve plotted by Kaplan-Meier method showed that up-regulation of AHNAK2 was related with poor prognosis of lung adenocarcinoma patients. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis and western blot were conducted to examine the expression level of genes in lung adenocarcinoma cells. Through functional in vitro experiments, cell proliferation, migration and invasion were all suppressed after AHNAK2 knockdown using Cell counting kit-8 (CCK-8) assay, wound-healing and transwell analysis. Reduction of AHNAK2 decreased the apoptosis rate using flow cytometry analysis. Moreover, the key markers of MAPK pathway, p-MEK, p-ERK and p-P90RSK were decreased due to the transfection of si-AHNAK2 in A549 cells. U0126, a MEK inhibitor, showed the similar effects on MAPK-related protein levels with si-AHNAK2. To sum up, AHNAK2 is significantly increased in lung adenocarcinoma and plays a carcinogenic role by activating the MAPK signaling pathway, providing a novel insight and raising possibility for lung adenocarcinoma treatment.

Stimulating Ca<sup>2+</sup> photoactivation of nerve cells by near-infrared light

Calcium ions (Ca<sup>2+</sup>) play a key role of the nerve cells generating universal intracellular signals and controlling important functions. Ca<sup>2+</sup> activation is of great significance for explaining the subcellular-level biological process. Light stimulated nerve cells to control intracellular signals and membrane activities has become a main method in neuroscience, and the photoactivation is one of the main ways to study intracellular Ca<sup>2+</sup> transmission. Nerve cells can be directly stimulated by light to produce action potentials, but such techniques are inaccurate in the delivered light energy. To improve this, here in this work we show that gold nanorods (GNRs) can be conjugated to ligands to bound to human neuroblast cells (SH-SY5Y), and introduce an optical method of stimulating and monitoring Ca<sup>2+</sup> signal in nerve cells in which the plasmonic excitation of GNRs is used. In this paper, we use confocal microscopy to display the 488 nm continuous wave laser irradiating SH-SY5Y cells with Ca<sup>2+</sup> indicator (Fluo-4, AM) to check fluorescence. Near-infrared pulsed light at the plasmon resonance absorption peak of GNRs is used to stimulate Ca<sup>2+</sup> signal transduction in SH-SY5Y labeled with GNRs, and Fluo-4, AM is used for two-photon excited fluorescence imaging. In addition, we use the pulsed laser with power of 0.5 mW and a wavelength of 800 nm. The Ca<sup>2+</sup> activation can be achieved in 10 s on average. The release rate of Ca<sup>2+</sup> from SH-SY5Y cells labeled with GNRs is 6 times that without GNRs. Next, in order to determine the source of changes in Ca<sup>2+</sup>, we use the BPATA to deplete the intracellular Ca<sup>2+</sup>, after 5 min, 200 μmol/L Ca<sup>2+</sup> solution is added, and its Δ<i>F</i>/<i>F</i> is found to be more than 1.5 times that without GNRs. Thus, we believe that GNRs could enhance photoactivation through local surface plasmon resonance induced membrane depolarization and generate an action potential. The results prove the feasibility of using GNRs to enhance the activation of Ca<sup>2+</sup> in nerve cells, and provide an optical means of lower photodamage and more precise for studying nerve cell ion channels. Our study demonstrates that enhancing photoactivation by GNRs could provide an outlook of basic research in neuroscience.

Adequate expression of Globin1 is required for development and maintenance of nervous system in Drosophila

Neurogenesis is driven by spatially and temporally regulated proliferation of neuronal progenitor cells that generates enormous number of assorted neurons to drive the complex behavior of an organism. Drosophila nervous system provides an advantageous model for identification and elucidation of the functional significance of the novel gene(s) involved in neurogenesis. The present study attempts to investigate the role(s) of globin1 (glob1) in the development and maintenance of the nervous system in Drosophila. It is increasingly clear now that globin genes play important role(s) in the various biological phenomena. The vertebrate neuroglobin has been reported to profoundly express in neuronal tissues and provides neuroprotection. We noted ubiquitous presence of Glob1 in the developing neuronal tissues with enhanced concentration throughout the VNC which comprises of midline cell clusters, which subsequently forms numerous types of progenitor cells and finally differentiate into specific neurons of the nervous system. Ubiquitous or pan-neuronal downregulation of glob1 causes partial lethality and mis-positioning of various neural-progenitor cells present in the embryonic midline cell clusters. Subsequently, profound expression of Glob1 was noted in the outer proliferation center of larval brain and photoreceptor axons of optic stalk. The overall arrangement of photoreceptor axons and stereotype positioning of neuroblast cells present in the central region of the brain were severally affected due to reduced expression of glob1. In addition, such larvae and surviving adults develop significant neuro-muscular disabilities. For the first time, our study suggests a novel role of glob1 in development and maintenance of the nervous system adding a new dimension to the functional significance of the multi-tasking glob1 gene in Drosophila.

Pyridoxine Preferentially Induces Auditory Neuropathy Through Mitochondrial Dysfunction and Endoplasmic Reticulum Stress-Mediated Apoptosis.

Auditory neuropathy due to toxicity mechanism of pyridoxine has not yet been fully documented. Therefore, the present study explored a direct mechanism underlying the effects of pyridoxine on auditory neuropathy in organ of Corti (OC) explants ex vivo and cochlear neuroblast cell line, VOT-33 in vitro. Primary OC explants containing spiral ganglion neurons and cultured VOT-33 cells were treated with pyridoxine. In nerve fiber of primary OC explants, pyridoxine decreased staining for NF200, a neuro-cytoskeletal protein. We also found that pyridoxine-induced VOT-33 apoptosis, as indicated by accumulation of the sub-G0/G1 fraction, caspase-3 activation, and PARP cleavage. In addition, pyridoxine induced reactive oxygen species (ROS) generation and alteration of mitochondrial membrane potential transition (MPT), including Bcl-2 family protein expression and consequently Ca2+ accumulation and changes of endoplasmic reticulum (ER) stress-related protein expression such as phospho-PERK, caspase-12, Grp78, and CHOP. Pyridoxine preferentially induced severe cell death on nerve fiber in primary OC explants and markedly increased apoptotic cell death via mitochondria-mediated ER stress in VOT-33 cells.

Abstract TMP33: Modulation of Receptor Protein Tyrosine Phosphatase Sigma Promotes Neuroregeneration and Functional Recovery After Ischemic Stroke

Introduction: Due to the limitation in current treatment window for stroke, the development of delayed stroke treatment is needed. The two main cellular processes of neural repair after stroke are axonal sprouting and neurogenesis. Inhibitory chondroitin sulfate proteoglycans (CSPGs) are increased within the glial scar and perineuronal net after ischemic brain injury, which might play a critical role in blocking neuroblasts migration, axonal regrowth and sprouting. Recently, a novel small peptide memetic of the CSPG receptor (PTP σ) wedge region named ISP (Intracellular Sigma Peptide) is generated and has capabilities to target PTP σ and relieve CSPGs inhibition in the models of spinal cord injury. Here, we aimed to determine whether post-stroke ISP treatment could improve functional recovery in a mouse model of ischemia. Methods: Mice were subjected to tMCAO followed by treatment of ISP or vehicle daily for 4 weeks beginning 24h or 7 days post-tMCAO. Motor and sensorimotor functions were measured prior to MCAO and at 1, 2, 3 and 4 weeks post-stroke using locomotion analysis and adhesion removal test. Cognitive function was also assessed with Barnes maze test at 4 weeks post-stroke. Neurogenesis and axonal sprouting was evaluated using genetic labeling and immunohistochemical analysis. Results: Our results show that continuous ISP treatment starting at 24 hours post stroke improves both survival and long-term functional recovery after stroke and 7 day post-stroke treatment improves the functional recovery. Correlated with the improved behavior measurements, immunohistochemical analysis revealed that post-stroke ISP treatment increased the total number of newly generated neuroblast cells migrating towards the peri-infarct area as well as a robust sprouting/regeneration of serotonergic axons around the stroke peri-infarct area. Conclusions: Our data suggest that inhibition of PTP σ/CSPGs signaling by ISP stimulates functional recovery after stroke via promoting neuroblast migration and axonal growth. Pharmacological targeting of the PTP σ/CSPGs interaction is therefore a potential novel therapeutic strategy for stroke treatment, which may provide a prolonged treatment window for improving functional recovery after stroke.

Ginsenoside Rg1 Prevents Chemotherapy-Induced Cognitive Impairment: Associations with Microglia-Mediated Cytokines, Neuroinflammation, and Neuroplasticity.

Chemotherapy-induced cognitive impairment, also known as "chemobrain," is a common side effect. The purpose of this study was to examine whether ginsenoside Rg1, a ginseng-derived compound, could prevent chemobrain and its underlying mechanisms. A mouse model of chemobrain was developed with three injections of docetaxel, adriamycin, and cyclophosphamide (DAC) in combination at a 2-day interval. Rg1 (5 and 10mg/kg daily) was given 1week prior to DAC regimen for 3weeks. An amount of 10mg/kg Rg1 significantly improved chemobrain-like behavior in water maze test. In vivo neuroimaging revealed that Rg1 co-treatment reversed DAC-induced decreases in prefrontal and hippocampal neuronal activity and ameliorated cortical neuronal dendritic spine elimination. It normalized DAC-caused abnormalities in the expression of multiple neuroplasticity biomarkers in the two brain regions. Rg1 suppressed DAC-induced elevation of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but increased levels of the anti-inflammatory cytokines IL-4 and IL-10 in multiple sera and brain tissues. Rg1 also modulated cytokine mediators and inhibited DAC-induced microglial polarization from M2 to M1 phenotypes. In in vitro experiments, while impaired viability of PC12 neuroblastic cells and hyperactivation of BV-2 microglial cells, a model of neuroinflammation, were observed in the presence of DAC, Rg1 co-treatment strikingly reduced DAC's neurotoxic effects and neuroinflammatory response. These results indicate that Rg1 exerts its anti-chemobrain effect in an association with the inhibition of neuroinflammation by modulating microglia-mediated cytokines and the related upstream mediators, protecting neuronal activity and promoting neuroplasticity in particular brain regions associated with cognition processing.

KDM2B regulates choline kinase expression and neuronal differentiation of neuroblastoma cells.

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.