Embryonic Rat Brain Research Articles

The Role of Nde1 phosphorylation in interkinetic nuclear migration and neural migration during cortical development.

Nde1 is a cytoplasmic dynein regulatory protein with important roles in vertebrate brain development. One noteworthy function is in the nuclear oscillatory behavior in neural progenitor cells, the control and mechanism of which remain poorly understood. Nde1 contains multiple phosphorylation sites for the cell cycle-dependent protein kinase CDK1, though the function of these sites is not well understood. To test their role in brain development, we expressed phosphorylation-state mutant forms of Nde1 in embryonic rat brains using in utero electroporation. We find that Nde1 T215 and T243 phosphomutants block apical interkinetic nuclear migration (INM) and, consequently, mitosis in radial glial progenitor cells. Another Nde1 phosphomutant at T246 also interfered with mitotic entry without affecting INM, suggesting a more direct role for Nde1 T246 in mitotic regulation. We also found that the Nde1 S214F mutation, which is associated with schizophrenia, inhibits Cdk5 phosphorylation at an adjacent residue which causes alterations in neuronal lamination. These results together identify important new roles for Nde1 phosphorylation in neocortical development and disease, and represent the first evidence for Nde1 phosphorylation roles in INM and neuronal lamination.

A simple method for preservation of embryonic rat brain tissue for primary neuron culture

Aims/Purpose: In this study we aim to investigate the effect of short‐term storage of embryonic rat brain tissue in Hibernate‐E medium to be used in the isolation of primary neurons for in vitro biocompatibility and electrophysiology tests of retinal and optic nerve prosthesis. Functionally viable neuron cells will be an alternative method for regular dissection from tissue for further experiments.Methods: Hippocampal tissues from E15‐E17 embryos of Wistar Albino rat were dissecting for primary neuron culture. Half of the tissues were isolated on the same day and used as a control group for further experiments. The test group was stored in a Hibernate‐E medium for 1 week and the same isolation method was performed for the test group. For tissue preservation, half medium replacement with fresh Hibernate‐E medium on the 4th day was enhanced cell viability. Morphology, characterization and synapse formation were determined with immunoflorescence staining.Results: Comparison of neuronal survival between cultures prepared from stored hippocampi and freshly isolated hippocampi resulted in similar neuronal survival rates in both preparations with cell viability assays. Cell viability rate of hippocampus tissue isolated from rat embryo brain was demonstrated as 96% and cell viability rate of preserved tissue in Hibernate‐E Medium for 7 days was revealed as 90.22% by trypan blue cell viability test (n = 4). Morphological and immunocytochemical characterizations of stored and fresh hippocampal cultures were similar by observing beta‐III Tubulin neuron cytoskeleton biomarker.Conclusions: As a conclusion, our method has the potential to increase repeatability of primary neuron cultures within 1 week and reduce the number of isolation steps in research. In addition, it will provide a great convenience in the experimental timelines of the researchers and contribute to the reduction of the workload despite any obstacles during primary neuron culture.

Low Density Lipoprotein Receptor-related Protein 2 Expression and Function in Cultured Astrocytes and Microglia.

Activation of glial cells, astrocytes and microglia, has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Amyloid β (Aβ), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be produced by neurons and to activate glial cells. Clearance of Aβ from the brain via active transport system is important to prevent the accumulation and aggregation. Low density lipoprotein receptor-related protein 2 (LRP2/megalin) is an Aβ transporter. However, expression and contribution of LRP2 in astrocytes and microglia remain to be clarified. In the present study, we examined the expression of LRP2 and its roles in cultured astrocytes prepared from rat embryonic brain cortex and mouse microglial cell line BV-2. Both cultured rat astrocytes and BV-2 cells expressed LRP2 mRNA detected by RT-PCR. When lipopolysaccharide (LPS) or all-trans retinoic acid (ATRA) were added to BV-2 cells, LRP2 mRNA expression and uptake of microbeads, Aβ and insulin were increased. On the other hand, LPS decreased LRP2 expression and uptake of Aβ and insulin in cultured astrocytes. Knockdown of LRP2 using siRNA attenuated the LPS- or ATRA-increased uptake of microbeads, Aβ and insulin in BV-2 cells. These results suggest that LRP2 was expressed in both astrocytes and microglia and might be involved in endocytosis activities. Adequate control of LRP2 expression and function in astrocytes and microglia might regulate Aβ and insulin levels in brain and would be a potential target in AD pathology.

Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii.

ABSTRACTHuman toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T. gondii cell cycle mechanism, the G1 restriction checkpoint (R-point), that operates the switch between parasite growth and differentiation. Apicomplexans lack conventional R-point regulators, suggesting adaptation of alternative factors. We showed that Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2, and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. Examination of cyclins verified the correlation of cyclin expression with growth dependence and development capacity of RH and ME49 strains. We demonstrated that rapidly dividing RH tachyzoites were dependent on TgCycP1 expression, which interfered with bradyzoite differentiation. Using the conditional knockdown model, we established that TgCycP2 regulated G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally incompetent RH tachyzoites. We tested the functions of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models. Based on functional and global gene expression analyses, we determined that TgCycP2 also regulated bradyzoite replication, while signal-induced TgCyc5 was critical for efficient tissue cyst maturation. In conclusion, we identified the central machinery of the T. gondii restriction checkpoint comprised of TgCrk2 kinase and three atypical T. gondii cyclins and demonstrated the independent roles of TgCycP1, TgCycP2, and TgCyc5 in parasite growth and development.IMPORTANCE Toxoplasma gondii is a virulent and abundant human pathogen that puts millions of silently infected people at risk of reactivation of the chronic disease. Encysted bradyzoites formed during the chronic stage are resistant to current therapies. Therefore, insights into the mechanism of tissue cyst formation and reactivation are major areas of investigation. The fact that rapidly dividing parasites differentiate poorly strongly suggests that there is a threshold of replication rate that must be crossed to be considered for differentiation. We discovered a cell cycle mechanism that controls the T. gondii growth-rest switch involved in the conversion of dividing tachyzoites into largely quiescent bradyzoites. This switch operates the T. gondii restriction checkpoint using a set of atypical and parasite-specific regulators. Importantly, the novel T. gondii R-point network was not present in the parasite's human and animal hosts, offering a wealth of new and parasite-specific drug targets to explore in the future.

Prenatal exposure to valproic acid induces alterations in the expression and activity of purinergic receptors in the embryonic rat brain.

Purinergic signalling is involved in the control of several processes related to brain development, such as neurogenesis and gliogenesis, migration and differentiation of neuronal precursors, synaptogenesis and synaptic elimination to achieve a fully wired and efficient mature brain. Therefore, any deregulation of purine-dependent signalling mediated by stimulation of specific adenosine and purinergic receptor subtypes: P1, P2X, or P2Y, can lead to functional deficits and the development of neuropsychiatric disorders, including autism spectrum disorders (ASD). In this study, we investigated the changes in expression and activity of selected purinergic receptors during rat brain development in an animal model of ASD. Pregnant dams received an intraperitoneal injection of valproic acid (VPA; 450 mg/kg body weight) at embryonic day (ED) 12.5, around the time of neural tube closure. Subsequently, changes in the expression and activity of specific purinergic receptor subtypes were analysed at ED19, an important prenatal stage of brain development. Our results suggest that prenatal VPA exposure leads to a significant increase in the level and activity of adenosinergic receptors A1, A2b and A3, which are involved in the regulation of progenitor cell proliferation and nerve growth, and upregulation of purinergic P2X2/P2X3 receptors, which in turn may contribute to the postnatal neuroanatomical abnormalities and synaptic dysfunction. Conversely, the significant downregulation of P2Y1 and P2X7 receptors, together with their reduced activity in the embryonic VPA brain, may indicate disturbances in the processes of neuronal precursor migration and differentiation, dendritic and axonal formation, and glutamate/GABA imbalance, thereby altering neuronal excitability. In conclusion, defects in purinergic signalling induced by prenatal VPA exposure could have a profound impact on brain development during embryogenesis and on intellectual and behavioural functions after birth. These observations could provide clues for future implementation of potential therapeutic strategies for ASD.

Identification of a novel mutation in MEF2C gene in an atypical patient with frontotemporal lobar degeneration.

The MEF2C gene encodes a transcription factor known to play a crucial role in molecular pathways affecting neuronal development. MEF2C mutations were described as a genetic cause of developmental disease (MRD20), and several reports sustain its involvement in dementia-related conditions, such as Alzheimer's disease and amyotrophic lateral sclerosis. These pathologies and frontotemporal degeneration (FTLD) are thought to share common physiopathological pathways. In this exploratory study, we searched for alterations in the DNA sequence of exons and boundaries, including 5'- and 3'-untranslated regions (5'UTR, 3'UTR), of MEF2C gene in 11 patients with clinical phenotypes related with MRD20 or FTLD. We identified a heterozygous deletion of 13 nucleotides in the 5'UTR region of a 69 years old FTLD patient. This alteration was absent in 200 healthy controls, suggesting a contribution to this patient's disease phenotype. In silico analysis of the mutated sequence indicated changes in mRNA secondary structure and stability, thus potentially affecting MEF2C protein levels. Furthermore, in vitro functional analysis of this mutation revealed that the presence of this deletion abolished the transcriptional activity of the gene in human embryonic cells and rat brain neurons, probably by modifying MEF2C expression. Altogether, our results provide evidence for the involvement of MEF2C in FTLD manifesting with seizures.

Valproic acid alters the expression of HIF‐1α in embryonic brains in a valproic acid rodent model of autism

Although studies have investigated the role of Valproic acid (VPA) in impaired neurogenesis in rodent models of autism, the molecular mechanisms that underlie neural developmental changes during embryonic brain development in this model are poorly understood. Here, we employed a VPA rat model of autism to investigate the effects of VPA treatment on key enzymes of glucose oxidative metabolism namely lactate dehydrogenase (LDH), and genes of the adaptive responses including hypoxia induced factor 1 alpha (HIF-1α) in embryonic rat brain. Pregnant Wistar rats were administered 600 mg/kg of VPA by intraperitoneal injection on day 12.5 of gestation and embryonic (E) day 15, E17, E20 rat brains were assayed for the activity of LDH. E15, E17 and E20 male and female rat models of VPA-induced autism showed significantly increased whole brain activities of LDH compared to the corresponding values in control rats. In addition, valproic acid altered the expression of HIF-1α at the different embryonic time points. Our results suggest at least some of the mechanism underlying impaired neurogenesis in the VPA model maybe partly due to changes in activity of LDH and alteration of HIF-1α. The altered expression of HIF-1α regulates the expression of several enzymes and growth factors that may have pathophysiological/pathogenic implications in autism and may ultimately lead to the discovery of new therapeutic target(s) for autism.

Comparative Analysis of the Expression of Chondroitin Sulfate Subtypes and Their Inhibitory Effect on Axonal Growth in the Embryonic, Adult, and Injured Rat Brains.

Chondroitin sulfate glycosaminoglycans (CS-GAGs) are the primary inhibitory GAGs for neuronal growth after central nervous system (CNS) injury. However, the inhibitory or permissive activity of CS-GAG subtypes is controversial and depends on the physiological needs of CNS tissues. In this study, we investigated the characteristics and effects of CS-GAGs on axonal growth, which was isolated from the brain cortices of normal rat embryo at E18, normal adult rat brain and injured adult rat brain. Isolated CS-GAGs from embryo, normal adult, and injured adult rat brains were used for analyzing their effect on attachment and axonal growth using modified spot assay with dorsal root ganglion (DRG) explants and cerebellar granule neurons (CGNs). CS-GAGs were separated using high performance liquid chromatography (HPLC), and the subtypes of CS-GAGs were analyzed. CS-GAGs of all three groups inhibited CGN attachment and axonal growth of DRGs. However, CS-GAGs of normal adult rat brain exhibited higher inhibitory activity than those of the other groups in both assays. When subtypes of CS-GAGs were analyzed using HPLC, CS-A (4S) was the most abundant in all three groups and found in largest amount in normal adult rat brain. In contrast, unsulfated CS (CS0) and CS-C (6S) were more abundant by 3-4-folds in E18 group than in the two adult groups. When compared with the normal adult rat brain, injured rat brain showed relatively similar patterns to that of embryonic rat brain at E18 in the expression of CS subtypes and their inhibitory effect on axonal growth. This phenomenon could be due to differential expression of CS-GAGs subtypes causing decrease in the amount of CS-A and mature-type CS proteoglycan core proteins.

Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures.

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer's disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague-Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses.

Cortical cells are altered by factors including bone morphogenetic protein released from a placental barrier model under altered oxygenation.

Episodes of hypoxia and hypoxia/reoxygenation during foetal development have been associated with increased risk of neurodevelopmental conditions presenting in later life.The mechanism for this is not understood; however, several authors have suggested that the placenta plays an important role. Previously we found both placentas from a maternal hypoxia model and pre-eclamptic placentas from patients release factors lead to a loss of dendrite complexity in rodent neurons. Here to further explore the nature and origin of these secretions we exposed a simple in vitro model of the placental barrier, consisting of a barrier of human cytotrophoblasts, to hypoxia or hypoxia/reoxygenation. We then exposed cortical cultures from embryonic rat brains to the conditioned media (CM) from below these exposed barriers and examined changes in cell morphology, number, and receptor presentation.The barriers released factors that reduced dendrite and astrocyte process lengths, decreased GABAB1 staining, and increased astrocyte number. The changes in astrocytes required the presence of neurons and were prevented by inhibition of the SMAD pathway and by neutralising Bone Morphogenetic Proteins (BMPs) 2/4. Barriers exposed to hypoxia/reoxygenation also released factors that reduced dendrite lengths but increased GABAB1 staining. Both oxygen changes caused barriers to release factors that decreased GluN1, GABAAα1 staining and increased GluN3a staining. We find that hypoxia in particular will elicit the release of factors that increase astrocyte number and decrease process length as well as causing changes in the intensity of glutamate and GABA receptor staining. There is some evidence that BMPs are released and contribute to these changes.

Valproic Acid Alters Activities of Key Enzymes of Glucose Oxidative Metabolism, Citric Acid Cycle and Neurotransmitter Synthesis in Embryonic Rat Brain

Although studies have investigated the role of gamma‐aminobutyric acid (GABA)ergic signaling in rodent neural development and behaviors relevant to autism, the energetics that underlie neural developmental changes are poorly understood. Here, we employed a valproic acid (VPA) rat model of autism to investigate the effects of VPA treatment on key enzymes of glucose oxidative metabolism and citric acid cycle (namely lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH)) in rat brain. Pregnant Wistar rats were administered 600 mg/kg of VPA by intraperitoneal injection on day 12.5 of gestation and embryonic (E) day 15 and E17 rat brains were assayed for the activities of the enzymes. Both E15 and E17 male and female rat models of VPA‐induced autism showed significantly changed whole brain activities of the enzymes studied compared to the corresponding values in control rats. The dysregulated GDH activity could alter the excitatory‐inhibitory balance in the brain of embryonic rats during development. Thus, our findings may have pathophysiological/pathogenic implications in autism and may ultimately lead to the discovery of new therapeutic target(s) for autism.Support or Funding InformationSeed Grant Idaho State University College of Pharmacy

Dynamic Changes in the Localization of Neuronatin-Positive Cells during Neurogenesis in the Embryonic Rat Brain

Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.

RETRACTED: Overexpression of cortistatin alleviates oxygen/glucose-deprivation-induced ER stress and prompts neural stem cell proliferation via SSTR2

Cerebral infarction (CI), a blood circulatory disorder, causes a high mortality and disability rate worldwide. Intriguingly, a newly discovered neuropeptide, Cortistatin (CST), has been indicated to inhibit the cortical activity. In our research, we aimed to explore the functional relevance of CST in neural stem cells (NSCs) in CI rats. The expression of CST was determined in NSCs induced by oxygen-glucose deprivation (OGD). NSCs isolated from the embryonic rat brain were treated with OGD to establish an in vitro CI model while dithiothreitol (DTT) was introduced to induce endoplasmic reticulum stress (ERS), which were evaluated by assessment of GRP94, caspase-12 and CHOP expression. Then CST expression was restored by transfection of oe-CST, followed by assessment of NSC proliferation ability and cytotoxicity. Finally, the expression of CST and its receptor Somatostatin receptor subtype 2 (SSTR2) was quantified for mechanism exploration. CST was downregulated in CI, which was further confirmed in NSCs under OGD treatment. Overexpressed CST was found to promote cell activity and attenuate OGD-induced cytotoxicity of NSCs. Meanwhile, it was observed that the injured proliferation ability of NSCs was restored by CST overexpression. Besides, lower expression of GRP94, caspase-12 and CHOP was indicative of suppressed occurrence of ERS by CST. Mechanically, CST inhibited ERS through SSTR2. CST could facilitate the proliferation of NSCs in CI induced by OGD, ultimately highlighting a novel therapeutic target for CI treatment.

Neuronal Trans-differentiation by Transcription Factors Ascl1 and Nurr1: Induction of a Dopaminergic Neurotransmitter Phenotype in Cortical GABAergic Neurons.

Neurons with a desired neurotransmitter phenotype can be differentiated from induced pluripotent stem cells or from somatic cells only through tedious protocols with relatively low yield. Readily available cortical neurons isolated from embryonic rat brain, which have already undergone a complete neuronal differentiation process, might serve as alternative template source. These cultures consist of 85% glutamatergic and 15% GABAergic neurons, and we attempted to trans-differentiate them into dopaminergic neurons. Transcription factors Nurr1, Lmx1A and Pitx3, essential determinants of a dopaminergic cell fate during CNS development, were not sufficient to induce tyrosine hydroxylase expression in a significant number of cells. Combining Nurr1 with the generic neuronal differentiator and re-programming factor Ascl1, however, resulted in generation of neurons which express dopaminergic markers TH, AADC, VMAT2 and DAT. Only neurons of GABAergic phenotype could be trans-differentiated towards a dopaminergic neurotransmitter phenotype, while for glutamatergic neurons, this process proved to be neurotoxic. Intriguingly, GABAergic neurons isolated from embryonal midbrain could not be trans-differentiated into dopaminergic neurons by Ascl1 and Nurr1. Thus, in principle, post-mitotic embryonal neurons can serve as templates for neurons with a desired neurotransmitter phenotype. However, neurotransmitter phenotype plasticity critically depends on the differentiation history of the template neurons, which can result in relatively low yields of dopaminergic neurons.

Exposure of Rat Neural Stem Cells to Ethanol Affects Cell Numbers and Alters Expression of 28 Proteins.

Developing brain cells express many proteins but little is known of how their protein composition responds to chronic exposure to alcohol and/or how such changes might relate to alcohol toxicity. We used cultures derived from embryonic rat brain (previously shown to contain mostly neural stem cells; rat NSC, rNSC), exposed them to ethanol (25-100mM) for up to 96h and studied how they reacted. Ethanol (50 and 100mM) reduced cell numbers indicating either compromised cell proliferation, cytotoxicity or both. Increased lipid peroxidation was consistent with the presence of oxidative stress accompanying alcohol-induced cytotoxicity. Proteomics revealed 28 proteins as altered by ethanol (50mM for 96h). Some were constituents of cytoskeleton, others were involved in transcription/translation, signal transduction and oxidative stress. Nucleophosmin (NPM1) and dead-end protein homolog 1 (DND1) were further studied by immunological techniques in cultured neurons and astrocytes (derived from brain tissue at embryonic ages E15 and E20, respectively). In the case of DND1 (but not NPM1) ethanol induced similar pattern of changes in both types of cells. Given the critical role of the protein NPM1 in cell proliferation and differentiation, its reduced expression in the ethanol-exposed rNSC could, in part, explain the lower cells numbers. We conclude that chronic ethanol profoundly alters protein composition of rNSC to the extent that their functioning-including proliferation and survival-would be seriously compromised. Translated to humans, such changes could point the way towards mechanisms underlying the fetal alcohol spectrum disorder and/or alcoholism later in life.

Somatic Depdc5 deletion recapitulates electroclinical features of human focal cortical dysplasia type IIA.

Epileptogenic mechanisms in focal cortical dysplasia (FCD) remain elusive, as no animal models faithfully recapitulate FCD seizures, which have distinct electrographic features and a wide range of semiologies. Given that DEPDC5 plays significant roles in focal epilepsies with FCD, we used in utero electroporation with clustered regularly interspaced short palindromic repeats gene deletion to create focal somatic Depdc5 deletion in the rat embryonic brain. Animals developed spontaneous seizures with focal pathological and electroclinical features highly clinically relevant to FCD IIA, paving the way toward understanding its pathogenesis and developing mechanistic-based therapies. Ann Neurol 2018;83:140-146.

Post-operative unadjuvanted therapeutic xenovaccination with chicken whole embryo vaccine suppresses distant micrometastases and prolongs survival in a murine Lewis lung carcinoma model.

Immunotherapy in the form of anticancer vaccination relies on the mobilization of the patient's immune system against specific cancer antigens. Instead of focusing on an autologous cell lysate, which is not always available in clinical practice, the present study investigates vaccines utilizing xenogeneic foetal tissue that are rich in oncofoetal antigens. Lewis lung carcinoma (LLC)-challenged C57BL/6 mice were treated with either a xenogeneic vaccine made from chicken whole embryo, or a xenogeneic vaccine made from rat embryonic brain tissue, supplemented with a Bacillus subtilis protein fraction as an adjuvant. Median and overall survival, size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared with untreated control mice. Following primary tumour removal, a course of three subcutaneous vaccinations with xenogeneic chicken embryo vaccine led to significant increase in overall survival rate (100% after 70 days of follow-up vs. 40% in untreated control mice), significant increase in circulating CD8a+ T cells (18.18 vs. 12.6% in untreated control mice), and a significant decrease in the area and incidence of metastasis foci. The xenogeneic rat brain tissue-based vaccine did not improve any of the investigated parameters, despite promising reports in other models. We hypothesize that the proper selection of antigen source (tissue) can constitute an effective immunotherapeutic product.

Developmental stage-specific expression of genes for sphingomyelin synthase in rat brain.

Sphingomyelin synthase genes (Sgms1 and Sgms2) encode the vital enzymes that participate in the processes of membrane transport, cell proliferation and apoptosis. We previously determined the exon-intron structure of Sgms1 and some features of its expression in human and rodent tissues. The circular RNAs (circRNAs) emerging from exons of the 5'-untranslated region (5'-UTR) of Sgms1 were determined. These circRNAs are represented at a high level in the adult brain. Here, we demonstrate that, in contrast to Sgms1, Sgms2 does not contain the multi-exon 5'-UTR but encodes circRNAs, which are composed of the coding region of the gene and are expressed at a low level. We present a study of the expression of sphingomyelin synthase genes in rat brain at embryonic days 7, 9, 13, 17 and 21 and in adult rat brain. In contrast to Sgms1, Sgms2 is expressed at a significantly low level in adult brain. In embryonic rat brain, the mRNA expression of sphingomyelin synthase genes is varied in a developmental stage-specific manner. The level of Sgms1 mRNAs, differing by 5'-UTR-in the formation of which alternative promoters can participate-changes significantly during the process of embryonic development. The expression of circRNAs of Sgms1 was significantly raised during rat embryonic brain development. We assume that the circRNAs are involved in the regulation of sphingomyelin synthase activity in rat brain in different developmental stages.

Lin28b and Sox2 regulate anesthesia-induced neural degeneration in neural stem cell derived neurons

Ketamine, a commonly used anesthetic compound, may cause neurotoxicity in immature or developing brains. In this study, we indented to understand the molecular expression and functional role of Lin-28 Homolog B (lin28b) in regulating ketamine-induced neurotoxicity in neural stem cells (NSCs)-differentiated neurons. NSCs from embryonic rat brains were cultured in vitro, and induced toward neuronal differentiation. NSCs-differentiated neurons were treated with various concentrations of ketamine for 24h to evaluate the concentration-dependent effect of ketamine on endogenous lin28b mRNA level. QRT-PCR showed that lin28b was downregulated by ketamine in NSCs-differentiated neurons, in concentration-dependent manner. Neurons were then transfected with adenovirus to ectopically upregulate lin28b. We found that ketamine-induced apoptosis and neurite retraction in NSCs-differentiated neurons were significantly reduced by adenovirus-mediated lin28b upregulation. Expression of sex determining region Y box 2 (Sox2) mRNA was examined in ketamine-injured and lin28b-upregulated NSCs-differentiated neurons. It was found Sox2 was downregulated by ketamine, and overexpressed by lin28b upregulation. Finally, Sox2 was downregulated by siRNA in NSCs-differentiated neurons. And we discovered that Lin28b-upregulation-associated neural protection was severely hampered by Sox2 downregulation in ketamine-injured neurons. Thus, Lin28b and Sox2 are important molecular components in ketamine-induced neurotoxicity.

Turning Death to Growth: Hematopoietic Growth Factors Promote Neurite Outgrowth through MEK/ERK/p53 Pathway.

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) are the essential hematopoietic growth factors to control hematopoiesis. However, the role of SCF and G-CSF in the central nervous system remains poorly understood. Here, we have demonstrated the involvement of MEK/ERK/p53 signaling in SCF+G-CSF-enhanced neurite extension. Cortical neurons dissected from embryonic rat brains were seeded onto the membranes of transwell inserts, and neurite outgrowth was determined by using both the neurite outgrowth quantification assay kit and immunostaining of β III tubulin. Quantitative RT-PCR and western blotting were used for determining gene and protein expression of ERK and p53, respectively. p53 small interfering RNA (siRNAs) were introduced into neurons for examining the involvement of p53 in SCF+G-CSF-mediated neurite outgrowth. We observed that both SCF and G-CSF alone increased activation of MEK/ERK and gene expression of p53, while SCF+G-CSF synergistically activated the MEK/ERK signaling and upregulated p53 expression. MEK specific inhibitors (PD98059 and U0126) blocked the SCF+G-CSF-increased ERK phosphorylation and p53 gene and protein expression, and the MEK specific inhibitors also eliminated the SCF+G-CSF-promoted neurite outgrowth. p53 siRNAs knocked down the SCF+G-CSF-elevated p53 protein and prevented the SCF+G-CSF-enhanced neurite outgrowth. These findings suggest that activation of MEK/ERK/p53 signaling is required for SCF+G-CSF-promoted neurite outgrowth. Through the pro-apoptotic pathway of the MEK/ERK/p53, SCF+G-CSF turns neuronal fate from apoptotic commitment toward neural network generation. This observation provides novel insights into the putative role of SCF+G-CSF in supporting generation of neural connectivity during CNS development and in brain repair under pathological or neurodegenerative conditions.