Primary Neural Cell Cultures Research Articles

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)G93A, revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord-derived EVs, from NTg and SOD1G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.

Multiple effects of the herbicide glufosinate-ammonium and its main metabolite on neural stem cells from the subventricular zone of newborn mice

The globally used herbicide glufosinate-ammonium (GLA) is structurally analogous to the excitatory neurotransmitter glutamate, and is known to interfere with cellular mechanisms involved in the glutamatergic system. In this report, we used an in vitro model of murine primary neural stem cell culture to investigate the neurotoxicity of GLA and its main metabolite, 4-methylphosphinico-2-oxobutanoic acid (PPO). We demonstrated that GLA and PPO disturb ependymal wall integrity in the ventricular-subventricular zone (V-SVZ) and alter the neuro-glial differentiation of neural stem cells. GLA and PPO impaired the formation of cilia, with reduced Celsr2 expression after PPO exposure. GLA promoted the differentiation of neuronal and oligodendroglial cells while PPO increased B1 cell population and impaired neuronal fate of neural stem cells. These results confirm our previous in vivo report that developmental exposure to GLA alters neurogenesis in the SVZ, and neuroblast migration along the rostral migratory stream. They also highlight the importance of investigating the toxicity of pesticide degradation products. Indeed, not only GLA, but also its metabolite PPO disrupts V-SVZ homeostasis and provides a novel cellular mechanism underlying GLA-induced neurodevelopmental toxicity. Furthermore, we were able to demonstrate a neurotoxic activity of a metabolite of GLA different from that of GLA active substance for the very first time.

Zika virus E protein alters the properties of human fetal neural stem cells by modulating microRNA circuitry.

Zika virus (ZV) infects neural stem cells (NSCs) and causes quiescence in NSCs, reducing the pool of brain cells, leading to microcephaly. Despite conscientious efforts, the molecular mechanisms for ZV-mediated effects on NSCs lack clarity. This study aimed to explore the underlying mechanisms for ZV-mediated induction of quiescence in the primary cultures of human fetal neural stem cells (fNSCs). We demonstrate that expression of ZV envelope (E) protein displays maximum quiescence in human fNSCs by accumulating cells in the G0/G1 phase of the cell cycle as compared to other non-structural proteins, viz. NS2A, NS4A and NS4B. E protein induces immature differentiation by induction of pro-neuronal genes in proliferating fNSCs, induces apoptosis in differentiating fNSCs 3 days post differentiation, and disrupts migration of cells from differentiating neurospheres. In utero electroporation of mouse brain with E protein shows drastic downregulation of proliferating cells in ventricular and subventricular zone regions. Global microRNA sequencing suggests that E protein modulates miRNA circuitry. Among differentially expressed miRNAs, we found 14 upregulated and 11 downregulated miRNAs. Mir-204-3p and mir-1273g-3p directly regulate NOTCH2 and PAX3 expression, respectively, by binding to their 3'UTR. Bioinformatic analysis using GO analysis for the targets of differentially expressed miRNAs revealed enrichment of cell cycle and developmental processes. Furthermore, WNT, CCKR, PDGF, EGF, p53, and NOTCH signaling pathways were among the top enriched pathways. Thus, our study provides evidence for the involvement of ZV E protein and novel insights into the molecular mechanism through identification of miRNA circuitry. Art work depicting the effect of Zika virus E protein on human fetal neural stem cells.

Modulation of the intrinsic neuronal excitability by multifunctional liposomes tailored for the treatment of Alzheimer's disease.

PurposeNanotechnologies turned out to be promising in the development of diagnostic and therapeutic approaches toward neurodegenerative disorders. However, only a very scant number of nanodevices until now proved to be effective on preclinical animal models. Although specific tests in vivo are available to assess the potential toxicity of these nanodevices on cognitive functions, those to evaluate their biosafety in vitro on neurons are still to be improved.Materials and methodsWe utilized the patch-clamp technique on primary cultures of cortical neural cells isolated from neonatal rats, aiming to evaluate their electrical properties after the incubation with liposomes (mApoE-PA-LIPs), previously proved able to cross the blood–brain barrier and to be effective on mouse models of Alzheimer’s disease (AD), both in the absence and in the presence of β-amyloid peptide oligomers.ResultsData show a high degree of biocompatibility, evaluated by lactate dehydrogenase (LDH) release and MTT assay, and the lack of cellular internalization. After the incubation with mApoE-PA-LIPs, neuronal membranes show an increase in the input resistance (from 724.14±76 MΩ in untreated population to 886.06±86 MΩ in the treated one), a reduction in the rheobase current (from 29.6±3 to 24.2±3 pA in untreated and treated, respectively), and an increase of the firing frequency, consistent with an ultimate increase in intrinsic excitability. Data obtained after co-incubation of mApoE-PA-LIPs with β-amyloid peptide oligomers suggest a retention of liposome efficacy.ConclusionThese data suggest the ability of liposomes to modulate neuronal electrical properties and are compatible with the previously demonstrated amelioration of cognitive functions induced by treatment of AD mice with liposomes. We conclude that this electrophysiological approach could represent a useful tool for nanomedicine to evaluate the effect of nanoparticles on intrinsic neuronal excitability.

Paraquat and Maneb Exposure Alters Rat Neural Stem Cell Proliferation by Inducing Oxidative Stress: New Insights on Pesticide-Induced Neurodevelopmental Toxicity.

Pesticide exposure has been linked to the pathogenesis of neurodevelopmental and neurodegenerative disorders including autism spectrum disorders, attention deficit/hyperactivity, and Parkinson's disease (PD). Developmental exposure to pesticides, even at low concentrations not harmful for the adult brain, can lead to neuronal loss and functional deficits. It has been shown that prenatal or early postnatal exposure to the herbicide paraquat (PQ) and the fungicide maneb (MB), alone or in combination, causes permanent toxicity in the nigrostriatal dopamine system, supporting the idea that early exposure to these pesticides may contribute to the pathophysiology of PD. However, the mechanisms mediating PQ and MB developmental neurotoxicity are not yet understood. Therefore, we investigated the neurotoxic effect of low concentrations of PQ and MB in primary cultures of rat embryonic neural stem cells (NSCs), with particular focus on cell proliferation and oxidative stress. Exposure to PQ alone or in combination with MB (PQ + MB) led to a significant decrease in cell proliferation, while the cell death rate was not affected. Consistently, PQ + MB exposure altered the expression of major genes regulating the cell cycle, namely cyclin D1, cyclin D2, Rb1, and p19. Moreover, PQ and PQ + MB exposures increased the reactive oxygen species (ROS) production that could be neutralized upon N-acetylcysteine (NAC) treatment. Notably, in the presence of NAC, Rb1 expression was normalized and a normal cell proliferation pattern could be restored. These findings suggest that exposure to PQ + MB impairs NSCs proliferation by mechanisms involving alterations in the redox state.

Layers and Multilayers of Self-Assembled Polymers: Tunable Engineered Extracellular Matrix Coatings for Neural Cell Growth

Growing primary cells and tissue in long-term cultures, such as primary neural cell culture, presents many challenges. A critical component of any environment that supports neural cell growth in vivo is an appropriate 2-D surface or 3-D scaffold, typically in the form of a thin polymer layer that coats an underlying plastic or glass substrate and aims to mimic critical aspects of the extracellular matrix. A fundamental challenge to mimicking a hydrophilic, soft natural cell environment is that materials with these properties are typically fragile and are difficult to adhere to and stabilize on an underlying plastic or glass cell culture substrate. In this review, we highlight the current state of the art and overview recent developments of new artificial extracellular matrix (ECM) surfaces for in vitro neural cell culture. Notably, these materials aim to strike a balance between being hydrophilic and soft while also being thick, stable, robust, and bound well to the underlying surface to provide an effective surface to support long-term cell growth. We focus on improved surface and scaffold coating systems that can mimic the natural physicochemical properties that enhance neuronal survival and growth, applied as soft hydrophilic polymer coatings for both in vitro cell culture and for implantable neural probes and 3-D matrixes that aim to enhance stability and longevity to promote neural biocompatibility in vivo. With respect to future developments, we outline four emerging principles that serve to guide the development of polymer assemblies that function well as artificial ECMs: (a) design inspired by biological systems and (b) the employment of principles of aqueous soft bonding and self-assembly to achieve (c) a high-water-content gel-like coating that is stable over time in a biological environment and possesses (d) a low modulus to more closely mimic soft, compliant real biological tissue. We then highlight two emerging classes of thick material coatings that have successfully captured these guiding principles: layer-by-layer deposited water-soluble polymers (LbL) and silk fibroin (SF) materials. Both materials can be deposited from aqueous solution yet transition to a water-insoluble coating for long-term stability while retaining a softness and water content similar to those of biological materials. These materials hold great promise as next-generation biocompatible coatings for tissue engineers and for chemists and biologists within the biomedical field.

Vitamin D Improves Neurogenesis and Cognition in a Mouse Model of Alzheimer\u2019s Disease

The impairment of hippocampal neurogenesis at the early stages of Alzheimer’s disease (AD) is believed to support early cognitive decline. Converging studies sustain the idea that vitamin D might be linked to the pathophysiology of AD and to hippocampal neurogenesis. Nothing being known about the effects of vitamin D on hippocampal neurogenesis in AD, we assessed them in a mouse model of AD. In a previous study, we observed that dietary vitamin D supplementation in female AD-like mice reduced cognitive decline only when delivered during the symptomatic phase. With these data in hand, we wondered whether the consequences of vitamin D administration on hippocampal neurogenesis are stage-dependent. Male wild-type and transgenic AD-like mice (5XFAD model) were fed with a diet containing either no vitamin D (0VD) or a normal dose of vitamin D (NVD) or a high dose of vitamin D (HVD), from month 1 to month 6 (preventive arm) or from month 4 to month 9 (curative arm). Working memory was assessed using the Y-maze, while amyloid burden, astrocytosis, and neurogenesis were quantified using immunohistochemistry. In parallel, the effects of vitamin D on proliferation and differentiation were assayed on primary cultures of murine neural progenitor cells. Improved working memory and neurogenesis were observed when high vitamin D supplementation was administered during the early phases of the disease, while a normal dose of vitamin D increased neurogenesis during the late phases. Conversely, an early hypovitaminosis D increased the number of amyloid plaques in AD mice while a late hypovitaminosis D impaired neurogenesis in AD and WT mice. The observed in vivo vitamin D-associated increased neurogenesis was partially substantiated by an augmented in vitro proliferation but not an increased differentiation of neural progenitors into neurons. Finally, a sexual dimorphism was observed. Vitamin D supplementation improved the working memory of males and females, when delivered during the pre-symptomatic and symptomatic phases, respectively. Our study establishes that (i) neurogenesis is improved by vitamin D in a male mouse model of AD, in a time-dependent manner, and (ii) cognition is enhanced in a gender-associated way. Additional pre-clinical studies are required to further understand the gender- and time-specific mechanisms of action of vitamin D in AD. This may lead to an adaptation of vitamin D supplementation in relation to patient’s gender and age as well as to the stage of the disease.

In Vitro Maintenance of Multipotent Neural Crest Stem Cells as Crestospheres.

Neural crest cells are a critical source of many cell types of the vertebrate body. However, as a stem cell population they are peculiar because of the transient nature of their stem cell niche; soon after the multipotent neural crest cells are specified in the neuroepithelium, they become mesenchymal cells that migrate into various destinations in early embryos. These rapid in vivo changes during neural crest development complicate the studies on their stem cell properties. Crestospheres are in vitro maintained primary cultures of premigratory neural crest cells that maintain a mixture of neural crest stem and progenitor cells for weeks without spontaneous differentiation, including the multipotent neural crest stem cells. Here, we describe how crestosphere cultures are initiated from either cranial or trunk levels of chick embryos. Alternatively, the same culture conditions can be used to maintain human embryonic stem cell-derived neural crest cells as crestospheres. Thus, crestospheres provide a useful tool for studies on neural crest stemness.

Proneurogenic Effects of Trazodone in Murine and Human Neural Progenitor Cells.

Several antidepressants increase adult hippocampal neurogenesis (ahNG) in rodents, primates, and, potentially, humans. This effect may at least partially account for their therapeutic activity. The availability of antidepressants whose mechanism of action involves different neurotransmitter receptors represents an opportunity for increasing our knowledge on their distinctive peculiarities and for dissecting the contribution of receptor subtypes in ahNG modulation. The aim of this study was to evaluate, in vitro, the effects of the antidepressant trazodone (TZD) on ahNG by using primary cultures of murine adult hippocampal neural progenitor cells (ahNPCs) and human induced pluripotent stem cell (iPSC)-derived NPCs. We demonstrated that TZD enhances neuronal differentiation of murine as well as human NPCs. TZD is a multimodal antidepressant, which binds with high affinity to 5-HT2a, α1, and 5-HT1a and with lower affinity to 5-HT2c, α2and 5-HTT. We demonstrated that TZD proneurogenic effects were mediated by 5-HT2a antagonism both in murine and in human NPCs and by 5-HT2c antagonism in murine cells. Moreover NF-κB p50 nuclear translocation appeared to be required for TZD-mediated proneurogenic effects. Interestingly, TZD had no proneurogenic effects in 5-HT depleted ahNPCs. The TDZ bell-shaped dose-response curve suggested additional effects. However, in our model 5-HT1a and α1/α2 receptors had no role in neurogenesis. Overall, our data also demonstrated that serotoninergic neurotransmission may exert both positive and negative effects on neuronal differentiation of ahNPCs in vitro.

GPE Promotes the Proliferation and Migration of Mouse Embryonic Neural Stem Cells and Their Progeny In Vitro.

This study was designed to investigate a possible role of the N-terminal tripeptide of insulin-like growth factor-1 (IGF-I), Gly-Pro-Glu (GPE), physiologically generated in neurons following IGF-I-specific cleavage, in promoting neural regeneration after an injury. Primary cultures of mouse neural stem cells (NSCs), obtained from 13.5 Days post-conception (dpc) mouse embryos, were challenged with either GPE, growth hormone (GH), or GPE + GH and the effects on cell proliferation, migration, and survival were evaluated both under basal conditions and in response to a wound healing assay. The cellular pathways activated by GPE were also investigated by using specific chemical inhibitors. The results of the study indicate that GPE treatment promotes the proliferation and the migration of neural stem cells in vitro through a mechanism that involves the activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase PI3K-Akt pathways. Intriguingly, both GPE effects and the signaling pathways activated were similar to those observed after GH treatment. Based upon the results obtained from this study, GPE, as well as GH, may be useful in promoting neural protection and/or regeneration after an injury.

Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA.

Abstract TP104: A Novel in vitro Model of Cerebral Ischemia Using Patient-derived Induced Pluripotent Stem Cells

Stroke constitutes the 5th cause of death in the United States and an important cause of morbidity. During ischemic stroke injury, the blood-brain barrier (BBB, part of the neurovascular unit) is compromised as brain microvascular endothelial cells (BMECs) loose their tight junctions complexes, resulting in the loss of the barrier function, unrestricted entrance of water and ions inside the brain parenchyma and neuronal cell death by excitotoxicity and cerebral edema formation. Despite the important effort to find neuroprotective agents capable to partially improve neuronal cell survival and restore the BBB function, our ability to translate findings from animal studies to clinical trials is still hampered by an important failure rate. One of the possible caveats we hypothesize that contributes to such important failure rate is the absence of a pre-clinical human model capable to ease the translation. We speculate that a human in vitro model of the neurovascular unit encompassing human BMECs, neurons and astrocytes may help improve such translation. In this study, we describe a novel in vitro model based on human induced pluripotent stem (iPSCs) cells. Using IMR90-c4 as an iPSC line, we were capable to differentiate BMECs, astrocytes and neurons. Using oxygen-glucose deprivation (OGD stress), we were able to demonstrate the ability of our iPSC-derived BMECs to respond to such injury by a loss of the cell barrier function similarly, such cellular response was similar to hCMEC/D3 monolayers (an immortalized human BMEC line commonly used). In contrast, iPSC-derived astrocytes showed a remarkable compliance to prolonged OGD stress, showing some consistency with the previous literature. In the other hand, preliminary data obtained with iPSC-derived neurons displayed a rapid decrease in cell viability and increase in cell death suggesting similarities with studies using primary neural cell cultures. Taken together, we document a differentiation protocol capable to obtain pure iPSC-derived BMECs, astrocytes and neurons lineages allowing us to investigate the cellular and molecular response to stroke injury in vitro. Such model may provide an interesting alternative model to improve our probability to identify novel stroke neuroprotective drug candidates.

Long-term culture and spontaneous recordings of primary neurons on conducting polymer microelectrode arrays

Event Abstract Back to Event Long-term culture and spontaneous recordings of primary neurons on conducting polymer microelectrode arrays Jolien Pas1, Dimitrios A. Koutsouras1 and George Malliaras1 1 Ecole Nationale Supérieure des Mines de Saint-Etienne, Bioelectronics, France Microelectrode arrays (MEAs) have widely been used as diagnostic tool to improve our understanding of the beautiful yet extremely complex nervous system. MEAs are used to record and stimulate brain tissue or neuronal cells, thereby providing a platform for both in vivo and in vitro experiments. To circumvent the complexity of the nervous system, in vitro measurements enable us to simplify the biological environment and help us to attain specific essential neuronal knowledge. By using one of the most promising conducting polymers poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT: PSS), unique MEAs are developed[1] with an ideal dual character for neuronal interfacing due to their potential to conduct both electronic and ionic carriers. Nevertheless, it remains a challenge to culture and maintain a healthy primary neural cell culture over a long period of time to record their spontaneous electrical cell activity. Here we show long-term survival of primary cortex cells on PEDOT:PSS coated MEAs and show high quality recording of electrophysiological signals. We find that the combination of dropcasting poly-D-lysine (PDL, 50 µg/mL) and laminin (20 µg/mL) provide a vital adhesive support for E18 cortical rat neurons. This enables culture of these cells for more than 28 days which is followed by simultaneous multichannel recordings of spontaneous signals at 1 kHz with an average amplitude of 100 µV. Moreover, with the addition of bicuculline (50 µM) as a GABAA-antagonist during recording, bursts of action potentials are obtained similar to previously demonstrated results of Arnold et al[2]. It means that we have created the necessary cell-friendly environment for culture of such fragile primary neurons on state-of-the art MEAs. This work thereby paves the way towards a more diagnostic and eventually potentially therapeutic PEDOT:PSS platform to enable the study of neural electrophysiology and neuropathology.

Neuronal Regulation of Neuroprotective Microglial Apolipoprotein E Secretion in Rat In Vitro Models of Brain Pathophysiology.

Apolipoprotein E (ApoE) is mainly secreted by glial cells and is involved in many brain functions, including neuronal plasticity, β-amyloid clearance, and neuroprotection. Microglia--the main immune cells of the brain--are one source of ApoE, but little is known about the physiologic regulation of microglial ApoE secretion by neurons and whether this release changes under inflammatory or neurodegenerative conditions. Using rat primary neural cell cultures, we show that microglia release ApoE through a Golgi-mediated secretion pathway and that ApoE progressively accumulates in neuroprotective microglia-conditioned medium. This constitutive ApoE release is negatively affected by microglial activation both with lipopolysaccharide and with ATP. Microglial ApoE release is stimulated by neuron-conditioned media and under coculture conditions. Neuron-stimulated microglial ApoE release is mediated by serine and glutamate through N-methyl-D-aspartate receptors and is differently regulated by activation states (i.e. lipopolysaccharide vs ATP) and by 6-hydroxydopamine. Microglial ApoE silencing abrogated protection of cerebellar granule neurons against 6-hydroxydopamine toxicity in cocultures, indicating that microglial ApoE release is neuroprotective. Our findings shed light on the reciprocal cross-talk between neurons and microglia that is crucial for normal brain functions. They also open the way for the identification of possible pharmacologic targets that can modulate neuroprotective microglial ApoE release under pathologic conditions.

Variation in growth pattern and morphological appearance of primary monolayer cultures of chondrocytes and neural cells isolated from the chick embryo at different stages

Aim: This study was an attempt at investigating the variation in growth and morphology of primary avian chondrocyte and neural cell cultures established under standard laboratory conditions. Methods: For establishing chondrocyte cultures, the tibia were dissected from chick embryos that were 12 (E12), 14 (E14) and 15 (E15) days old. For the neural cell culture initiation, the two lobes of the forebrain were dissected from chick embryos that were 4 (E4), 8 (E8) and 12 (E12) days old. The final characterization of the cells was performed by H&E and CFV staining. Results: In the chondrocyte cultures, two forms of morphology were observed which is reversible in process.The primary variation evaluated in chondrocytes cultures was the effect of the media on the state of the cells. Based on whether the cells were cultured in DMEM or MEM, there was a difference in the transformation of the attached cells from the differentiated to de-differentiated forms. The primary variation examined in neuron cultures was the embryonic age of the tissue and the effect that it had on the proliferation and neurite formation of the cells. E8 embryo neural cells cultures result in well-developed cells with neurite formation along with axonal and dendritic outgrowths with highly complex interconnections between them. Conclusion: Ultimately, this study demonstrated the composition of culture media had an effect on the morphological appearance of the chondrocytes, as well as, confirmed that the culture of primary neurons is best performed using cells from an E8 embryo.

FOXO3 Shares Common Targets with ASCL1 Genome-wide and Inhibits ASCL1-Dependent Neurogenesis

FOXO transcription factors are central regulators of longevity from worms to humans. FOXO3, the FOXO isoform associated with exceptional human longevity, preserves adult neural stem cell pools. Here, we identify FOXO3 direct targets genome-wide in primary cultures of adult neural progenitor cells (NPCs). Interestingly, FOXO3-bound sites are enriched for motifs for bHLH transcription factors, and FOXO3 shares common targets with the proneuronal bHLH transcription factor ASCL1/MASH1 in NPCs. Analysis of the chromatin landscape reveals that FOXO3 and ASCL1 are particularly enriched at the enhancers of genes involved in neurogenic pathways. Intriguingly, FOXO3 inhibits ASCL1-dependent neurogenesis in NPCs and direct neuronal conversion in fibroblasts. FOXO3 also restrains neurogenesis in vivo. Our study identifies a genome-wide interaction between the prolongevity transcription factor FOXO3 and the cell-fate determinant ASCL1 and raises the possibility that FOXO3's ability to restrain ASCL1-dependent neurogenesis may help preserve the neural stem cell pool.

In vitro imaging of primary neural cell culture from Drosophila

Cell culture systems are widely used for molecular, genetic and biochemical studies. Primary cell cultures of animal tissues offer the advantage that specific cell types can be studied in vitro outside of their normal environment. We provide a detailed protocol for generating primary neural cell cultures derived from larval brains of Drosophila melanogaster. The developing larval brain contains stem cells such as neural precursors and intermediate neural progenitors, as well as fully differentiated and functional neurons and glia cells. We describe how to analyze these cell types in vitro by immunofluorescent staining and scanning confocal microscopy. Cell type-specific fluorescent reporter lines and genetically encoded calcium sensors allow the monitoring of developmental, cellular processes and neuronal activity in living cells in vitro. The protocol provides a basis for functional studies of wild-type or genetically manipulated primary neural cells in culture, both in fixed and living samples. The entire procedure takes ∼3 weeks.

Microwave-assisted synthesis of hydroxyphenyl nitrones with protective action against oxidative stress

Oxidative stress plays an important role in neuronal death in neurodegenerative disorders such as Parkinson's disease (PD). Hydroxyphenyl nitrones, derivatives of the nitrone spin trap alpha-phenyl-N-tert-butylnitrone (PBN), were synthesized and their antioxidant, anti-inflammatory and neuroprotective activity in neural cells evaluated. These hydroxyphenyl nitrones 5–7 were synthesized by reaction of the corresponding hydroxybenzaldehyde with N-tert-butyl hydroxylamine under microwave irradiation. They showed good peroxyl free radical scavenger capacities, analyzed by oxygen radical absorbance capacity (ORAC). Also inhibited peroxynitrite-mediated tyrosine nitration of alpha-synuclein in vitro and protected human neuroblastoma (SH-SY5Y) cells against SIN-1 and 6-OHDA toxicity when micromolar concentrations were used. Besides, the hydroxyphenyl nitrones evaluated showed anti-inflammatory activity modulating nitrite production in primary neural cell cultures of astrocytes and microglia treated with lipopolysaccharide (LPS), a potent inflammatory agent. These experimental data suggest a potential therapeutic use of these hydroxyphenyl nitrones against oxygen and nitrogen reactive species involved in neurodegenerative pathology.

Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo.

Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia

BackgroundMagnetic nanoparticles (MNPs) offer a large range of applications in life sciences. Applications in neurosciences are one focus of interest. Unfortunately, not all groups have access to nanoparticles or the possibility to develop and produce them for their applications. Hence, they have to focus on commercially available particles. Little is known about the uptake of nanoparticles in primary cells. Previously studies mostly reported cellular uptake in cell lines. Here we present a systematic study on the uptake of magnetic nanoparticles (MNPs) by primary cells of the nervous system.ResultsWe assessed the internalization in different cell types with confocal and electron microscopy. The analysis confirmed the uptake of MNPs in the cells, probably with endocytotic mechanisms. Furthermore, we compared the uptake in PC12 cells, a rat pheochromocytoma cell line, which is often used as a neuronal cell model, with primary neuronal cells. It was found that the percentage of PC12 cells loaded with MNPs was significantly higher than for neurons. Uptake studies in primary mixed neuronal/glial cultures revealed predominant uptake of MNPs by microglia and an increase in their number. The number of astroglia and oligodendroglia which incorporated MNPs was lower and stable. Primary mixed Schwann cell/fibroblast cultures showed similar MNP uptake of both cell types, but the Schwann cell number decreased after MNP incubation. Organotypic co-cultures of spinal cord slices and peripheral nerve grafts resembled the results of the dispersed primary cell cultures.ConclusionsThe commercial MNPs used activated microglial phagocytosis in both disperse and organotypic culture systems. It can be assumed that in vivo application would induce immune system reactivity, too. Because of this, their usefulness for in vivo neuroscientific implementations can be questioned. Future studies will need to overcome this issue with the use of cell-specific targeting strategies. Additionally, we found that PC12 cells took up significantly more MNPs than primary neurons. This difference indicates that PC12 cells are not a suitable model for natural neuronal uptake of nanoparticles and qualify previous results in PC12 cells.