Embryonic Rat Cortical Neurons Research Articles

Metformin alters dendrite development and synaptic plasticity in rat cortical neurons

Synaptic plasticity is crucial as it dynamically molds the strength and connectivity of neural circuits, influencing learning, memory, and the development of neurological disorders. Metformin, a widely prescribed anti-diabetic medication, has been shown to readily cross the blood-brain barrier (BBB) and the placenta. However, its prolonged impact on neuronal morphology and functions remains underexplored. In this study, we investigated the influence of metformin on dendrite development and synaptic plasticity in embryonic brains and primary rat cortical neurons. Our findings reveal a negative modulation of dendrite development by metformin, as evidenced by altered dendritic arborization, impaired dendritic spine morphology and disruptions in synaptic plasticity, suggesting a potential link between metformin exposure and aberrations in neuronal connectivity. In addition, we extend our insights to the impact of maternal metformin exposure on embryonic brains, revealing a significant inhibition of dendrite development in E18.5 rat brains. In conclusion, this study adds to the expanding knowledge base on the non-metabolic effects of metformin, emphasizing the significance of assessing its potential influence on both neuronal structure and function. There is an urgent need for further investigations into the enduring impact of prolonged metformin administration on the structural and functional aspects of neurons.

Enzyme-Mediated Proximity Labeling Identifies Small RNAs in the Endoplasmic Reticulum Lumen.

In eukaryotic cells, the subcellular targeting of RNA controls many fundamental aspects of cellular physiology. Despite broad distributions throughout the cytoplasm, RNA molecules are conventionally believed to be excluded from the secretory pathway compartments including the endoplasmic reticulum (ER). Recent discovery of RNA N-glycan modification (glycoRNAs) has challenged this view, but direct evidence of RNA localization in the ER lumen has been lacking. In this study, we applied enzyme-mediated proximity labeling to profile the ER lumen-localized RNAs in human embryonic kidney 293T cells and rat cortical neurons. Our data set reveals the presence of small non-coding RNAs in the ER lumen, including U RNAs and Y RNAs, which raises interesting questions regarding their transport mechanism and biological functions in the ER.

SCO-spondin-derived peptide NX210 rescues neurons from cerebral ischemia/reperfusion injury through modulating the Integrin-β1 mediated PI3K/Akt pathway

Ischemic stroke is a common condition with high morbidity and mortality, causing irreversible neuronal damage and seriously affecting neurological function. There has been no ideal effective treatment so far. The NX210 peptide is derived from the thrombospondin type 1 repeat (TSR) sequence of SCO-spondin, and has been reported to exert various neurogenic properties. This study investigated whether NX210 had therapeutic effects and possible underlying mechanisms against cerebral ischemia/reperfusion (I/R). Therefore, primary embryonic rat cortical neurons and Sprague–Dawley (SD) rats that were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion/reperfusion (MCAO/R) injuries, respectively, were treated with or without NX210. We found that NX210 reduced OGD/R-induced cell viability loss and cytotoxicity. NX210 decreased cerebral infarct volume and brain edema, ameliorated neurological dysfunction, attenuated oxidative stress damage, and diminished neuronal apoptosis in MCAO/R rats. Furthermore, western blot analysis shown that treatment with NX210 up-regulated the expression of Integrin-β1, phosphorylated-PI3K (p-PI3K) and phosphorylated-Akt (p-Akt). The Integrin-β1 specific inhibitor, ATN-161, was used to identify pathways involved. The anti-oxidation activities and anti-apoptosis of NX210 was reversed by treatment with ATN-161. Overall, our results indicated that NX210 prevents oxidative stress and neuronal apoptosis in cerebral I/R via upregulation of the Integrin-β1/PI3K/Akt signaling pathway. These results indicated that NX210 may be a promising therapeutic candidate for ischemic stroke.

Comparative microelectrode array data of the functional development of hPSC-derived and rat neuronal networks

We present a dataset of microelectrode array (MEA) recordings from human pluripotent stem cell (hPSC)-derived and rat embryonic cortical neurons during their in vitro maturation. The data were prepared to assess extracellularly recorded spontaneous activity and to compare the functional development of these neuronal networks. In addition to recordings of spontaneous activity, we provide pharmacological responses of hPSC-derived and rat cortical cultures at their mature stage. Together with the recorded electrode raw data, we share the analysis code to form a comprehensive dataset including spike times, spike waveforms, burst activity and network synchronization metrics calculated with two different connectivity estimators. Moreover, we provide the analysis code that produced the key scientific findings published previously with this dataset. This large dataset enables investigation of the functional aspects of maturing cortical neuronal networks and provides substantial parameters to assess the differences and similarities between hPSC-derived and rat cortical networks in vitro. This publicly available dataset will be beneficial, especially for experimental and computational neuroscientists.

An ESCRT/VPS4 Envelopment Trap To Examine the Mechanism of Alphaherpesvirus Assembly and Transport in Neurons.

The assembly and egress of alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), within neurons is poorly understood. A key unresolved question is the structure of the viral particle that moves by anterograde transport along the axon, and two alternative mechanisms have been described. In the "married" model, capsids acquire their envelopes in the cell body and then traffic along axons as enveloped virions within a bounding organelle. In the "separate" model, nonenveloped capsids travel from the cell body into and along the axon, eventually encountering their envelopment organelles at a distal site, such as the nerve cell terminal. Here, we describe an "envelopment trap" to test these models using the dominant negative terminal endosomal sorting complex required for transport (ESCRT) component VPS4-EQ. Green fluorescent protein (GFP)-tagged VPS4-EQ was used to arrest HSV-1 or PRV capsid envelopment, inhibit downstream trafficking, and GFP-label envelopment intermediates. We found that GFP-VPS4-EQ inhibited trafficking of HSV-1 capsids into and along the neurites and axons of mouse CAD cells and rat embryonic primary cortical neurons, consistent with egress via the married pathway. In contrast, transport of HSV-1 capsids was unaffected in the neurites of human SK-N-SH neuroblastoma cells, consistent with the separate mechanism. Unexpectedly, PRV (generally thought to utilize the married pathway) also appeared to employ the separate mechanism in SK-N-SH cells. We propose that apparent differences in the methods of HSV-1 and PRV egress are more likely a reflection of the host neuron in which transport is studied rather than true biological differences between the viruses themselves. IMPORTANCE Alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), are pathogens of the nervous system. They replicate in the nerve cell body and then travel great distances along axons to reach nerve termini and spread to adjacent epithelial cells; however, key aspects of how these viruses travel along axons remain controversial. Here, we test two alternative mechanisms for transport, the married and separate models, by blocking envelope assembly, a critical step in viral egress. When we arrest formation of the viral envelope using a mutated component of the cellular ESCRT apparatus, we find that entry of viral particles into axons is blocked in some types of neurons but not others. This approach allows us to determine whether envelope assembly occurs prior to entry of viruses into axons or afterwards and, thus, to distinguish between the alternative models for viral transport.

Neuroinflammation induced by amyloid beta 1-42 oligomers contributes to neuronal loss in an in vitro model of Alzheimer's disease.

Alzheimer's disease is characterized by the extracellular accumulation of senile plaques composed of amyloid beta and the intracellular deposition of neurofibrillary tangles composed of hyperphosphorylated tau. Soluble oligomers of Aβ 1-42 peptide (AβO) are neurotoxic and participate directly in the pathophysiology of Alzheimer's disease. Here, we studied the contribution of microglial activation in the human 1-42 AβO-induced neurotoxicity, in an in vitro model of Alzheimer's disease. Using primary mixed cultures of rat embryonic cortical neurons and microglial cells, AβO toxicity was investigated in presence or absence of TLR4 antagonist (TAK-242). Survival of cortical neurons, integrity of their neurite network and activation of microglial cells were studied by immunostaining. In addition, proteomic changes were investigated using an immuno-based protein assay covering 1300 proteins including inflammatory related proteins, immune cell marker, checkpoint molecules as well as mediators and signaling molecules related to apoptosis and necroptosis among many others. We showed that AβO application led to an activation of microglial cells, a release of pro-inflammatory cytokines and a chronic neurotoxicity. Neuronal loss was significantly reduced but not abolished in presence of an inhibitor of TLR4. In addition, protein expression analysis revealed major enrichments in protein pathways related to cytokine signaling. Altogether, these findings showed that the toxicity of AβO is partially due to the activation of microglial cells and the release of pro-inflammatory cytokines. It highlights the close pathological connection between AβO microglial cells, neuroinflammation and neuronal death in Alzheimer's disease.

The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Significant Elevation in Potassium Concentration Surrounding Stimulated Excitable Cells Revealed by an Aptamer-Modified Nanowire Transistor.

Recording ion fluctuations surrounding biological cells with a nanoelectronic device offers seamless integration of nanotechnology into living organisms and is essential for understanding cellular activities. The concentration of potassium ion in the extracellular fluid () is a critical determinant of cell membrane potential and must be maintained within an appropriate range. Alteration in can affect neuronal excitability, induce heart arrhythmias, and even trigger seizure-like reactions in the brain. Therefore, monitoring local fluctuations in real time provides an early diagnosis of the occurrence of the K+-induced pathophysiological responses. Here, we modified the surface of a silicon nanowire field-effect transistor (SiNW-FET) with K+-specific DNA-aptamers (AptK+) to monitor the real-time variations of in primary cultured rat embryonic cortical neurons or human embryonic stem cell-derived cardiomyocytes. The binding affinity of AptK+ to K+, determined by measuring the dissociation constant of the AptK+-K+ complex (Kd = 10.1 ± 0.9 mM), is at least 38-fold higher than other ions (e.g., Na+, Ca2+, and Mg2+). By placing cultured cortical neurons over an AptK+/SiNW-FET device, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation raised the dose-dependently to 16 mM when AMPA concentration was >10 μM; this elevation could be significantly suppressed by an AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. Likewise, the stimulation of isoproterenol to cardiomyocytes raised the to 6-8 mM, with a concomitant increase in the beating rate. This study utilizing a robust nanobiosensor to detect real-time ion fluctuations surrounding excitable cells underlies the importance of ion homeostasis and offers the feasibility of developing an implant device for real-time monitoring.

Synthesis of New Water Soluble β-Cyclodextrin@Curcumin Conjugates and In Vitro Safety Evaluation in Primary Cultures of Rat Cortical Neurons.

Self-aggregation of Curcumin (Cur) in aqueous biological environment decreases its bioavailability and in vivo therapeutic efficacy, which hampers its clinical use as candidate for reducing risk of neurodegenerative diseases. Here, we focused on the design of new Cur- β-Cyclodextrin nanoconjugates to improve the solubility and reduce cell toxicity of Cur. In this study, we described the synthesis, structural characterization, photophysical properties and neuron cell toxicity of two new water soluble β-CD/Cur nanoconjugates as new strategy for reducing risks of neurodegenerative diseases. Cur was coupled to one or two β-CD molecules via triazole rings using CuAAC click chemistry strategy to yield β-CD@Cur and (β-CD)2@Cur nanoconjugates, respectively. The synthesized nanoconjugates were found to be able to self-assemble in aqueous condition and form nano-aggregates of an average diameter size of around 35 and 120 nm for β-CD@Cur and (β-CD)2@Cur, respectively. The photophysical properties, water solubility and cell toxicity on rat embryonic cortical neurons of the designed nanoconjugates were investigated and compared to that of Cur alone. The findings revealed that both new nanoconjugates displayed better water solubility and in vitro biocompatibility than Cur alone, thus making it possible to envisage their use as future nano-systems for the prevention or risk reduction of neurodegenerative diseases.

Zn2+-Depletion Enhances Lysosome Fission in Cultured Rat Embryonic Cortical Neurons Revealed by a Modified Epifluorescence Microscopic Technique.

Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.

Biomechanical Forces Regulate Gene Transcription During Stretch-Mediated Growth of Mammalian Neurons.

At birth, there are 100 billion neurons in the human brain, with functional neural circuits extending through the spine to the epidermis of the feet and toes. Following birth, limbs and vertebrae continue to grow by several orders of magnitude, forcing established axons to grow by up to 200 cm in length without motile growth cones. The leading regulatory paradigm suggests that biomechanical expansion of mitotic tissue exerts tensile force on integrated nervous tissue, which synchronizes ongoing growth of spanning axons. Here, we identify unique transcriptional changes in embryonic rat DRG and cortical neurons while the corresponding axons undergo physiological levels of controlled mechanical stretch in vitro. Using bioreactors containing cultured neurons, we recapitulated the peak biomechanical increase in embryonic rat crown-rump-length. Biologically paired sham and “stretch-grown” DRG neurons spanned 4.6- and 17.2-mm in length following static or stretch-induced growth conditions, respectively, which was associated with 456 significant changes in gene transcription identified by genome-wide cDNA microarrays. Eight significant genes found in DRG were cross-validated in stretch-grown cortical neurons by qRT-PCR, which included upregulation of Gpat3, Crem, Hmox1, Hpse, Mt1a, Nefm, Sprr1b, and downregulation of Nrep. The results herein establish a link between biomechanics and gene transcription in mammalian neurons, which elucidates the mechanism underlying long-term growth of axons, and provides a basis for new research in therapeutic axon regeneration.

Neurotoxicity and hyperphosphorylation of tau protein induced by the amyloid peptide oligomers involve different glutamate dependent and independent mechanisms

AbstractBackgroundAlzheimer’s disease is characterized by the extracellular accumulation of senile plaques composed of amyloid beta and the intracellular deposition of neurofibrillary tangles composed of hyperphosphorylated tau (Tp). Aβ oligomers (AβO) induced sequential damages starting with synapses loss, followed by the neurite network disorganization and finally the neuronal death. In this study, we deeply investigated the role of glutamate and its receptors (mGluR5 and NMDARs) in the AβO neurotoxicity and in the Tp.MethodUsing primary cultures of rat embryonic cortical neurons, the AβO toxicity was investigated in presence or absence of glutamate receptor antagonists (MPEP, Ifenprodil, MK801, memantine...). The AβO effects on the glutamate release and on the Ca2+ flux were studied in presence or absence of BAPTA in the medium. In addition, the AβO effect on the hyperphosphorylation of Tau was studied. Different sites of hyperphosphorylation were investigated (AT100, AT8 and Thr231 involving the DAPK1 and GSK3β).ResultWe showed that 5 min after AβO application, a large release of glutamate was seen in the extracellular medium. This release was immediately followed by a Ca2+ entrance in the neurons. In absence of extracellular Ca2+, the caspase‐3 activation was abolished as well as the neuronal death. Similarly, in presence of NMDAR or mGluR5 antagonists, the neuronal death was abolished, by contrast, Tau hyperphosphorylation and aggregation was still observed.ConclusionAltogether, these findings showed the crucial role of glutamate and Ca2+ in the AβO acute neuronal death, and demonstrated that the effects on Tp might not involve the NMDAR and the mGluR5.

The calcium-activated protease calpain regulates netrin-1 receptor deleted in colorectal cancer-induced axon outgrowth in cortical neurons.

During development, neurons extend axons toward their appropriate synaptic targets to establish functional neuronal connections. The growth cone, a highly motile structure at the tip of the axon, is capable of recognizing extracellular guidance cues and translating them into directed axon outgrowth through modulation of the actin cytoskeleton. Netrin-1 mediates its attractive function through the receptor deleted in colorectal cancer (DCC) to promote axon outgrowth and guidance. The calcium-activated protease calpain is involved in the cleavage of cytoskeletal proteins, which plays an important role during adhesion turnover and cell migration. However, its function during neuronal development is less understood. Here we demonstrate that netrin-1 activated calpain in embryonic rat cortical neurons in an extracellular-regulated kinase 1/2-dependent manner. In addition, we found that netrin-1 stimulation led to an increase in calpain-1 localization in the axon, whereas its endogenous inhibitor calpastatin was decreased in the growth cones of cortical neurons by indirect immunofluorescence. Interestingly, calpain-1 was able to cleave DCC in vitro. Furthermore, netrin-1 induced the cleavage of the cytoskeletal proteins spectrin and focal adhesion kinase concomitantly with the intracellular domain of DCC in a calpain-dependent manner in embryonic rat cortical neurons. Cortical neurons over-expressing calpastatin or calpain-depleted neurons displayed increased basal axon length and were unresponsive to netrin-1 stimulation. Altogether, we propose a novel model whereby netrin-1/DCC-mediated axon outgrowth is modulated by calpain-mediated proteolysis of DCC and cytoskeletal targets in embryonic cortical neurons. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Retrograde axonal transport of BDNF and proNGF diminishes with age in basal forebrain cholinergic neurons

Basal forebrain cholinergic neurons (BFCNs) are critical for learning and memory and degenerate early in Alzheimer's disease (AD). BFCNs depend for their survival and function on nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are retrogradely transported from BFCN targets. Age is the greatest risk factor for developing AD, yet the influence of age on BFCN axonal transport is poorly understood. To model aging, embryonic rat basal forebrain or cortical neurons were cultured in microfluidic chambers. Senescence-associated beta-galactosidase staining indicated an aging phenotype only in BFCNs cultured for 18+ days in vitro. BDNF axonal transport impairments were observed exclusivley in aged BFCNs. BFCNs displayed robust proNGF transport, which also diminished with in vitro age. The expression of NGF receptor tropomyosin-related kinase-A and BDNF receptor tropomyosin-related kinase-B also decreased significantly with in vitro age in BFCNs only. These results suggest a unique vulnerability of BFCNs to age-induced transport deficits. These deficits, coupled with the reliance of BFCNs on neurotrophin transport, may explain their vulnerability to age-related neurodegenerative disorders like AD.

Varying Effects of the G‐Protein Coupled Estrogen Receptor During Development in Embryonic Rat Hippocampal and Cortical Neurons

Estrogen has been known to play a role in enhancing brain development for over half a century. Most studies of estrogen's effect on the nervous system have focused on the traditional estrogen receptors Estrogen Receptor‐α (ERα) and Estrogen Receptor‐β (ERβ). The recent discovery of the G‐Protein Coupled Estrogen Receptor (GPER) suggests knowledge of estrogen's effects may be incomplete. While evidence has suggested the roles GPER plays in the nervous system include neuroprotection, neuronal recovery from stroke and degeneration, neuron proliferation, as well as many others. A caveat is that most studies have looked only at the effect in mature neurons and not the effect GPER has during early neuronal development. Here we sought to study the role of GPER activity in neurite outgrowth and synapse formation during early development.Embryonic day 18 rat cortical and hippocampal neurons were cultured in estrogen‐ and phenol red‐ free medium in the presence of a nonselective ER agonist (E2), a selective GPER agonist (G‐1), and a selective GPER antagonist (G‐15). Neurite growth was measured at 20, 48, 72, and 96 hours in culture using the ImageJ plugin NeuronJ. Synapses were visualized at 7 and 14 days in culture by using immunofluorescence of the pre‐ and post‐synaptic markers, synaptophysin and PSD‐95, respectively. Synapse formation was measured by measuring the overlap between these markers in ImageJ. Our data revealed activation of GPER promoted neurite growth in hippocampal neurons, but not in cortical neurons, but blocking the receptor inhibited neurite growth in cortical neurons. In addition, low levels of G1 (1μM) inhibited neurite outgrowth in cortical neurons. Cortical neurons showed no significant difference in synapse formation, while in hippocampal neurons the effect varied based on time period and dosage. This provides the first evidence that GPER activation may play different roles in brain regions, as well as having specific dosage based effects in specific regions. Future experiments will investigate the molecular and cellular mechanisms underlying the pharmacological differences in GPER effects between developing cortical and hippocampal neurons.Support or Funding InformationThis project was supported by the Saint Louis University Start‐up Fund for Dr. Xu lab and the Sigma XI Grant‐In‐Aid of Research awarded to Mr. Kyle Pemberton.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Soluble laminin polymers enhance axon growth of primary neurons in vitro.

A substrate of laminin polymers formed at pH 4 (acidic pH-induced laminin; aLam) promotes neurite growth of embryonic rat cortical neurons better than a substrate of similar but structurally different laminin polymers formed at neutral pH (neutral pH-induced laminin; nLam). We investigated the effects of these laminin polymers, used as soluble supplements, on neurite growth of cultured adult rat primary dorsal root ganglion neurons. When added to the culture medium, aLam was found to promote neurite growth about twofold better than nLam. Immunoblocking experiments revealed that aLam elicited neurite growth to a similar extent through the α1 or α3 integrin subunit, while nLam required the availability of the α1 integrin subunit to elicit neurite growth. With aLam, but not nLam, immunoblocking of the α1 or α3 subunit resulted in an increase in the protein level of the alternative subunit. The presence of a mature focal adhesion complex, which is associated with neurite growth, was elevated in neurons in the presence of aLam relative to nLam or culture medium. Our data indicated that the two types of laminin polymers promote neurite growth of adult rat primary sensory neurons to a different degree, likely through different ligand-receptor interactions. These findings support the potential of soluble laminin polymers as injectable therapeutics for eliciting axon growth after nervous system injury. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2372-2381, 2018.

Persistent 6-OH-BDE-47 exposure impairs functional neuronal maturation and alters expression of neurodevelopmentally-relevant chromatin remodelers.

Polybrominated diphenyl ethers (PBDEs) are a pervasive class of brominated flame retardants that are present in the environment at particularly high levels, especially in the United States. Their environmental stability, propensity for bioaccumulation, and known potential for neurotoxicity has evoked interest regarding their effects on the developing nervous system. Exposure to PBDEs has been strongly associated with neurodevelopmental disorders. However, the details of their mechanistic roles in such disorders are incompletely understood. Here, we report the effects of one of the most prevalent congeners, BDE-47, and its hydroxylated metabolites on the maturation and function of embryonic rat cortical neurons. Prolonged exposure to 6OH-BDE-47 produces the strongest effects amongst the parent BDE-47 congener and its tested hydroxylated metabolites. These effects include: i) disruption of transcriptional responses to neuronal activity, ii) dysregulation of multiple genes associated with neurodevelopmental disorders, and intriguingly, iii) altered expression of several subunits of the developmentally-relevant BAF (Brg1-associated factors) chromatin remodeling complex, including the key subunit BAF170. Taken together, our data indicate that persistent exposure to 6OH-BDE-47 may interfere with neurodevelopmental chromatin remodeling mechanisms and gene transcription programs, which in turn are likely to interfere with downstream processes such as synapse development and overall functional maturity of neurons. Results from this study have identified a novel aspect of 6OH-BDE-47 toxicity and open new avenues to explore the effects of a ubiquitous environmental toxin on epigenetic regulation of neuronal maturation and function.

A novel 3D-microstructured multielectrode array configuration for high-yield and high-fidelity electrophysiological recordings

A novel 3D-microstructured multielectrode array configuration for high-yield and high-fidelity electrophysiological recordings

Central Nervous System Neuron-Glia co-Culture Models and Application to Neuroprotective Agents.

Glial cell activation plays an important role in the pathogenesis of various neurodegenerative disorders. This article presents a protocol for the preparation of cultures consisting of rat embryonic cortical neurons grown in the presence of cortical microglia, in which the glia are present in physical contact with the neurons or separated by a semipermeable membrane barrier. An example of how such systems can be used to evaluate potential neuroprotective agents will also be described.

Ribosomal stress and Tp53-mediated neuronal apoptosis in response to capsid protein of the Zika virus

We report here that in rat and human neuroprogenitor cells as well as rat embryonic cortical neurons Zika virus (ZIKV) infection leads to ribosomal stress that is characterized by structural disruption of the nucleolus. The anti-nucleolar effects were most pronounced in postmitotic neurons. Moreover, in the latter system, nucleolar presence of ZIKV capsid protein (ZIKV-C) was associated with ribosomal stress and apoptosis. Deletion of 22 C-terminal residues of ZIKV-C prevented nucleolar localization, ribosomal stress and apoptosis. Consistent with a casual relationship between ZIKV-C-induced ribosomal stress and apoptosis, ZIKV-C-overexpressing neurons were protected by loss-of-function manipulations targeting the ribosomal stress effector Tp53 or knockdown of the ribosomal stress mediator RPL11. Finally, capsid protein of Dengue virus, but not West Nile virus, induced ribosomal stress and apoptosis. Thus, anti-nucleolar and pro-apoptotic effects of protein C are flavivirus-species specific. In the case of ZIKV, capsid protein-mediated ribosomal stress may contribute to neuronal death, neurodevelopmental disruption and microcephaly.