functional-evaluation-biological-neurotoxins-networked-cultures-stem

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene- and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediator-dependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.

A Mediator-responsive form of metazoan RNA polymerase II.

A common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture.

Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis.

There is extensive data to support the use of commercial transport media as a stabilizer for known clinical samples; however, there is little information to support their use outside of controlled conditions specified by the manufacturer. Furthermore, there is no data to determine the suitability of said media for biological pathogens, specifically those of interest to the US military. This study evaluates commercial off-the-shelf (COTS) transport media based on sample recovery, viability, and quality of nucleic acids and peptides for nonpathogenic strains of Bacillus anthracis, Yersinia pestis, and Venezuelan equine encephalitis virus, in addition to ricin toxin. Samples were stored in COTS, PBST, or no media at various temperatures over an extended test period. The results demonstrate that COTS media, although sufficient for the preservation of nucleic acid and proteinaceous material, are not capable of maintaining an accurate representation of biothreat agents at the time of collection.

Suitability of Commercial Transport Media for Biological Pathogens under Nonideal Conditions.

Simultaneous use of cell-permeant and impermeant fluorescent nuclear dyes is a common method to study cell viability and cell death progression. Although these assays are usually conducted as end-point studies, time-lapse imaging offers a powerful technique to distinguish temporal changes in cell viability at single-cell resolution. SYTO 13 and Hoechst 33342 are two commonly used cell-permeant nuclear dyes; however their suitability for live imaging has not been well characterized. We compare end-point assays with time-lapse imaging studies over a 6 h period to evaluate the compatibility of these two dyes with longitudinal imaging, using both control neurons and an apoptotic neuron model.

Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death.

Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available.

High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research.

Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species.

Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2.

Im Mai 2011 blutigen ein Enteroaggregative Escherichia coli verursachten O104:H4-Stamm, der eine Shiga-Toxin 2-konvertierende Phage erworben hatte einen gro&szlig;en Ausbruch der Durchfall in Europa bekannt f&uuml;r seine hohe Pr&auml;valenz des H&auml;molytisch-ur&auml;misches Syndrom F&auml;llen war. Mehrere Studien haben beschrieben, die genomische Inventar und am&ouml;benartigen St&auml;mme, die im Zusammenhang mit des Ausbruchs und eine Sammlung von historischen E. Coli O104:H4 isolieren, Entwurf des Genoms Assemblys verwenden. Wir pr&auml;sentieren die komplette, geschlossene Genomsequenzen ein Isolat von 2011 Ausbruch (2011C-3493) und zwei Isolate aus F&auml;llen von blutigen Durchfall, die in der Republik Georgien in 2009 (2009EL-2050 und 2009EL-2071) aufgetreten. Vergleichende Genomanalyse bedeutet, dass w&auml;hrend der georgischen St&auml;mme die n&auml;chsten Nachbarn sind 2011 Ausbruch isoliert bis jetzt sequenzierten, strukturelle und Nukleotid-Niveau-Unterschiede offensichtlich in den Stx2 Phage Genomen, Mer/Tet Antibiotikaresistenz Insel, und in der Prophage und Plasmid-Profile der Sorten sind, einschlie&szlig;lich eine bisher unbeschriebene Plasmid mit Homologie zu den pMT Virulenz Plasmid von Yersinia Pestis. Dar&uuml;ber hinaus zeigte die multiphenotype Analyse, 2009EL-2071 besessen Widerstandsf&auml;higkeit auch Polymyxin und Membran-Unterbrechung des Agenten. Schlie&szlig;lich zeigen wir Beweise durch Elektronenmikroskopie des Vorhandenseins einer gemeinsamen Phage Morphotype unter den europ&auml;ischen und georgischen St&auml;mme und eine zweite Phage Morphotype unter der georgischen St&auml;mme. Die Anwesenheit von mindestens zwei stx2 Phage Genotypen in Host genetische Hintergr&uuml;nde, die von einem letzten gemeinsamen Vorfahren der Isolate 2011 Ausbruch abgeleitet werden k&ouml;nnen zeigt, dass die Entstehung von stx2 Phage-haltigen E. Coli O104:H4 St&auml;mme wahrscheinlich mehr als einmal aufgetreten, oder, dass der aktuelle Ausbruch isoliert m&ouml;glicherweise das Ergebnis einer j&uuml;ngsten &Uuml;bertragung ein neues stx2 Phage Element in eine bereits vorhandene stx2-positivem genetischen Hintergrund.

Genomischer Vergleich von Escherichia coli O104:H4 isoliert von 2009 und 2011 enth&uuml;llt Plasmid und Prophage Heterogenit&auml;t, einschlie&szlig;lich der Shiga-Toxin Codierung Phage stx2.

Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as in vitro cell models to study neuronal functions and neurotoxicity. The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells.

Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid.

Glutamate receptor (GluR)-mediated neurotoxicity is implicated in a variety of disorders ranging from ischemia to neural degeneration. Under conditions of elevated glutamate, the excessive activation of GluRs causes internalization of pathologic levels of Ca(2+), culminating in bioenergetic failure, organelle degradation, and cell death. Efforts to characterize cellular and molecular aspects of excitotoxicity and conduct therapeutic screening for pharmacologic inhibitors of excitogenic progression have been hindered by limitations associated with primary neuron culture. To address this, we evaluated glutamate-induced neurotoxicity in highly enriched glutamatergic neurons (ESNs) derived from murine embryonic stem cells. As of 18 days in vitro (DIV 18), ESNs were synaptically coupled, exhibited spontaneous network activity with neurotypic mEPSCs and expressed NMDARs and AMPARs with physiological current:voltage behaviors. Addition of 0.78-200 μM glutamate evoked reproducible time- and dose-dependent metabolic failure in 6 h, with a calculated EC50 value of 0.44 μM at 24 h. Using a combination of cell viability assays and electrophysiology, we determined that glutamate-induced toxicity was specifically mediated by NMDARs and could be inhibited by addition of NMDAR antagonists, increased extracellular Mg(2+) or substitution of Ba(2+) for Ca(2+). Glutamate treatment evoked neurite fragmentation and focal swelling by both immunocytochemistry and scanning electron microscopy. Presentation of morphological markers of cell death was dose-dependent, with 0.78-200 μM glutamate resulting in apoptosis and 3000 μM glutamate generating a mixture of necrosis and apoptosis. Addition of neuroprotective small molecules reduced glutamate-induced neurotoxicity in a dose-dependent fashion. These data indicate that ESNs replicate many of the excitogenic mechanisms observed in primary neuron culture, offering a moderate-throughput model of excitotoxicity that combines the verisimilitude of primary neurons with the flexibility and scalability of cultured cells. ESNs therefore offer a physiologically relevant platform that exhibits characteristic NMDAR responses, and appears suitable to evaluate molecular mechanisms of glutamate-induced excitotoxicity and screen for candidate therapeutics.

Novel application of stem cell-derived neurons to evaluate the time- and dose-dependent progression of excitotoxic injury.

Using paired-end RNA sequencing, we have quantified the deep transcriptional changes that occur during differentiation of murine embryonic stem cells into a highly enriched population of glutamatergic cortical neurons. These data provide a detailed and nuanced account of longitudinal changes in the transcriptome during neurogenesis and neuronal maturation, starting from mouse embryonic stem cells and progressing through neuroepithelial stem cell induction, radial glial cell formation, neurogenesis, neuronal maturation and cortical patterning. Understanding the transcriptional mechanisms underlying the differentiation of stem cells into mature, glutamatergic neurons of cortical identity has myriad applications, including the elucidation of mechanisms of cortical patterning; identification of neurogenic processes; modeling of disease states; detailing of the host cell response to neurotoxic stimuli; and determination of potential therapeutic targets. In future work we anticipate correlating changes in longitudinal gene expression to other cell parameters, including neuronal function as well as characterizations of the proteome and metabolome. In this data article, we describe the methods used to produce the data and present the raw sequence read data in FASTQ files, sequencing run statistics and a summary flatfile of raw counts for 22,164 genes across 31 samples, representing 3-5 biological replicates at each timepoint. We propose that this data will be a valuable contribution to diverse research efforts in bioinformatics, stem cell research and developmental neuroscience studies.

Article	Year
A Mediator-responsive form of metazoan RNA polymerase II. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16769904	2006
Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis. BMC cancer\| PubMed ID: 18474089	2008
Suitability of Commercial Transport Media for Biological Pathogens under Nonideal Conditions. International journal of microbiology\| PubMed ID: 22121364	2011
Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death. BMC research notes\| PubMed ID: 22892216	2012
High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research. BMC neuroscience\| PubMed ID: 23095170	2012
Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species. BMC microbiology\| PubMed ID: 23126230	2012
Genomic Comparison of Escherichia coli O104:H4 Isolates from 2009 and 2011 Reveals Plasmid, and Prophage Heterogeneity, Including Shiga Toxin Encoding Phage stx2. PloS one\| PubMed ID: 23133618	2012
Morphological and functional differentiation in BE(2)-M17 human neuroblastoma cells by treatment with Trans-retinoic acid. BMC neuroscience\| PubMed ID: 23597229	2013
Novel application of stem cell-derived neurons to evaluate the time- and dose-dependent progression of excitotoxic injury. PloS one\| PubMed ID: 23691214	2013
Longitudinal RNA sequencing of the deep transcriptome during neurogenesis of cortical glutamatergic neurons from murine ESCs. F1000Research\| PubMed ID: 24358889	2013

Kyle Hubbard

Biography

JoVE Journal Publications

Other Publications