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

BACKGROUND: 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. METHODS: With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC) lines (R1, C57BL/6 and D3) adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX), and botulinum neurotoxin (BoNT), were also evaluated. RESULTS: Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs) per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs) expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons. CONCLUSIONS: These findings demonstrate a cost-effective, scalable and flexible method to produce a highly enriched glutamatergic neuron population. The functional characterization of pathophysiological responses to neurotropic toxins and the compatibility with multi-well plating formats were used to demonstrate the suitability of ESNs as a discovery platform for molecular mechanisms of action, moderate-throughput analytical approaches and diagnostic screening. Furthermore, for the first time we demonstrate a cell-based model that is sensitive to all seven BoNT serotypes with EC50 values comparable to those reported in primary neuron populations. These data providing compelling evidence that ESNs offer a neuromimetic platform suitable for the evaluation of molecular mechanisms of neurotoxicity.

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs.

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research.

There are no pharmacological treatments to rescue botulinum neurotoxin (BoNT)-mediated paralysis of neuromuscular signaling. In part, this failure can be attributed to the lack of a cell culture model system that is neuron-based, allowing detailed elucidation of the mechanisms underlying BoNT pathogenesis, yet still compatible with modern cellular and molecular approaches. We have developed a method to derive highly enriched, glutamatergic neurons from suspension-cultured murine embryonic stem (ES) cells. Hypothesizing that ES cell-derived neurons (ESNs) might comprise a novel platform to investigate the neurotoxicology of BoNTs, we evaluated the susceptibility of ESNs to BoNT/A and BoNT/E using molecular and functional assays. ESNs express neuron-specific proteins, develop synapses and release glutamate in a calcium-dependent manner under depolarizing conditions. They express the BoNT substrate SNARE proteins SNAP25, VAMP2 and syntaxin, and treatment with BoNT/A and BoNT/E holotoxin results in proteolysis of SNAP25 within 24 h with EC50s of 0.81 and 68.6 pM, respectively. Intoxication with BoNT/A results in the functional inhibition of potassium-induced, calcium-dependent glutamate release. ESNs remain viable and susceptible to intoxication for up to 90 days after plating, enabling longitudinal screens exploring toxin-specific mechanisms underlying persistence of synaptic blockade. The evidence suggests that derived neurons are a novel, biologically relevant model system that combines the verisimilitude of primary neurons with the genetic tractability and scalable expansion of a continuous cell line, and thus should significantly accelerate BoNT research and drug discovery while dramatically decreasing animal use.

Genetic enhancement of the Lis1+/- phenotype by a heterozygous mutation in the adenomatous polyposis coli gene.

Hemizygous Lis1 mutations cause type 1 lissencephaly, a neuronal migration disorder in humans. The Lis1+/- mouse is a model for lissencephaly; mice exhibit neuronal migration defects but are viable and fertile. On an inbred genetic background, 20% of Lis1+/- mice develop hydrocephalus and die prematurely. Lis1 functions with the microtubule motor cytoplasmic dynein. Because dynactin, a dynein regulator, interacts with end-binding protein 1 (EB1) and beta-catenin, two known binding partners of the adenomatous polyposis coli (APC) protein, we looked for a genetic interaction between Lis1 and APC. Mice with a heterozygous truncating mutation in APC (Min mutation) do not exhibit neuronal migration defects or develop hydrocephalus. However, the presence of the APC mutation increases the migration deficit and the incidence of hydrocephalus in Lis1+/- animals. Lis1 and dynein distribution is altered in cells derived from Min mice, and both Lis1 and dynein interact with the C terminus of APC in vitro. Together, our findings point to a previously unknown interaction between APC and Lis1 during mammalian brain development.

Regulation of cytoplasmic dynein ATPase by Lis1.

Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport.

Effect of dexamethasone administered with magnesium sulfate on inflammation-mediated degradation of the blood-brain barrier using an in vitro model.

Patients at risk for preterm delivery are frequently administered both antenatal steroids for fetal maturation and magnesium sulfate for neuroprotection. In this study, we investigate whether steroids coadministered with magnesium sulfate preserve blood-brain barrier integrity in neuroinflammation. Human umbilical vein endothelial cells were grown in astroglial conditioned media in a 2-chamber cell culture apparatus. Treatment with tumor necrosis factor-α (TNF-α) or catalytically active recombinant matrix metalloproteinase 9 (MMP-9) simulated neuroinflammation. Membrane integrity was assessed by zona occludens 1 (ZO-1) immunoreactivity, permeability to fluorescently conjugated dextran, and transendothelial electrical resistance (TEER). The TNF-α and MMP-9 treatment increased the rate of dextran transit, decreased TEER, and decreased ZO-1 immunoreactivity at junctional interfaces. Dexamethasone pretreatment alone or in combination with 0.5 mmol/L magnesium sulfate preserved monolayer integrity after inflammatory insult. Magnesium sulfate alone was not protective. This study supports a possible interaction between steroids and magnesium in neuroprotection.

Alpha-latrotoxin rescues SNAP-25 from BoNT/A-mediated proteolysis in embryonic stem cell-derived neurons.

The botulinum neurotoxins (BoNTs) exhibit zinc-dependent proteolytic activity against members of the core synaptic membrane fusion complex, preventing neurotransmitter release and resulting in neuromuscular paralysis. No pharmacologic therapies have been identified that clinically relieve botulinum poisoning. The black widow spider venom α-latrotoxin (LTX) has the potential to attenuate the severity or duration of BoNT-induced paralysis in neurons via the induction of synaptic degeneration and remodeling. The potential for LTX to antagonize botulinum poisoning was evaluated in embryonic stem cell-derived neurons (ESNs), using a novel screening assay designed around the kinetics of BoNT/A activation. Exposure of ESNs to 400 pM LTX for 6.5 or 13 min resulted in the nearly complete restoration of uncleaved SNAP-25 within 48 h, whereas treatment with 60 mM K(+) had no effect. Time-lapse imaging demonstrated that LTX treatment caused a profound increase in Ca(2+) influx and evidence of excitotoxicity, though ESNs remained viable 48 h after LTX treatment. This is the first instance of a cell-based treatment that has shown the ability to eliminate BoNT activity. These data suggest that LTX treatment may provide the basis for a new class of therapeutic approach to BoNT intoxication and may contribute to an improved understanding of long-term mechanisms of BoNT intoxication and recovery. They further demonstrate that ESNs are a novel, responsive and biologically relevant model for LTX research and BoNT therapeutic drug discovery.

Lis1 and Ndel1 influence the timing of nuclear envelope breakdown in neural stem cells.

Lis1 and Ndel1 are essential for animal development. They interact directly with one another and with cytoplasmic dynein. The developing brain is especially sensitive to reduced Lis1 or Ndel1 levels, as both proteins influence spindle orientation, neural cell fate decisions, and neuronal migration. We report here that Lis1 and Ndel1 reduction in a mitotic cell line impairs prophase nuclear envelope (NE) invagination (PNEI). This dynein-dependent process facilitates NE breakdown (NEBD) and occurs before the establishment of the bipolar spindle. Ndel1 phosphorylation is important for this function, regulating binding to both Lis1 and dynein. Prophase cells in the ventricular zone (VZ) of embryonic day 13.5 Lis1(+/-) mouse brains show reduced PNEI, and the ratio of prophase to prometaphase cells is increased, suggesting an NEBD delay. Moreover, prophase cells in the VZ contain elevated levels of Ndel1 phosphorylated at a key cdk5 site. Our data suggest that a delay in NEBD in the VZ could contribute to developmental defects associated with Lis1-Ndel1 disruption.