CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

Mechanisms Underlying Chronic Binge Alcohol Exposure-Induced Uterine Artery Dysfunction in Pregnant Rat.

BACKGROUND: A cardinal feature of fetal alcohol syndrome is growth restriction. Maternal uterine artery adaptations to pregnancy correlate with birthweight and survival. We hypothesized that gestational binge alcohol exposure impairs maternal uterine vascular function, affecting endothelial nitric oxide (NO)-mediated vasodilation. METHODS: Pregnant rats grouped as pair-fed control or binge alcohol exposed received a once-daily, orogastric gavage of isocaloric maltose-dextrin or alcohol, respectively. On gestational day 20, primary uterine arteries were isolated, cannulated, and connected to a pressure transducer, and functional studies were conducted by dual-chamber arteriography. Uterine arteries maintained at constant intramural pressure (90 mm Hg) were maximally constricted with thromboxane, and a dose-response for acetylcholine (Ach) was recorded. RESULTS: The alcohol group exhibited significantly impaired endothelium-dependent, Ach-induced uterine artery relaxation (↓∼30%). Subsequently, a dose-response was recorded following inhibition of endothelium-derived hyperpolarizing factor (apamin and TRAM-34) and prostacyclin (indomethacin). Ach-induced relaxation in the pair-fed control decreased by ~46%, and interestingly, relaxation in alcohol group further decreased by an additional ~48%, demonstrating that gestational binge alcohol impairs the NO system in the primary uterine artery. An endothelium-independent sodium nitroprusside effect was not observed. Immunoblotting indicated that alcohol decreased the level of endothelial excitatory P-Ser1177 endothelial NO synthase (eNOS) (p < 0.05) and total eNOS expression (p < 0.05) compared to both the normal and pair-fed controls. P-Ser1177 eNOS level was also confirmed by immunofluorescence imaging. CONCLUSIONS: This is the first study to demonstrate maternal binge alcohol consumption during pregnancy disrupts uterine artery vascular function via impairment of the eNOS vasodilatory system.

Placental Proteomics Reveal Insights into Fetal Alcohol Spectrum Disorders.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) describe many of the well-known neurodevelopmental deficits afflicting children exposed to alcohol in utero. The effects of alcohol on the maternal-fetal interface, especially the placenta, have been less explored. We herein hypothesized that chronic binge alcohol exposure during pregnancy significantly alters the placental protein profile in a rat FASD model. METHODS: Pregnant rats were orogastrically treated daily with alcohol (4.5 g/kg, gestational day [GD] 5 to 10; 6.0 g/kg, GD 11 to 19) or 50% maltose dextrin (isocalorically matched pair-fed controls). On GD 20, placentae were collected, flash-frozen, and stored until tissues were homogenized. Protein lysates were denatured, reduced, captured on a 10-kDa spin filter, and digested. Peptides were eluted, reconstituted, and analyzed by a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer. RESULTS: Mass spectrometry (MS) analysis identified 2,285 placental proteins based on normalized spectral counts and 2,000 proteins by intensity-based absolute quantification. Forty-five placental proteins were significantly (p < 0.05) altered by gestational alcohol exposure by both quantification approaches. These included proteins directly related to alcohol metabolism; specific isoforms of alcohol dehydrogenase and aldehyde dehydrogenase were up-regulated in the alcohol group. Ingenuity analysis identified ethanol degradation as the most significantly altered canonical pathway in placenta, and fetal/organ development as most altered function, with increased risk for metabolic, neurological, and cardiovascular diseases. Physiological roles of the significantly altered proteins were related to early pregnancy adaptations, implantation, gestational diseases, fetal organ development, neurodevelopment, and immune functions. CONCLUSIONS: We conclude that the placenta is a valuable organ not only to understand FASD etiology but it may also serve as a diagnostic tool to identify novel biomarkers for detecting the outcome of fetal alcohol exposure. Placental MS analysis can offer sophisticated insights into identifying alcohol metabolism-related enzymes and regulators of fetal development.

Warning Signs of Potential Black Swan Outbreaks in Infectious Disease.

Black swan events in infectious disease describe rare but devastatingly large outbreaks. While experts are skeptical that such events are predictable, it might be possible to identify the warning signs of a black swan event. Specifically, following the initiation of an outbreak, key differentiating features could serve as alerts. Such features could be derived from meta-analyses of large outbreaks for multiple infectious diseases. We hypothesized there may be common features among the pathogen, environment, and host epidemiological triad that characterize an infectious disease black swan event. Using Los Alamos National Laboratory's tool, Analytics for Investigation of Disease Outbreaks, we investigated historical disease outbreak information and anomalous events for several infectious diseases. By studying 32 different infectious diseases and global outbreaks, we observed that in the past 20-30 years, there have been potential black swan events in the majority of infectious diseases analyzed. Importantly, these potential black swan events cannot be attributed to the first introduction of the disease to a susceptible host population. This paper describes our observations and perspectives and illustrates the value of broad analysis of data across the infectious disease realm, providing insights that may not be possible when we focus on singular infectious agents or diseases. Data analytics could be developed to warn health authorities at the beginning of an outbreak of an impending black swan event. Such tools could complement traditional epidemiological modeling to help forecast future large outbreaks and facilitate timely warning and effective, targeted resource allocation for mitigation efforts.

Optimization of Application-Driven Development of In Vitro Neuromuscular Junction Models.

Neuromuscular junctions (NMJs) are specialized synapses responsible for signal transduction between motor neurons (MNs) and skeletal muscle tissue. Malfunction at this site can result from developmental disorders, toxic environmental exposures, and neurodegenerative diseases leading to severe neurological dysfunction. Exploring these conditions in human or animal subjects is restricted by ethical concerns and confounding environmental factors. Therefore, in vitro NMJ models provide exciting opportunities for advancements in tissue engineering. In the last two decades, multiple NMJ prototypes and platforms have been reported, and each model system design is strongly tied to a specific application: exploring developmental physiology, disease modeling, or high-throughput screening. Directing the differentiation of stem cells into mature MNs and/or skeletal muscle for NMJ modeling has provided critical cues to recapitulate early-stage development. Patient-derived inducible pluripotent stem cells provide a personalized approach to investigating NMJ disease, especially when disease etiology cannot be resolved down to a specific gene mutation. Having reproducible NMJ culture replicates is useful for high-throughput screening to evaluate drug toxicity and determine the impact of environmental threat exposures. Cutting-edge bioengineering techniques have propelled this field forward with innovative microfabrication and design approaches allowing both two-dimensional and three-dimensional NMJ culture models. Many of these NMJ systems require further validation for broader application by regulatory agencies, pharmaceutical companies, and the general research community. In this summary, we present a comprehensive review on the current state-of-art research in NMJ models and discuss their ability to provide valuable insight into cell and tissue interactions. Impact statement In vitro neuromuscular junction (NMJ) models reveal the specialized mechanisms of communication between neurons and muscle tissue. This site can be disrupted by developmental disorders, toxic environmental exposures, or neurodegenerative diseases, which often lead to fatal outcomes and is therefore of critical importance to the medical community. Many bioengineering approaches for in vitro NMJ modeling have been designed to mimic development and disease; other approaches include in vitro NMJ models for high-throughput toxicology screening, providing a platform to limit or replace animal testing. This review describes various NMJ applications and the bioengineering advancements allowing for human NMJ characteristics to be more accurately recapitulated. While the extensive range of NMJ device structures has hindered standardization attempts, there is still a need to harmonize these devices for broader application and to continue advancing the field of NMJ modeling.

Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation.

BACKGROUND: Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. RESULTS: Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-ß signaling pathway and consistent activation of sonic hedgehog, Wnt/ß-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. CONCLUSIONS: Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

Resources to Discover and Use Short Linear Motifs in Viral Proteins.

Viral proteins evade host immune function by molecular mimicry, often achieved by short linear motifs (SLiMs) of three to ten consecutive amino acids (AAs). Motif mimicry tolerates mutations, evolves quickly to modify interactions with the host, and enables modular interactions with protein complexes. Host cells cannot easily coordinate changes to conserved motif recognition and binding interfaces under selective pressure to maintain critical signaling pathways. SLiMs offer potential for use in synthetic biology, such as better immunogens and therapies, but may also present biosecurity challenges. We survey viral uses of SLiMs to mimic host proteins, and information resources available for motif discovery. As the number of examples continues to grow, knowledge management tools are essential to help organize and compare new findings.

Regional dysregulation of taurine and related amino acids in the fetal rat brain following gestational alcohol exposure.

The fetal brain exhibits exquisite alcohol-induced regional neuronal vulnerability. A candidate mechanism for alcohol-mediated brain deficits is disruption of amino acid (AA) bioavailability. AAs are vitally important for proper neurodevelopment, as they comprise the most abundant neurotransmitters in the brain and act as neurotransmitter precursors, nitric oxide donors, antioxidants, and neurotrophic factors, which induce synaptogenesis, neuronal proliferation, and migration. We hypothesized that gestational alcohol alters brain AA concentrations, disrupts AAs associated with neuropathogenesis, and that alterations are region-specific. We assigned pregnant Sprague-Dawley rats to either a pair-fed control or a binge alcohol treatment group on gestational day (GD) 4. Alcohol animals were acclimatized via a once-daily orogastric gavage of a 4.5 g/kg alcohol dose from GD 5-10, and progressed to a 6 g/kg alcohol dose from GD 11-20. Pair-fed animals received isocaloric maltose dextrin (once daily; GD 5-20). Fetal cerebral cortex, cerebellum, and hippocampus were collected on GD 21. Following collection, Fluorometric High Performance Liquid Chromatography (HPLC) involving pre-column derivatization with o-phthaldialdehyde quantified regional content of 22 AAs. Chronic binge alcohol administration to pregnant dams regionally altered AA concentrations in all three structures, with the cerebral cortex exhibiting the least vulnerability and the hippocampus exhibiting maximal vulnerability. We conjecture that the AA imbalances observed in this study are critically implicated in pathological and compensatory processes occurring in the brain in response to gestational alcohol exposure.

Fetal regional brain protein signature in FASD rat model.

Fetal alcohol spectrum disorders (FASD) describe neurodevelopmental deficits in children exposed to alcohol in utero. We hypothesized that gestational alcohol significantly alters fetal brain regional protein signature. Pregnant rats were binge-treated with alcohol or pair-fed and nutritionally-controlled. Mass spectrometry identified 1806, 2077, and 1456 quantifiable proteins in the fetal hippocampus, cortex, and cerebellum, respectively. A stronger effect of alcohol exposure on the hippocampal proteome was noted: over 600 hippocampal proteins were significantly (P < .05) altered, including annexin A2, nucleobindin-1, and glypican-4, regulators of cellular growth and developmental morphogenesis. In the cerebellum, cadherin-13, reticulocalbin-2, and ankyrin-2 (axonal growth regulators) were significantly (P < .05) altered; altered cortical proteins were involved in autophagy (endophilin-B1, synaptotagmin-1). Ingenuity analysis identified proteins involved in protein homeostasis, oxidative stress, mitochondrial dysfunction, and mTOR as major pathways in the cortex and hippocampus significantly (P < .05) affected by alcohol. Thus, neurodevelopmental protein changes may directly relate to FASD neuropathology.

Chronic binge alcohol consumption during pregnancy alters rat maternal uterine artery pressure response.

We aimed to investigate pressure-dependent maternal uterine artery responses and vessel remodeling following gestational binge alcohol exposure. Two groups of pregnant rats were used: the alcohol group (28.5% wt/v, 6.0 g/kg, once-daily orogastric gavage in a binge paradigm between gestational day (GD) 5-19) and pair-fed controls (isocalorically matched). On GD20, excised, pressurized primary uterine arteries were studied following equilibration (60 mm Hg) using dual chamber arteriograph. The uterine artery diameter stabilized at 20 mm Hg, showed passive distension at 40 mm Hg, and redeveloped tone at 60 mm Hg. An alcohol effect (P = 0.0025) was observed on the percent constriction of vessel diameter with greater pressure-dependent myogenic constriction. Similar alcohol effect was noted with lumen diameter response (P = 0.0020). The percent change in media:lumen ratio was higher in the alcohol group (P < 0.0001). Thus, gestational alcohol affects pressure-induced uterine artery reactivity, inward-hypotrophic remodeling, and adaptations critical for nutrient delivery to the fetus.