Western Zika Virus in Human Fetal Neural Progenitors Persists Long Term with Partial Cytopathic and Limited Immunogenic Effects.

The recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.

A quantitative framework to evaluate modeling of cortical development by neural stem cells.

Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease.

Clinical neurogenetics: behavioral management of inherited neurodegenerative disease.

Psychiatric symptoms often manifest years before overt neurologic signs in patients with inherited neurodegenerative disease. The most frequently cited example of this phenomenon is the early onset of personality changes in "presymptomatic" Huntington patients. In some cases the changes in mood and cognition are even more debilitating than their neurologic symptoms. The goal of this article is to provide the neurologist with a concise primer that can be applied in a busy clinic or private practice.

[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-dimethyl phenylamino)pyrazin-2(1H)-one: introduction of N3-phenylpyrazinones as potential CRF-R1 PET imaging agents.

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [(18)F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.

RBFOX1 regulates both splicing and transcriptional networks in human neuronal development.

RNA splicing plays a critical role in the programming of neuronal differentiation and, consequently, normal human neurodevelopment, and its disruption may underlie neurodevelopmental and neuropsychiatric disorders. The RNA-binding protein, fox-1 homolog (RBFOX1; also termed A2BP1 or FOX1), is a neuron-specific splicing factor predicted to regulate neuronal splicing networks clinically implicated in neurodevelopmental disease, including autism spectrum disorder (ASD), but only a few targets have been experimentally identified. We used RNA sequencing to identify the RBFOX1 splicing network at a genome-wide level in primary human neural stem cells during differentiation. We observe that RBFOX1 regulates a wide range of alternative splicing events implicated in neuronal development and maturation, including transcription factors, other splicing factors and synaptic proteins. Downstream alterations in gene expression define an additional transcriptional network regulated by RBFOX1 involved in neurodevelopmental pathways remarkably parallel to those affected by splicing. Several of these differentially expressed genes are further implicated in ASD and related neurodevelopmental diseases. Weighted gene co-expression network analysis demonstrates a high degree of connectivity among these disease-related genes, highlighting RBFOX1 as a key factor coordinating the regulation of both neurodevelopmentally important alternative splicing events and clinically relevant neuronal transcriptional programs in the development of human neurons.

DISC1: a schizophrenia gene with multiple personalities.

Two papers address the contribution of DISC1 to neural development and schizophrenia risk in this issue of Neuron. These complementary studies elegantly bridge the gap between genetic and cellular studies of schizophrenia, providing a level of functional validation that is often lacking in the field.

Genome-wide analysis of a Wnt1-regulated transcriptional network implicates neurodegenerative pathways.

Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of ß-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.

Functional genomic analyses identify pathways dysregulated by progranulin deficiency, implicating Wnt signaling.

Progranulin (GRN) mutations cause frontotemporal dementia (FTD), but GRN's function in the CNS remains largely unknown. To identify the pathways downstream of GRN, we used weighted gene coexpression network analysis (WGCNA) to develop a systems-level view of transcriptional alterations in a human neural progenitor model of GRN-deficiency. This highlighted key pathways such as apoptosis and ubiquitination in GRN deficient human neurons, while revealing an unexpected major role for the Wnt signaling pathway, which was confirmed by analysis of gene expression data from postmortem FTD brain. Furthermore, we observed that the Wnt receptor Fzd2 was one of only a few genes upregulated at 6 weeks in a GRN knockout mouse, and that FZD2 reduction caused increased apoptosis, while its upregulation promoted neuronal survival in vitro. Together, these in vitro and in vivo data point to an adaptive role for altered Wnt signaling in GRN deficiency-mediated FTD, representing a potential therapeutic target.

Regulation of MET by FOXP2, genes implicated in higher cognitive dysfunction and autism risk.

Autism spectrum disorder (ASD) is a highly heritable, behaviorally defined, heterogeneous disorder of unknown pathogenesis. Several genetic risk genes have been identified, including the gene encoding the receptor tyrosine kinase MET, which regulates neuronal differentiation and growth. An ASD-associated polymorphism disrupts MET gene transcription, and there are reduced levels of MET protein expression in the mature temporal cortex of subjects with ASD. To address the possible neurodevelopmental contribution of MET to ASD pathogenesis, we examined the expression and transcriptional regulation of MET by a transcription factor, FOXP2, which is implicated in regulation of cognition and language, two functions altered in ASD. MET mRNA expression in the midgestation human fetal cerebral cortex is strikingly restricted, localized to portions of the temporal and occipital lobes. Within the cortical plate of the temporal lobe, the pattern of MET expression is highly complementary to the expression pattern of FOXP2, suggesting the latter may play a role in repression of gene expression. Consistent with this, MET and FOXP2 also are reciprocally expressed by differentiating normal human neuronal progenitor cells (NHNPs) in vitro, leading us to assess whether FOXP2 transcriptionally regulates MET. Indeed, FOXP2 binds directly to the 5' regulatory region of MET, and overexpression of FOXP2 results in transcriptional repression of MET. The expression of MET in restricted human neocortical regions, and its regulation in part by FOXP2, is consistent with genetic evidence for MET contributing to ASD risk.

Siomycin A targets brain tumor stem cells partially through a MELK-mediated pathway.

Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.

Endogenous Wnt signaling maintains neural progenitor cell potency.

Wnt signaling regulates neural stem cell (NSC) function and development throughout an individual's lifetime. Intriguingly, adult hippocampal progenitors (AHPs) produce several Wnts, and the intracellular machinery necessary to respond to them, creating the potential for an active autocrine-signaling loop within this stem cell niche. However, the standard luciferase-based Wnt assay failed to detect this signaling loop. This assay is inherently less temporally sensitive to activity among a population of unsynchronized proliferating cells because it relies on the rapidly degrading reporter luciferase. We circumvented this limitation using a promoter assay that employs green fluorescent protein (GFP), as a relatively long-lived reporter of canonical Wnt activity. We found that at baseline, AHPs secreted functional Wnt that self-stimulates low-level canonical Wnt signaling. Elimination baseline Wnt activity, via application of an extracellular Wnt antagonist promoted neurogenesis, based on a combination of unbiased gene expression analysis and cell-fate analysis. A detailed clonal analysis of progenitors transduced with specific intracellular antagonists of canonical signaling, either Axin or truncated cadherin (beta-catenin sequestering), revealed that loss of baseline signaling depletes the population of multipotent precursors, thereby driving an increasing fraction to assume a committed cell fate (i.e., unipotent progenitors). Similarly, baseline Wnt signaling repressed differentiation of human NSCs. Although the specific Wnts produced by neural precursors vary with age and between species, their effects remain remarkably consistent. In sum, this study establishes that autonomous Wnt signaling is a conserved feature of the neurogenic niche that preserves the delicate balance between NSC maintenance and differentiation.

Markers of adult neural stem cells.

I provide detailed protocols for conduction and troubleshooting the key steps in our three most used experimental designs: (1) prospectively counting and sorting of human neural stem cells (NSCs)/committed progenitors before placing them in culture; (2) high-throughput methods of quantifying changes in NSC/progenitor proliferation, in vitro; and (3) retrovirally tagging NSCs before differentiation to assess cell fates in individual clones. Detailed troubleshooting of immunohistochemical and fluorescence-activated cell sorting staining is described. Some of these techniques overlap with other chapters in this volume. Ultimately, this provision of complementary technical information should help ensure the reader's experimental success.

Mechanism of uptake and retention of F-18 BMS-747158-02 in cardiomyocytes: a novel PET myocardial imaging agent.

BACKGROUND: BMS-747158-02 is a novel fluorine 18-labeled pyridazinone derivative designed for cardiac imaging. The uptake and retention mechanisms of F-18 BMS-747158-02 in cardiac myocytes were studied in vitro, and the biodistribution of F-18 BMS-747158-02 was studied in vivo in mice. METHODS AND RESULTS: Fluorine 19 BMS-747158-01 inhibited mitochondrial complex I (MC-I) in bovine heart submitochondrial particles with an IC(50) of 16.6 +/- 3 nmol/L that was comparable to the reference inhibitors of MC-1, rotenone, pyridaben, and deguelin (IC(50) of 18.2 +/- 6.7 nmol/L, 19.8 +/- 2.6 nmol/L, and 23.1 +/- 1.5 nmol/L, respectively). F-18 BMS-747158-02 had high uptake in monolayers of neonatal rat cardiomyocytes (10.3% +/- 0.7% of incubated drug at 60 minutes) that was inhibited by 200 nmol/L of rotenone (91% +/- 2%) and deguelin (89% +/- 3%). In contrast, an inactive pyridaben analog, P-070 (IC(50) value >4 micromol/L in MC-1 assay), did not inhibit the binding of F-18 BMS-747158-02 in cardiomyocytes. Uptake and washout kinetics for F-18 BMS-747158-02 in rat cardiomyocytes indicated that the time to half-maximal (t((1/2))) uptake was very rapid (approximately 35 seconds), and washout t((1/2)) for efflux of F-18 BMS-747158-02 was greater than 120 minutes. In vivo biodistribution studies in mice showed that F-18 BMS-747158-02 had substantial myocardial uptake (9.5% +/- 0.5% of injected dose per gram) at 60 minutes and heart-to-lung and heart-to-liver ratios of 14.1 +/- 2.5 and 8.3 +/- 0.5, respectively. Positron emission tomography imaging in the mouse allowed clear cardiac visualization and demonstrated sustained myocardial uptake through 55 minutes. CONCLUSIONS: F-18 BMS-747158-02 is a novel positron emission tomography cardiac tracer targeting MC-I in cardiomyocytes with rapid uptake and slow washout. These characteristics allow fast and sustained accumulation in the heart.

Gene expression profiling in frataxin deficient mice: microarray evidence for significant expression changes without detectable neurodegeneration.

Friedreich's ataxia (FRDA) is caused by reduction of frataxin levels to 5-35%. To better understand the biochemical sequelae of frataxin reduction, in absence of the confounding effects of neurodegeneration, we studied the gene expression profile of a mouse model expressing 25-36% of the normal frataxin levels, and not showing a detectable phenotype or neurodegenerative features. Despite having no overt phenotype, a clear microarray gene expression phenotype was observed. This phenotype followed the known regional susceptibility in this disease, most changes occurring in the spinal cord. Additionally, gene ontology analysis identified a clear mitochondrial component, consistent with previous findings. We were able to confirm a subset of changes in fibroblast cell lines from patients. The identification of a core set of genes changing early in the FRDA pathogenesis can be a useful tool in both clarifying the disease process and in evaluating new therapeutic strategies.

Antiangiogenesis and anticancer efficacy of TA138, a novel alphavbeta3 antagonist.

BACKGROUND: Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. MATERIALS AND METHODS: The antiangiogenic efficacy of the novel alphavbeta3 antagonist TA138 was examined using in vivo and in vitro model systems. RESULTS: The in vitro studies demonstrated the ability of TA138 and RP747 (conjugated TA138) to inhibit endothelial cell migration toward vitronectin, with an IC50=0.04 and 0.045 microM, respectively. Furthermore, utilizing the chick chorioallantoic membrane models, TA138 inhibited basic fibroblast growth factor-induced neovascularization. CONCLUSION: TA138 might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or other pathological neovascularization processes. RP747 demonstrated antitumor efficacy in 1 spontaneous tumor model (c-neu oncomouse model, alphavbeta3 positive cells) and in 1 xenograft model (HCT116 human tumor colon carcinoma, alphavbeta3 negative cells) injected subcutaneously into nude mice.

Antisense oligonucleotides selectively regulate aspartyl beta-hydroxylase and its truncated protein isoform in vitro but distribute poorly into A549 tumors in vivo.

Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.

Where, oh where, have my stem cells gone?

During the summer of 2001, Americans were treated to high political drama courtesy of the debate over embryonic and adult stem cell research. The popular press was flush with predictions about how neural stem cells would reverse, almost by magic, the devastation caused by diseases such as Alzheimer's, Parkinson's, stroke or spinal cord injury. Unfortunately, this promise remains unfulfilled because we have such a poor understanding of how stem cells function. With regard to adult stem cells, we are not even completely sure where they are, or how or when they got there. A provocative study by Ourednik et al. published in Science suggests that in primates, adult neural stem cells are allocated during early corticogenesis. The study also provides evidence for the existence of stem cells dispersed throughout the frontal cortex and striatum.

An objective procedure for ischemic area evaluation of the stroke intraluminal thread model in the mouse and rat.

Computer-assisted procedures are used to measure infarct areas in animal stroke models, but this approach usually follows the less objective manual tracing of the boundaries of the infarct. Building on previously reported methodology using scanned images of triphenyltetrazolium chloride (TTC)-stained rat brains in the intraluminal thread model, we developed an objective method to assess ischemic damage in both the mouse and rat brains. The unique addition to our approach is the use of sham-treated animals, which thereby permits the removal of normal brain white matter from the ipsilateral injured brain. All brain sections per animal were scanned simultaneously using a Microtek Scanmaker 4 flatbed scanner. Color segmentation on full color images of 2 mm coronal brain sections was performed. Using Image Pro Plus (4.0) and color segmentation, ischemic and normal white matter areas were measured in the green channel and the entire brain area in the red channel. The percent of unstained tissue was calculated for sham-treated animals and for those with cerebral ischemia. By subtracting the average unstained area of the sham-treated group from the average unstained area from the ischemic group, the ischemic area was calculated. This methodology was validated using mouse and rat permanent and transient, focal ischemia models and MK-801 in the permanent ischemia models. MK-801, dosed at 3 mg/kg i.p. prior to the injury, reduced the injury by 75% in the mouse and 44% in the rat permanent occlusion models. The benefits of this methodology include: objectivity of the analysis of the ischemic injury, use of readily available software so that costs can be contained and removal of normal subcortical white matter from the calculation. This method should allow more consistent evaluation of changes in the infarct size, therefore, resulting in reduced variability and higher productivity.