Building a growing genomic data repository for maternal and fetal health through the PING Consortium.

Background: Prenatally transmitted viruses can cause severe damage to the developing brain. There is unexplained variability in prenatal brain injury and postnatal neurodevelopmental outcomes, suggesting disease modifiers. Discordant outcomes among dizygotic twins could be explained by genetic susceptibly or protection. Among several well-recognized threats to the developing brain, Zika is a mosquito-borne, positive-stranded RNA virus that was originally isolated in Uganda and spread to cause epidemics in Africa, Asia, and the Americas. In the Americas, the virus caused congenital Zika syndrome and a multitude of neurodevelopmental disorders. As of now, there is no preventative treatment or cure for the adverse outcomes caused by prenatal Zika infection. The Prenatal Infection and Neurodevelopmental Genetics (PING) Consortium was initiated in 2016 to identify factors modulating prenatal brain injury and postnatal neurodevelopmental outcomes for Zika and other prenatal viral infections. Methods: The Consortium has pooled information from eight multi-site studies conducted at 23 research centers in six countries to build a growing clinical and genomic data repository. This repository is being mined to search for modifiers of virally induced brain injury and developmental outcomes. Multilateral partnerships include commitments with Children's National Hospital (USA), Instituto Nacional de Salud (Colombia), the Natural History of Zika Virus Infection in Gestation program (Brazil), and Zika Instituto Fernandes Figueira (Brazil), in addition to the Centers for Disease Control and Prevention and the National Institutes of Health. Discussion: Our goal in bringing together these sets of patient data was to test the hypothesis that personal and populational genetic differences affect the severity of brain injury after a prenatal viral infection and modify neurodevelopmental outcomes. We have enrolled 4,102 mothers and 3,877 infants with 3,063 biological samples and clinical data covering over 80 phenotypic fields and 5,000 variables. There were several notable challenges in bringing together cohorts enrolled in different studies, including variability in the timepoints evaluated and the collected clinical data and biospecimens. Thus far, we have performed whole exome sequencing on 1,226 participants. Here, we present the Consortium's formation and the overarching study design. We began our investigation with prenatal Zika infection with the goal of applying this knowledge to other prenatal infections and exposures that can affect brain development.

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Retrotransposon insertions associated with risk of neurologic and psychiatric diseases.

Transposable elements (TEs) are active in neuronal cells raising the question whether TE insertions contribute to risk of neuropsychiatric disease. While genome-wide association studies (GWAS) serve as a tool to discover genetic loci associated with neuropsychiatric diseases, unfortunately GWAS do not directly detect structural variants such as TEs. To examine the role of TEs in psychiatric and neurologic disease, we evaluated 17,000 polymorphic TEs and find 76 are in linkage disequilibrium with disease haplotypes (P < 10-6 ) defined by GWAS. From these 76 polymorphic TEs, we identify potentially causal candidates based on having insertions in genomic regions of regulatory chromatin and on having associations with altered gene expression in brain tissues. We show that lead candidate insertions have regulatory effects on gene expression in human neural stem cells altering the activity of a minimal promoter. Taken together, we identify 10 polymorphic TE insertions that are potential candidates on par with other variants for having a causal role in neurologic and psychiatric disorders.

Fluconazole Is Neuroprotective via Interactions with the IGF-1 Receptor.

There is a continuing unmet medical need to develop neuroprotective strategies to treat neurodegenerative disorders. To address this need, we screened over 2000 compounds for potential neuroprotective activity in a model of oxidative stress and found that numerous antifungal agents were neuroprotective. Of the identified compounds, fluconazole was further characterized. Fluconazole was able to prevent neurite retraction and cell death in in vitro and in vivo models of toxicity. Fluconazole protected neurons in a concentration-dependent manner and exhibited efficacy against several toxic agents, including 3-nitropropionic acid, N-methyl D-aspartate, 6-hydroxydopamine, and the HIV proteins Tat and gp120. In vivo studies indicated that systemically administered fluconazole was neuroprotective in animals treated with 3-nitropropionic acid and prevented gp120-mediated neuronal loss. In addition to neuroprotection, fluconazole also induced proliferation of neural progenitor cells in vitro and in vivo. Fluconazole mediates these effects through upregulation and signaling via the insulin growth factor-1 receptor which results in decreased cAMP production and increased phosphorylation of Akt. Blockade of the insulin growth factor-1 receptor signaling with the selective inhibitor AG1024 abrogated the effects of fluconazole. Our studies suggest that fluconazole may be an attractive candidate for treatment of neurodegenerative diseases due to its protective properties against several categories of neuronal insults and its ability to spur neural progenitor cell proliferation.

SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Response of human macrophages to gamma radiation is mediated via expression of endogenous retroviruses.

Ionizing radiation-induced tissue damage recruits monocytes into the exposed area where they are differentiated to macrophages. These implement phagocytic removal of dying cells and elicit an acute inflammatory response, but can also facilitate tumorigenesis due to production of anti-inflammatory cytokines. Using primary human monocyte-derived macrophages (MDMs) and the THP1 monocytic cell line, we demonstrate that gamma radiation triggers monocyte differentiation toward the macrophage phenotype with increased expression of type I interferons (IFN-I) and both pro- and anti-inflammatory macrophage activation markers. We found that these changes correlate with significantly upregulated expression of 622 retroelements from various groups, particularly of several clades of human endogenous retroviruses (HERVs). Elevated transcription was detected in both sense and antisense directions in the HERV subgroups tested, including the most genetically homogeneous clade HML-2. The level of antisense transcription was three- to five-fold higher than of the sense strand levels. Using a proximity ligation assay and immunoprecipitation followed by RNA quantification, we identified an increased amount of the dsRNA receptors MDA-5 and TLR3 bound to an equivalent number of copies of sense and antisense chains of HERVK HML-2 RNA. This binding triggered MAVS-associated signaling pathways resulting in increased expression of IFN-I and inflammation related genes that enhanced the cumulative inflammatory effect of radiation-induced senescence. HML-2 knockdown was accompanied with reduced expression and secretion of IFNα, pro-inflammatory (IL-1ß, IL-6, CCL2, CCL3, CCL8, and CCL20) and anti-inflammatory (IL10) modulators in irradiated monocytes and MDMs. Taken together, our data indicate that radiation stress-induced HERV expression enhances the IFN-I and cytokine response and results in increased levels of pro-inflammatory modulators along with expression of anti-inflammatory factors associated with the macrophage tumorigenic phenotype.

Regulation of stem cell function and neuronal differentiation by HERV-K via mTOR pathway.

Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.

A Cluster of Autism-Associated Variants on X-Linked NLGN4X Functionally Resemble NLGN4Y.

Autism spectrum disorder (ASD) is more prevalent in males; however, the etiology for this sex bias is not well understood. Many mutations on X-linked cell adhesion molecule NLGN4X result in ASD or intellectual disability. NLGN4X is part of an X-Y pair, with NLGN4Y sharing ∼97% sequence homology. Using biochemistry, electrophysiology, and imaging, we show that NLGN4Y displays severe deficits in maturation, surface expression, and synaptogenesis regulated by one amino acid difference with NLGN4X. Furthermore, we identify a cluster of ASD-associated mutations surrounding the critical amino acid in NLGN4X, and these mutations phenocopy NLGN4Y. We show that NLGN4Y cannot compensate for the functional deficits observed in ASD-associated NLGN4X mutations. Altogether, our data reveal a potential pathogenic mechanism for male bias in NLGN4X-associated ASD.

Isoform-specific cleavage of neuroligin-3 reduces synapse strength.

The assembly and maintenance of synapses are dynamic processes that require bidirectional contacts between the pre- and postsynaptic structures. A network of adhesion molecules mediate this physical interaction between neurons. How synapses are disassembled and if there are distinct mechanisms that govern the removal of specific adhesion molecules remain unclear. Here, we report isoform-specific proteolytic cleavage of neuroligin-3 in response to synaptic activity and protein kinase C signaling resulting in reduced synapse strength. Although neuroligin-1 and neuroligin-2 are not directly cleaved by this pathway, when heterodimerized with neuroligin-3, they too undergo proteolytic cleavage. Thus protein kinase C-dependent cleavage is mediated through neuroligin-3. Recent studies on glioma implicate the neuroligin-3 ectodomain as a mitogen. Here we demonstrate: (1) there are mechanisms governing specific adhesion molecule remodeling; (2) neuroligin-3 is a key regulator of neuroligin cleavage events; and (3) there are two cleavage pathways; basal and activity-dependent that produce the mitogenic form of neuroligin-3.

Activated T cells induce proliferation of oligodendrocyte progenitor cells via release of vascular endothelial cell growth factor-A.

Neuroinflammatory diseases such as multiple sclerosis are characterized by infiltration of lymphocytes into the central nervous system followed by demyelination and axonal degeneration. While evidence suggests that activated T lymphocytes induce neurotoxicity and impair function of neural stem cells, the effect of T cells on oligodendrocyte progenitor cells (OPCs) is still uncertain, partly due to the difficulty in obtaining human OPCs. Here we studied the effect of activated T cells on OPCs using OPCs derived from human hematopoietic stem cells or from human fetal brain. OPCs were exposed to supernatants (sups) from activated T cells. Cell proliferation was determined by EdU incorporation and CellQuanti-Blue assays. Surprisingly, we found that sups from activated T cells induced OPC proliferation by regulating cell cycle progression. Vascular endothelial growth factor A (VEGF-A) transcripts were increased in T cells after activation. Immunodepletion of VEGF-A from activated T cell sups significantly attenuated its effect on OPC proliferation. Furthermore, VEGF receptor 2 (VEGFR2) was expressed on OPCs and its inhibition also attenuated activated T cell-induced OPC proliferation. Thus, activated T cells have a trophic role by promoting OPC proliferation via the VEGFR2 pathway.

Trailing-Edge Flap Control for Mitigating Rotor Power Fluctuations of a Large-Scale Offshore Floating Wind Turbine under the Turbulent Wind Condition.

A trailing-edge flap control strategy for mitigating rotor power fluctuations of a 5 MW offshore floating wind turbine is developed under turbulent wind inflow. The wind shear must be considered because of the large rotor diameter. The trailing-edge flap control strategy is based on the turbulent wind speed, the blade azimuth angle, and the platform motions. The rotor power is predicted using the free vortex wake method, coupled with the control strategy. The effect of the trailing-edge flap control on the rotor power is determined by a comparison with the rotor power of a turbine without a trailing-edge flap control. The optimal values of the three control factors are obtained. The results show that the trailing-edge flap control strategy is effective for improving the stability of the output rotor power of the floating wind turbine under the turbulent wind condition.

Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1ß.

BACKGROUND: The cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation. METHODS: Surface immunocytochemistry directly visualized protease-activated receptor-1 (PAR1) and interleukin-1 (IL-1) receptors on neurons in human postmortem cortex in patients with and without neuroinflammatory lesions. Viability of cultured neurons was determined after exposure to cerebrospinal fluid from patients with progressive multiple sclerosis or purified granzyme B and IL-1ß. Inhibitors of PAR1 activation and of PAR1-associated second messenger signaling were used to elucidate a mechanism of neurotoxicity. RESULTS: Immunohistochemistry of human post-mortem brain tissue demonstrated cells expressing higher amounts of PAR1 near and within subcortical lesions in patients with multiple sclerosis compared to control tissue. Human cerebrospinal fluid samples containing granzyme B and IL-1ß were toxic to human neuronal cultures. Granzyme B was neurotoxic through activation of PAR1 and subsequently the phospholipase Cß-IP3 second messenger system. Inhibition of PAR1 or IP3 prevented granzyme B toxicity. IL-1ß enhanced granzyme B-mediated neurotoxicity by increasing PAR1 expression. CONCLUSIONS: Neurons within the inflamed central nervous system are imperiled because they express more PAR1 and are exposed to a neurotoxic combination of both granzyme B and IL-1ß. The effects of these inflammatory mediators may be a contributing factor in the progressive brain atrophy associated with neuroinflammatory diseases. Knowledge of how exposure to IL-1ß and granzyme B act synergistically to cause neuronal death yields potential novel neuroprotective treatments for neuroinflammatory diseases.

Signal Construction-Based Dispersion Compensation of Lamb Waves Considering Signal Waveform and Amplitude Spectrum Preservation.

The results of Lamb wave identification for the aerospace structures could be easily affected by the nonlinear-dispersion characteristics. In this paper, dispersion compensation of Lamb waves is of particular concern. Compared with the similar research works on the traditional signal domain transform methods, this study is based on signal construction from the viewpoint of nonlinear wavenumber linearization. Two compensation methods of linearly-dispersive signal construction (LDSC) and non-dispersive signal construction (NDSC) are proposed. Furthermore, to improve the compensation effect, the influence of the signal construction process on the other crucial signal properties, including the signal waveform and amplitude spectrum, is considered during the investigation. The linear-dispersion and non-dispersion effects are firstly analyzed. Then, after the basic signal construction principle is explored, the numerical realization of LDSC and NDSC is discussed, in which the signal waveform and amplitude spectrum preservation is especially regarded. Subsequently, associated with the delay-and-sum algorithm, LDSC or NDSC is employed for high spatial resolution damage imaging, so that the adjacent multi-damage or quantitative imaging capacity of Lamb waves can be strengthened. To verify the proposed signal construction and damage imaging methods, the experimental and numerical validation is finally arranged on the aluminum plates.

Direct induction of human neural stem cells from peripheral blood hematopoietic progenitor cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses.

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

Interaction of paroxetine with mitochondrial proteins mediates neuroprotection.

There are severe neurological complications that arise from HIV infection, ranging from peripheral sensory neuropathy to cognitive decline and dementia for which no specific treatments are available. The HIV proteins secreted from infected macrophages, gp120 and Tat, are neurotoxic. The goal of this study was to screen, identify and develop neuroprotective compounds relevant to HIV-associated neurocognitive disorders (HAND). We screened more than 2000 compounds that included FDA approved drugs for protective efficacy against oxidative stress-mediated neurodegeneration and identified selective serotonin reuptake inhibitors (SSRIs) as potential neuroprotectants. Numerous SSRIs were then extensively evaluated as protectants against neurotoxicity as measured by changes in neuronal cell death, mitochondrial potential, and axodendritic degeneration elicited by HIV Tat and gp120 and other mitochondrial toxins. While many SSRIs demonstrated neuroprotective actions, paroxetine was potently neuroprotective (100 nM potency) against these toxins in vitro and in vivo following systemic administration in a gp120 neurotoxicity model. Interestingly, the inhibition of serotonin reuptake by paroxetine was not required for neuroprotection, since depletion of the serotonin transporter had no effect on its neuroprotective properties. We determined that paroxetine interacts selectively and preferentially with brain mitochondrial proteins and blocks calcium-dependent swelling but had less effect on liver mitochondria. Additionally, paroxetine induced proliferation of neural progenitor cells in vitro and in vivo in gp120 transgenic animals. Therefore, SSRIs such as paroxetine may provide a novel adjunctive neuroprotective and neuroregenerative therapy to treat HIV-infected individuals.

Derivation of neural stem cells from human adult peripheral CD34+ cells for an autologous model of neuroinflammation.

Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine.