Neuronal Precursor Cells Research Articles

Potential Role of Growth Factors Controlled Release in Achieving Enhanced Neuronal Trans-differentiation from Mesenchymal Stem Cells for Neural Tissue Repair and Regeneration.

With an increase in the incidence of neurodegenerative diseases, a need to replace incapable conventional methods has arisen. To overcome this burden, stem cells therapy has emerged as an efficient treatment option. Endeavours to accomplish this have paved the path to neural regeneration through efficient neuronal transdifferentiation. Despite their potential, the use of stem cells still entails several limitations, such as low differentiation efficiency and difficulties in guiding differentiation. The process of neural differentiation through the stem cells is achieved through the use of chemical inducers or growth factors and their direct introduction reduces their bioavailability in the system. To address these limitations, neural regeneration ventures require growth factors to be effectively implemented on stem cells in order to produce functional neuronal precursor cells. An efficient technique to achieve it is through the delivery of growth factors via microcarriers for their sustained release. It ensures the presence of commensurable concentration even at later stages of neuronal transdifferentiation. Nanofibers and nanoparticles, along with liposomes and such, have been used to implement this. The interaction between such carriers and the growth factors is mainly electrostatic. Such interaction enables them to form a stable assembly through immobilisation of the growth factor either onto their surfaces or within the core of their structures. The rate of sustained release depends upon the release kinetics associated with the polymeric structure employed and its interaction with the encapsulated growth factor. The sustained release ensures that the stem cells immerse under the effect of the growth factors for a prolonged period, ultimately aiding in the formation of cells showing ample characteristics of neuron precursors. This review analyses the various carriers that have been employed for the release of growth factors in an orderly fashion and their constituents, along with the advantages and the limitations they pose in delivering the growth factors for facilitating the process of neuronal transdifferentiation.

EXTH-51. DEVELOPMENT OF A NOVEL GENE THERAPY APPROACH TARGETING GLIOBLASTOMA FOLLOWING ARTIFICIAL INTELLIGENCE (AI)-DIRECTED IDENTIFICATION OF THE GBM STATE

Abstract BACKGROUND Profound heterogeneity has severely hampered therapeutic advancements in GBM. Remarkably, however, GBM exhibits broad clinical and histopathologic overlaps suggesting the presence of a common state. The GBM state embodies network restructuring forced by founding mutations and perpetuated in subclones of GBM stem-like cells (GSCs). Successful targeting of the GBM state promises to circumvent the heterogeneity. METHODS To decipher the GBM state, we applied NETZEN, an AI suite integrating a deep neural network with gene network-based ranking, to first generate the reference GBM gene network from TCGA’s entire GBM RNAseq collection, and then identify the altered master regulatory gene subnetwork in GBM using a dataset containing >30 diverse patient-derived GSC lines and their paired differentiated cells, 6 astrocyte and 3 neuronal precursor cell lines. To develop a gene therapy against the GBM state, we screened a rAAV capsid library through GBM patient-derived xenografts (PDX) to identify variants with specific tropism for GBM cells that can deliver targeting constructs intratumorally. RESULTS We discovered a putative GBM state anchored by developmentally restricted master regulators. To validate its critical role, we deconstructed it using shRNA in a panel of PDX and uniformly observed improved tumor control and survival compared to controls (p< 0.05 in all lines). More notably, when the core GBM state was forcibly reconstructed in astrocytes, transformation into GSC-like cells occurred, as measured by single-cell analysis, neurosphere formation, and most importantly, development of lethal infiltrating brain tumors in 15/15 mice. Finally, selected novel rAAV capsids with 10-40-fold higher specificity for GBM cells were utilized in a shRNA-based rAAV platform to target key master regulators of the validated GBM state. CONCLUSIONS The GBM state is established by a developmental master regulator subnetwork permitting the creation of a first-of-its-kind, heterogeneity-agnostic GBM therapy. This AI-directed R&D program can be expanded to target other tumors.

Mapping Proteoglycan Function Using Novel Genetic Strategies.

Multiple intrinsic and extrinsic factors contribute to stem and neuronal precursor cell maintenance and/or differentiation. Proteoglycans, major residents of the stem cell microenvironment, modulate key signaling cues and are of particular importance. The complexity and diversity of the glycan structure of proteoglycans make their functional characterization a challenging task. In order to test the functional role of glycosaminoglycans (GAGs) in cell self-renewal, maintenance, and differentiation, we have taken a loss-of-function approach by developing a library of both biosynthetic and degradative enzymes to specifically remodel the ECM.

ZIKV Infection and miRNA Network in Pathogenesis and Immune Response.

Over the years, viral infections have caused severe illness in humans. Zika Virus (ZIKV) is a flavivirus transmitted by mosquito vectors that leads to notable neurological impairment, whose most dramatic impact is the Congenital ZIKV Syndrome (CZS). ZIKV targets neuronal precursor cells leading to apoptosis and further impairment of neuronal development, causing microcephaly, lissencephaly, ventriculomegaly, and calcifications. Several regulators of biological processes are involved in CZS development, and in this context, microRNAs (miRNAs) seem to have a fundamental role. miRNAs are important regulators of protein translation, as they form the RISC silencing complex and interact with complementary mRNA target sequences to further post-transcriptional repression. In this context, little is known about their participation in the pathogenesis of viral infections. In this review, we discuss how miRNAs could relate to ZIKV and other flavivirus infections.

Maternal immune activation downregulates schizophrenia genes in the foetal mouse brain.

Susceptibility to schizophrenia is mediated by genetic and environmental risk factors. Maternal immune activation by infections during pregnancy is hypothesized to be a key environmental risk factor. However, little is known about how maternal immune activation contributes to schizophrenia pathogenesis. In this study, we investigated if maternal immune activation influences the expression of genes associated with schizophrenia in foetal mouse brains. We found that two sets of schizophrenia genes were downregulated more than expected by chance in the foetal mouse brain following maternal immune activation, namely those genes associated with schizophrenia through genome-wide association study (fold change = 1.93, false discovery rate = 4 × 10−4) and downregulated genes in adult schizophrenia brains (fold change = 1.51, false discovery rate = 4 × 10−10). We found that these genes mapped to key biological processes, such as neuronal cell adhesion. We also identified cortical excitatory neurons and inhibitory interneurons as the most vulnerable cell types to the deleterious effects of this interaction. Subsequently, we used gene expression information from herpes simplex virus 1 infection of neuronal precursor cells as orthogonal evidence to support our findings and to demonstrate that schizophrenia-associated cell adhesion genes, PCDHA2, PCDHA3 and PCDHA5, were downregulated following herpes simplex virus 1 infection. Collectively, our results provide novel evidence for a link between genetic and environmental risk factors in schizophrenia pathogenesis. These findings carry important implications for early preventative strategies in schizophrenia.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

Electroconvulsive Shock, but Not Transcranial Magnetic Stimulation, Transiently Elevates Cell Proliferation in the Adult Mouse Hippocampus.

Hippocampal plasticity is hypothesized to play a role in the etiopathogenesis of depression and the antidepressant effect of medications. One form of plasticity that is unique to the hippocampus and is involved in depression-related behaviors in animal models is adult neurogenesis. While chronic electroconvulsive shock (ECS) strongly promotes neurogenesis, less is known about its acute effects and little is known about the neurogenic effects of other forms of stimulation therapy, such as repetitive transcranial magnetic stimulation (rTMS). Here, we investigated the time course of acute ECS and rTMS effects on markers of cell proliferation and neurogenesis in the adult hippocampus. Mice were subjected to a single session of ECS, 10 Hz rTMS (10–rTMS), or intermittent theta burst stimulation (iTBS). Mice in both TMS groups were injected with BrdU 2 days before stimulation to label immature cells. One, 3, or 7 days later, hippocampi were collected and immunostained for BrdU + cells, actively proliferating PCNA + cells, and immature DCX + neurons. Following ECS, mice displayed a transient increase in cell proliferation at 3 days post-stimulation. At 7 days post–stimulation there was an elevation in the number of proliferating neuronal precursor cells (PCNA + DCX +), specifically in the ventral hippocampus. iTBS and rTMS did not alter the number of BrdU + cells, proliferating cells, or immature neurons at any of the post-stimulation time points. Our results suggest that neurostimulation treatments exert different effects on hippocampal neurogenesis, where ECS may have greater neurogenic potential than iTBS and 10–rTMS.

The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs.

SummaryThe generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.

Alterations in Dendritic Spine Maturation and Neurite Development Mediated by FAM19A1.

Neurogenesis and functional brain activity require complex associations of inherently programmed secretory elements that are regulated precisely and temporally. Family with sequence similarity 19 A1 (FAM19A1) is a secreted protein primarily expressed in subsets of terminally differentiated neuronal precursor cells and fully mature neurons in specific brain substructures. Several recent studies have demonstrated the importance of FAM19A1 in brain physiology; however, additional information is needed to support its role in neuronal maturation and function. In this study, dendritic spine morphology in Fam19a1-ablated mice and neurite development during in vitro neurogenesis were examined to understand the putative role of FAM19A1 in neural integrity. Adult Fam19a1-deficient mice showed low dendritic spine density and maturity with reduced dendrite complexity compared to wild-type (WT) littermates. To further explore the effect of FAM19A1 on neuronal maturation, the neurite outgrowth pattern in primary neurons was analyzed in vitro with and without FAM19A1. In response to FAM19A1, WT primary neurons showed reduced neurite complexity, whereas Fam19a1-decifient primary neurons exhibited increased neurite arborization, which was reversed by supplementation with recombinant FAM19A1. Together, these findings suggest that FAM19A1 participates in dendritic spine development and neurite arborization.

CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9−/− and corresponding C57BL/6 wild-type control mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9−/− mice showed an increased loss of neuronal nuclear protein+ mature neurons and doublecortin+ neuronal precursor cells and an increase in β-amyloid precursor protein+ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8+ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and embryonic stem cells and their derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.

Phenolic constituents isolated from Senna tora sprouts and their neuroprotective effects against glutamate-induced oxidative stress in HT22 and R28 cells

The consumption of sprouts has been steadily increasing due to their being an excellent source of nutrition. It is known that the bioactive constituents of legumes can be increased after germination. In this study, the extract from Senna tora sprouts is shown to exhibit improved radical scavenging activities and better neuroprotective effects in HT22 hippocampal neuronal (HT22) and R28 retina precursor (R28) cells than those from seeds due to an increased content of phenolic constituents, especially compounds 1 and 3–6. A phytochemical investigation of S. tora sprouts resulted in the isolation of two new naphthopyrone glycosides (1–2) with 27 previously reported compounds. Their structures were determined via interpreting spectroscopic data. Compounds 1 and 3–6 were found to possess radical scavenging activities and neuroprotective effects against oxidative stress in both neuronal cells. Hence, Senna tora sprouts and their constituents may be developed as natural neuroprotective agents via antioxidative effects.

(Invited) Biomimetic Nanocomposites for Implantable Devices

Materials with difficult-to-attain combination of multiple properties - mechanical, electrical, optical, chemical, transport, and biological – represent the quintessential bottleneck implantable and in particularly neuroprosthetic devices. Nanocomposites with molecular, nano-, meso-, and microscale levels of structural engineering can address these requirement, which can be demonstrated on the basis of layered biomimetic nanocomposites from nanoparticles, nanoshells, and carbon nanotubes. Besides mechanical robustness making possible their implantation, the biomimetic nanocomposites can exceed charge injection capacity and other key electrochemical characteristics of gold, IrOx and other materials used currently for neural stimulation. Small dimensions of the nanocomposite electrodes afford marked reduction of the scar tissue formation around implants. Photoactive multilayer from semiconductor particles were used to grow neuron precursor cells on them. It was found that light adsorbed in the nanoparticle layers results in the electrical excitation of the neurons making this system a functional analog of retina. Cartilage-like nanocomposites from aramid nanofibers also provide the long-term corrosion-resistant protection for a wide range of neuroprosthetic devices. New strides toward the design of the protective coatings and other nanomaterials for implantable devices using graph theory (GT) will be described.

Advances in congenital Zika syndrome and its pathogenesis

<p indent=0mm>Zika virus (ZIKV), originally discovered in a sentinel rhesus monkey in Zika valley in Uganda in 1947, has long been viewed as a classical mosquito-borne flavivirus. Due to its association with only a mild and self-limiting illness, ZIKV was a neglected pathogen following <sc>60 years</sc> since its identification. However, this virus became a serious public health concern in 2015−2016 outbreak in the Americas because of its association with congenital Zika syndrome (CZS). World Health Organization (WHO) declared the ZIKV outbreak a public health emergency of international concern in 2016. CZS is a pattern of birth defects found among fetuses and babies infected with ZIKV during pregnancy. The main clinical features of CZS are variable including severe microcephaly, decreased brain tissue, meningitis, damage to the back of the eye, hearing defects, arthrogryposis as well as hypertonia restricting body movement soon after birth. Benefiting from close collaboration of the international scientific community, intensive studies have been performed in development of experimental models to gain insights into the molecular basis of CZS in humans. These models include cultured neuroprogenitor stem cells, cortical organoids, especially mouse and nonhuman primate animal models which recapitulate many features of CZS in humans. Thanks to these models, questions related to how ZIKV infection causes congenital disease have been largely answered. For example, ZIKV was found to directly infects human neural progenitor cells (hNPCs) of the cerebral cortex, resulting in reduced hNPC proliferation; ZIKV also exhibits tropism for microglia in the brain, leading to production of inflammatory cytokines that inhibit neuronal precursor cell proliferation and differentiation. Additionally, studies have suggested that ZIKV can infect and trigger inflammasome pathways and innate antiviral immune responses of glial cells. While remarkable progress has been made to explain the associated underlying mechanisms, many unanswered key questions remain as follows: (1) How is the placenta in different developmental stage susceptible to ZIKV infection? (2) How is ZIKV transmitted across the placenta to infect the fetus? (3) What is the neurodevelopmental sequelae of congenital infection? (4) How does pre-existing maternal flavivirus immunity impact pregnancy-associated disease? Overall, due to collaborative and multidisciplinary efforts, a large body of knowledge of ZIKV biology has been obtained that has deepened our understanding of the CZS and informed the development of candidate vaccines and therapies. The lessons we have learned from ZIKV could enable effective preparation for the ultimate control of emergence of new congenital viral diseases in the future.

Aromatic-Turmerone Analogs Protect Dopaminergic Neurons in Midbrain Slice Cultures through Their Neuroprotective Activities.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The inflammatory activation of microglia participates in dopaminergic neurodegeneration in PD. Therefore, chemicals that inhibit microglial activation are considered to have therapeutic potential for PD. Aromatic (ar)-turmerone is a main component of turmeric oil extracted from Curcuma longa and has anti-inflammatory activity in cultured microglia. The aims of the present study are (1) to investigate whether naturally occurring S-enantiomer of ar-turmerone (S-Tur) protects dopaminergic neurons in midbrain slice cultures and (2) to examine ar-turmerone analogs that have higher activities than S-Tur in inhibiting microglial activation and protecting dopaminergic neurons. R-enantiomer (R-Tur) and two analogs showed slightly higher anti-inflammatory effects in microglial BV2 cells. S- and R-Tur and these two analogs reversed dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. Unexpectedly, this neuroprotection was independent of the inhibition of microglial activation. Additionally, two analogs more potently inhibited dopaminergic neurodegeneration triggered by a neurotoxin, 1-methyl-4-phenylpyridinium, than S-Tur. Taken together, we identified two ar-turmerone analogs that directly and potently protected dopaminergic neurons. An investigation using dopaminergic neuronal precursor cells suggested the possible involvement of nuclear factor erythroid 2-related factor 2 in this neuroprotection.

Unacylated ghrelin ameliorates muscle atrophy and contractile dysfunction in age‐associated loss of muscle mass and function

Sarcopenia, the progressive loss of muscle mass and dysfunction, universally affects the elderly and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation (acyl ghrelin, AG), which binds to its receptors (GHSR1a) in the brain. Growing studies demonstrate that AG and “unacyl” ghrelin (UnAG) both have direct impacts on proliferation independent of GHSR1a. Neuronal precursor cells and myoblast incubated with UnAG increases proliferation and protein synthesis rate by promoting differentiation and fusion. Thus, we hypothesized that UnAG ameliorates neurogenic atrophy and contractile dysfunction in aged skeletal muscle. Young (4 mo.) and old (18 mo.) female mice were treated with saline or UnAG. UnAG (100 µg/kg/day) via osmotic pump for 9-10 months. We assessed in vitro contractile properties of isolated extensor digitorum longus (EDL). UnAG treatment significantly increased terminal serum UnAG levels in the old animals. In contrast to previous studies using AG, UnAG did not affect food consumption or body weight. Muscle mass of gastrocnemius and quadriceps were ~20-30% decreased in old mice, but UnAG ameliorated the atrophy by 15-30% for both muscles. Specific force (force per cross-sectional are) of isolated EDL muscle was diminished by ~30% in Sod1KO, but UnAG restored the force deficit by ~80% in the KO mice. We find that upregulation of protein kinase A (PKA) and hyper-phosphorylation of FoxO3a signaling pathways may contribute to the protection. RNA-seq analysis further revealed that UnAG shifted mRNA expression of 366 genes towards younger states that are differentially regulated in old mice. Our data show a direct role of UnAG on sarcopenia independent of changes of food consumption or body weight, implicating UnAG as a potential therapeutic strategy that can enhance quality of life and healthspan of rapidly growing elderly population.

A Birst of LINE‐1 and IAP Retrotransposon Expression in the Mouse Brain Following Acute Behavioral Stress

Retrotransposons belong to mobile genetic elements that account for up to ~40% of the human genome and are propagated by being transcribed into RNA, then reverse-transcribed into DNA and inserted into a new site in the genome. Physiological regulation and possible effects of such retrotranspositions in cells of an adult organism are largely unknown. In the mouse genome LINE-1 (L1) and intracisternal A particle (IAP) are among functional and the most transpositionally active retrotransposons, capable of self-copying and insertion into DNA. Recent findings indicate that L1 is active in neuronal precursor cells during differentiation, and its de novo insertions can change expression of neuron-specific genes and cause somatic mosaicism in the adult brain. Furthermore, clinical studies have identified increased L1 expression and copy numbers in the brain of patients with psychiatric disorders. However, there has been limited understanding whether such retrotranspositions happen in the adult brain, when and how they occur, and what is their role in the CNS function. In our experiments we test the hypothesis that retrotranspositions can be activated in the brain by behavioral and particularly cognitive stimuli and that this process can influence adaptive neural plasticity. In previous experiments we showed an increase in L1 DNA copy number in hippocampus, frontal cortex and cerebellum of the mouse brain, but not in the muscular and testicular tissues after a prolonged stress. In this study we tested whether an acute stress can lead to L1 and IAP mRNA expression changes in the mouse brain. We used male C57Bl/6 mice at the age of 5 months. To assess the effect of acute stress, the animals were subjected to immobilization for 30 minutes or 2 hours. The frontal cortex and hippocampus were taken for qPCR studies. Compared to naive home cage controls, mice that underwent 30 min immobilization had significantly increased mRNA levels of L1 and IAP in frontal cortex and L1 in hippocampus. After 2 hours of immobilization IAP mRNA levels were significantly increased in both frontal cortex and hippocampus compare to control group. Therefore, L1 expression is up-regulated prior to IAP expression, perhaps, due to the differences in the transcription regulation of these two elements. Thus, we have shown that expression of both L1 and IAP is rapidly induced in several brain regions by behavioral stress. Since our previous data demonstrated a stress-induced increase of L1 and IAP DNA copy number in the same brain structures, together it points to a full retrotransposition event. We further suggest that reverse transcription involved in L1 and IAP retrotransposition by behavioral stimuli may also contribute to long-term plasticity and memory formation in the nervous system.

Developmental Neurotoxicity of Fipronil and Rotenone on a Human Neuronal In Vitro Test System

Pesticide exposure during in utero and early postnatal development can cause a wide range of neurological defects. However, relatively few insecticides have been recognized as developmental neurotoxicants, so far. Recently, discovery of the insecticide, fipronil, in chicken eggs has raised public concern. The status of fipronil as a potential developmental neurotoxicant is still under debate. Whereas several in vivo and in vitro studies suggest specific toxicity, other in vitro studies could not confirm this concern. Here, we tested fipronil and its main metabolic product, fipronil sulfone both at concentrations between 1.98 and 62.5 µM, alongside with the established developmental neurotoxicant, rotenone (0.004–10 µM) in vitro on the human neuronal precursor cell line NT2. We found that rotenone impaired all three tested DNT endpoints, neurite outgrowth, neuronal differentiation, and precursor cell migration in a dose-dependent manner and clearly separable from general cytotoxicity in the nanomolar range. Fipronil and fipronil sulfone specifically inhibited cell migration and neuronal differentiation, but not neurite outgrowth in the micromolar range. The rho-kinase inhibitor Y-27632 counteracted inhibition of migration for all three compounds (EC50 between 12 and 50 µM). The antioxidant, n-acetyl cysteine, could ameliorate the inhibitory effects of fipronil on all three tested endpoints (EC 50 between 84 and 164 µM), indicating the involvement of oxidative stress. Fipronil sulfone had a stronger effect than fipronil, confirming the importance to test metabolic products alongside original pesticides. We conclude that in vitro fipronil and fipronil sulfone display specific developmental neurotoxicity on developing human model neurons.

Reconditioning the Neurogenic Niche of Adult Non-human Primates by Antisense Oligonucleotide-Mediated Attenuation of TGF\u03b2 Signaling

Adult neurogenesis is a target for brain rejuvenation as well as regeneration in aging and disease. Numerous approaches showed efficacy to elevate neurogenesis in rodents, yet translation into therapies has not been achieved. Here, we introduce a novel human TGFβ-RII (Transforming Growth Factor—Receptor Type II) specific LNA-antisense oligonucleotide (“locked nucleotide acid”—“NVP-13”), which reduces TGFβ-RII expression and downstream receptor signaling in human neuronal precursor cells (ReNcell CX® cells) in vitro. After we injected cynomolgus non-human primates repeatedly i.th. with NVP-13 in a preclinical regulatory 13-week GLP-toxicity program, we could specifically downregulate TGFβ-RII mRNA and protein in vivo. Subsequently, we observed a dose-dependent upregulation of the neurogenic niche activity within the hippocampus and subventricular zone: human neural progenitor cells showed significantly (up to threefold over control) enhanced differentiation and cell numbers. NVP-13 treatment modulated canonical and non-canonical TGFβ pathways, such as MAPK and PI3K, as well as key transcription factors and epigenetic factors involved in stem cell maintenance, such as MEF2A and pFoxO3. The latter are also dysregulated in clinical neurodegeneration, such as amyotrophic lateral sclerosis. Here, we provide for the first time in vitro and in vivo evidence for a novel translatable approach to treat neurodegenerative disorders by modulating neurogenesis.

P2Y2 deficiency impacts adult neurogenesis and related forebrain functions.

Adult neurogenesis occurs particularly in the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle. This continuous addition of neurons to pre-existing neuronal networks is essential for intact cognitive and olfactory functions, respectively. Purinergic signaling modulates adult neurogenesis, however, the role of individual purinergic receptor subtypes in this dynamic process and related cognitive performance is poorly understood. In this study, we analyzed the role of P2Y2 receptor in the neurogenic niches and in related forebrain functions such as spatial working memory and olfaction using mice with a targeted deletion of the P2Y2 receptor (P2Y2-/- ). Proliferation, migration, differentiation, and survival of neuronal precursor cells (NPCs) were analyzed by BrdU assay and immunohistochemistry; signal transduction pathway components were analyzed by immunoblot. In P2Y2-/- mice, proliferation of NPCs in the SGZ and the SVZ was reduced. However, migration, neuronal fate decision, and survival were not affected. Moreover, p-Akt expression was decreased in P2Y2-/- mice. P2Y2-/- mice showed an impaired performance in the Y-maze and a higher latency in the hidden food test. These data indicate that the P2Y2 receptor plays an important role in NPC proliferation as well as in hippocampus-dependent working memory and olfactory function.