Regulation Of Neuronal Differentiation Research Articles

Unraveling the signaling network between dysregulated microRNA and mRNA expression in sevoflurane-induced developmental neurotoxicity in rat

Research has indicated that general anesthesia may cause neuroapoptosis and long-term cognitive dysfunction in developing animals, however, the precise mechanisms orchestrating these outcomes remain inadequately elucidated within scholarly discourse. The purpose of this study was to investigate the impact of sevoflurane on the hippocampus of developing rats by analyzing the changes in microRNA and mRNA and their interactions. Rats were exposed to sevoflurane for 4 h on their seventh day after birth, and the hippocampus was collected for analysis of neuroapoptosis by Western blot and immunohistochemistry. High-throughput sequencing was conducted to analyze the variances in miRNA and mRNA expression levels, and the Morris water maze was employed to assess long-term memory in rats exposed to sevoflurane after 8 weeks. The results showed that sevoflurane exposure led to dysregulation of 5 miRNAs and 306 mRNAs in the hippocampus. Bioinformatic analysis revealed that these dysregulated miRNA-mRNA target pairs were associated with pathological neurodevelopment and developmental disorders, such as regulation of axonogenesis, regulation of neuron projection development, regulation of neuron differentiation, transmission of nerve impulse, and neuronal cell body. Further analysis showed that these miRNAs formed potential network interactions with 44 mRNAs, and two important nodes were identified, miR-130b-5p and miR-449c-5p. Overall, this study suggests that the dysregulation of the miRNA-mRNA signaling network induced by sevoflurane may contribute to neurodevelopmental toxicity in the hippocampus of rats and be associated with long-term cognitive dysfunction.

18F]TRACK, a PET tracer for imaging of tropomyosin receptor kinases (Trk): Efficient radiosynthesis and preliminary evaluation in the TgF344‐AD rat model of Alzheimer’s disease

AbstractBackgroundA family of tropomyosin receptor kinases (TrkA/B/C) plays important roles in the regulation of neuronal differentiation, growth, and survival via their interactions with neurotrophins. A growing body of literature suggests the downregulation of Trk in many neurodegenerative conditions of the central nervous system (CNS), including Alzheimer’s disease (AD). We recently reported the development of [18F]TRACK, the PET tracers for in vivo imaging of TrkB and its first in‐human study. Here, we wish to report a fully automated GMP‐compliant radiosynthesis of [18F]TRACK at high radiochemical purity (RCP) and molar activity and its preliminary evaluation in wild‐type and a transgenic rat model of AD.MethodRadiosynthesis was performed using Scintomics GRP automated module via copper‐catalyzed 18F‐fluorination of the chiral boron pinacolate precursor. Brain imaging in wild‐type (n = 3) and TgF344‐AD rat models (n = 2) at 23 months was performed on microPET Concord R4 scanner.Result[18F]TRACK was synthesized with a 5.0±1.4% radiochemical yield not corrected for decay (activity range 95‐154 mCi) with >99% RCP and molar activities of 250 ± 75 GBq/µmol (n = 6). The synthesis was fully automated and all batches passed quality control procedures, including chemical purity, sterility and pyrogenicity. The tracer showed moderate brain uptake in wild‐type rats with the highest accumulation in the thalamus (SUVmax = 1.56 ± 0.28, 10 min post‐injection), consistent with the brain regional distribution in humans acquired in a parallel study. Preliminary data suggests lower tracer uptake in TgF344‐AD rat model of AD in thalamus, striatum and frontal cortex compared to wild‐type animals.ConclusionThis study demonstrated the feasibility of the GMP‐compliant production of [18F]TRACK in sufficient doses and molar activities for scanning multiple subjects per day or long range deliveries. Preliminary biodistribution studies showed moderate brain uptake of the tracer with preferential accumulation in the thalamus across species. These findings warrants further studies of Trk brain expression and its implication in neurodegenerative and psychiatric pathologies using [18F]TRACK.

MP20-04 ARHGEF2 REGULATES LINEAGE PLASTICITY PROMOTING NEUROENDOCRINE DIFFERENTIATION AND TREATMENT RESISTANCE IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-04 ARHGEF2 REGULATES LINEAGE PLASTICITY PROMOTING NEUROENDOCRINE DIFFERENTIATION AND TREATMENT RESISTANCE IN ADVANCED PROSTATE CANCER Shu Ning, Wei Lou, Alan Lombard, Masuda Sharifi, Zachary Schaaf, Christopher Evans, and Allen Gao Shu NingShu Ning More articles by this author , Wei LouWei Lou More articles by this author , Alan LombardAlan Lombard More articles by this author , Masuda SharifiMasuda Sharifi More articles by this author , Zachary SchaafZachary Schaaf More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Allen GaoAllen Gao More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Treatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies has been an aggressive variant of prostate cancer with unfavorable prognosis and limited therapeutic options. This study aims to identify potential neural lineage signature genes and evaluate whether they could serve as early biomarkers to stratify patients for optimal treatment and potential therapeutic targets in advanced prostate cancer. METHODS: We performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell lines and advanced prostate cancer patient databases to identify neural lineage signature (NLS) genes including ARHGEF2. The correlation of NLS genes with clinicopathologic features was determined. RNA sequencing analysis and viability of tumor organoids were performed on C4-2B MDVR and H660 cells with ARHGEF2 knocking down and gene set enrichment was analyzed. RESULTS: We identified a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency and neural stem cell differentiation, which may indicate a spectrum of early and intermediate stages of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine (NE) markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes. Differentially expressed NLS genes stratify small cell NEPC from prostate adenocarcinoma, which is closely associated with higher Gleason Score and metastasis status. Higher ARHGEF2 levels correlate with a shortened survival time in NEPC patients. ARHGEF2 regulates neuron differentiation and neural precursor cell proliferation which are significantly enriched in treatment-emergent NEPC patients and H660 cells. Upregulation of ARHGEF2 in both patient and experimental models positively correlates with NE markers. ARHGEF2 knocking down inhibited tumor organoids viability and NE markers in enzalutamide-resistant and NEPC cells. CONCLUSIONS: Our studies suggest that ARHGEF2 activation promotes neural lineage plasticity contributing to the survival and proliferation of enzalutamide-resistance and NEPC cells. ARHGEF2 could serve as a biomarker to stratify advanced prostate cancer patients for better treatment options and a potential therapeutic target in advanced prostate cancer. Source of Funding: NIH CA253605, CA 225836, CA250082, DOD PC180180 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e276 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shu Ning More articles by this author Wei Lou More articles by this author Alan Lombard More articles by this author Masuda Sharifi More articles by this author Zachary Schaaf More articles by this author Christopher Evans More articles by this author Allen Gao More articles by this author Expand All Advertisement PDF downloadLoading ...

Developmental Changes of Human Neural Progenitor Cells Grafted into the Ventricular System and Prefrontal Cortex of Mouse Brain in Utero

The transplantation of neural progenitors into a host brain represents a useful tool to evaluate the involvement of cell-autonomous processes and host local cues in the regulation of neuronal differentiation during the development of the mammalian brain. Human brain development starts at the embryonic stages, in utero, with unique properties at its neotenic stages. We analyzed the engraftment and differentiation of human neuronal progenitor cells (hNPCs) transplanted in utero into the mouse brain. The influence of the environment was studied by transplanting human NPCs within the lateral ventricles (LV), compared with the prefrontal cortex (PFC) of immunocompetent mice. We developed a semi-automated method to accurately quantify the number of cell bodies and the distribution of neuronal projections among the different mouse brain structures, at 1 and 3 months post-transplantation (MPT). Our data show that human NPCs can differentiate between immature "juvenile" neurons and more mature pyramidal cells in a reproducible manner. Depending on the injection site, LV vs. PFC, specific fetal local environments could modify the synaptogenesis processes while maintaining human neoteny. The use of immunocompetent mice as host species allows us to investigate further neuropathological conditions making use of all of the engineered mouse models already available.

EPO regulates neuronal differentiation of adult human neural-crest derived stem cells in a sex-specific manner

BackgroundSexual differences in the biology of human stem cells are increasingly recognized to influence their proliferation, differentiation and maturation. Especially in neurodegenerative diseases such as Alzheimers disease (AD), Parkinson’s disease (PD) or ischemic stroke, sex is a key player for disease progression and recovery of damaged tissue. Recently, the glycoprotein hormone erythropoietin (EPO) has been implicated as a regulator of neuronal differentiation and maturation in female rats.MethodsIn this study, we used adult human neural crest-derived stem cells (NCSCs) as a model system for exploring potential sex specific effects of EPO on human neuronal differentiation. We started with expression validation of the specific EPO receptor (EPOR) by performing PCR analysis in the NCSCs. Next, EPO mediated activation of nuclear factor-κB (NF-κB) via Immunocytochemistry (ICC) was performed, followed by investigating the sex-specific effects of EPO on neuronal differentiation by determining morphological changes in axonal growth and neurite formation accompanied by ICC.ResultsUndifferentiated male and female NCSCs showed a ubiquitous expression of the EPO receptor (EPOR). EPO treatment resulted in a statistically profound (male p = 0.0022, female p = 0.0012) nuclear translocation of NF-κB RELA in undifferentiated NCSCs of both sexes. But after one week of neuronal differentiation, we could show a highly significant (p = 0,0079) increase of nuclear NF-κB RELA in females only. In contrast, we observed a strong decrease (p = 0,0022) of RELA activation in male neuronal progenitors. Extending the view on the role of sex during human neuronal differentiation, here we demonstrate a significant increase of axon lengths in female NCSCs-derived neurons upon EPO-treatment (+ EPO: 167,73 (SD = 41,66) µm, w/o EPO: 77,68 (SD = 18,31) µm) compared to their male counterparts (+ EPO: 68,37 (SD = 11,97) µm, w/o EPO: 70,23 (SD = 12,89) µm).ConclusionOur present findings therefore show for the first time an EPO-driven sexual dimorphism in neuronal differentiation of human neural-crest derived stem cells and emphasize sex-specific variability as a crucial parameter in stem cell biology and for treating neurodegenerative diseases.

18F]TRACK, a PET tracer for imaging of tropomyosin receptor kinases (Trk): efficient radiosynthesis and preliminary evaluation in the TgF344‐AD rat model of Alzheimer’s disease.

AbstractBackgroundA family of tropomyosin receptor kinases (TrkA/B/C) plays important roles in the regulation of neuronal differentiation, growth, and survival via their interactions with neurotrophins. A growing body of literature suggests the downregulation of Trk in many neurodegenerative conditions of the central nervous system (CNS), including Alzheimer’s disease (AD). We recently reported the development of [18F]TRACK, the PET tracers for in vivo imaging of TrkB and its first in‐human study. Here, we wish to report a fully automated GMP‐compliant radiosynthesis of [18F]TRACK at high radiochemical purity (RCP) and molar activity and its preliminary evaluation in wild‐type and a transgenic rat model of AD.MethodRadiosynthesis was performed using Scintomics GRP automated module via copper‐catalyzed 18F‐fluorination of the chiral boron pinacolate precursor. Brain imaging in wild‐type (n = 3) and TgF344‐AD rat models (n = 2) at 23 months was performed on microPET Concord R4 scanner.Result[18F]TRACK was synthesized with a 5.0±1.4 % radiochemical yield not corrected for decay (activity range 95–154 mCi) with >99% RCP and molar activities of 250 ± 75 GBq/µmol (n = 6). The synthesis was fully automated and all batches passed quality control procedures, including chemical purity, sterility and pyrogenicity. The tracer showed moderate brain uptake in wild‐type rats with the highest accumulation in the thalamus (SUVmax = 1.56 ± 0.28, 10 min post‐injection), consistent with the brain regional distribution in humans acquired in a parallel study. Preliminary data suggests lower tracer uptake in TgF344‐AD rat model of AD in thalamus, striatum and frontal cortex compared to wild‐type animals.ConclusionThis study demonstrated the feasibility of the GMP‐compliant production of [18F]TRACK in sufficient doses and molar activities for scanning multiple subjects per day or long range deliveries. Preliminary biodistribution studies showed moderate brain uptake of the tracer with preferential accumulation in the thalamus across species. These findings warrants further studies of Trk brain expression and its implication in neurodegenerative and psychiatric pathologies using [18F]TRACK.

GPR137 Inhibits Cell Proliferation and Promotes Neuronal Differentiation in the Neuro2a Cells

The orphan receptor, G protein-coupled receptor 137 (GPR137), is an integral membrane protein involved in several types of cancer. GPR137 is expressed ubiquitously, including in the central nervous system (CNS). We established a GPR137 knockout (KO) neuro2A cell line to analyze GPR137 function in neuronal cells. KO cells were generated by genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and cultured as single cells by limited dilution. Rescue cells were then constructed to re-express GPR137 in GPR137 KO neuro2A cells using an expression vector with an EF1-alpha promoter. GPR137 KO cells increased cellular proliferation and decreased neurite outgrowth (i.e., a lower level of neuronal differentiation). Furthermore, GPR137 KO cells exhibited increased expression of a cell cycle regulator, cyclin D1, and decreased expression of a neuronal differentiation marker, NeuroD1. Additionally, GPR137 KO cells exhibited lower expression levels of the neurite outgrowth markers STAT3 and GAP43. These phenotypes were all abrogated in the rescue cells. In conclusion, GPR137 deletion increased cellular proliferation and decreased neuronal differentiation, suggesting that GPR137 promotes cell cycle exit and neuronal differentiation in neuro2A cells. Regulation of neuronal differentiation by GPR137 could be vital to constructing neuronal structure during brain development.Graphical

Effects of Importin α1/KPNA1 deletion and adolescent social isolation stress on psychiatric disorder-associated behaviors in mice.

Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Identification and validation of hub genes of synovial tissue for patients with osteoarthritis and rheumatoid arthritis

BackgroundOsteoarthritis (OA) and rheumatoid arthritis (RA) were two major joint diseases with similar clinical phenotypes. This study aimed to determine the mechanistic similarities and differences between OA and RA by integrated analysis of multiple gene expression data sets.MethodsMicroarray data sets of OA and RA were obtained from the Gene Expression Omnibus (GEO). By integrating multiple gene data sets, specific differentially expressed genes (DEGs) were identified. The Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein–protein interaction (PPI) network analysis of DEGs were conducted to determine hub genes and pathways. The “Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT)” algorithm was employed to evaluate the immune infiltration cells (IICs) profiles in OA and RA. Moreover, mouse models of RA and OA were established, and selected hub genes were verified in synovial tissues with quantitative polymerase chain reaction (qPCR).ResultsA total of 1116 DEGs were identified between OA and RA. GO functional enrichment analysis showed that DEGs were enriched in regulation of cell morphogenesis involved in differentiation, positive regulation of neuron differentiation, nuclear speck, RNA polymerase II transcription factor complex, protein serine/threonine kinase activity and proximal promoter sequence-specific DNA binding. KEGG pathway analysis showed that DEGs were enriched in EGFR tyrosine kinase inhibitor resistance, ubiquitin mediated proteolysis, FoxO signaling pathway and TGF-beta signaling pathway. Immune cell infiltration analysis identified 9 IICs with significantly different distributions between OA and RA samples. qPCR results showed that the expression levels of the hub genes (RPS6, RPS14, RPS25, RPL11, RPL27, SNRPE, EEF2 and RPL19) were significantly increased in OA samples compared to their counterparts in RA samples (P < 0.05).ConclusionThis large-scale gene analyses provided new insights for disease-associated genes, molecular mechanisms as well as IICs profiles in OA and RA, which may offer a new direction for distinguishing diagnosis and treatment between OA and RA.

Notch Signaling and MicroRNA: The Dynamic Duo Steering Between Neurogenesis and Glioblastomas.

Notch signaling is an evolutionary conserved pathway that plays a central role in development and differentiation of eukaryotic cells. It has been well documented that Notch signaling is inevitable for neuronal cell growth and homeostasis. It regulates process of differentiation from early embryonic stages to fully developed brain. To achieve this streamlined development of neuronal cells, a number of cellular processes are being orchestrated by the Notch signaling. Abrogated Notch signaling is related to several brain tumors, including glioblastomas. On the other hand, microRNAs are small molecules that play decisive role in mediating and modulating Notch signaling. This review discusses the crucial role of Notch signaling in development of nervous system and how this versatile pathway interplay with microRNAs in glioblastoma. This review sheds light on interplay between abrogated Notch signaling and miRNAs in the regulation of neuronal differentiation with special focus on miRNAs mediated regulation of tumorigenesis in glioblastoma. Furthermore, it discusses different aspects of neurogenesis modulated by the Notch signaling that could be exploited for the identification of new diagnostic tools and therapies for the treatment of glioblastoma.

Plantar Stimulations during 3-Day Hindlimb Unloading Prevent Loss of Neural Progenitors and Maintain ERK1/2 Activity in the Rat Hippocampus.

Adult neurogenesis is a flexible process that depends on the environment and correlates with cognitive functions. Cognitive functions are impaired by various factors including space flight conditions and reduced physical activity. Physically active life significantly improves both cognition and the hippocampal neurogenesis. Here, we analyzed how 3-day simulated microgravity caused by hindlimb unloading (HU) or dynamic foot stimulation (DFS) during HU can affect the hippocampal neurogenesis. Adult Wistar rats were recruited in the experiments. The results demonstrated a decrease in the number of doublecortine (DCX) positive neural progenitors, but proliferation in the subgranular zone of the dentate gyrus was not changed after 3-day HU. Analysis of the effects of DFS showed restoration of neural progenitor population in the subgranular zone of the dentate gyrus. Additionally, we analyzed activity of the cRaf/ERK1/2 pathway, which is one of the major players in the regulation of neuronal differentiation. The results demonstrated inhibition of cRaf/ERK1/2 signaling in the hippocampus of HU rats. In DFS rats, no changes in the activity of cRaf/ERK1/2 were observed. Thus, we demonstrated that the process of neurogenesis fading during HU begins with inhibition of the formation of immature neurons and associated ERK1/2 signaling activity, while DFS prevents the development of mentioned alterations.

Effects of artificial light at night (ALAN) on gene expression of Aquatica ficta firefly larvae.

Artificial light at night (ALAN) is a major driver of firefly population declines, but its physiological effects are not well understood. To investigate the impact of ALAN on firefly development, we exposed larval Aquatica ficta fireflies to ALAN for two weeks. High larval mortality was observed in the periods of 1-68 days and 106-134 days post-treatment, which may represent the short- and long-term impacts of ALAN. We then profiled the transcriptome of larval Aquatica ficta fireflies following two weeks of ALAN exposure. A total of 1262 (1.67% out of 75777 unigenes) were differentially expressed in the treatment group: 1157 were down-regulated, and 105 were up-regulated. Up-regulated unigenes were related to regulation of hormone levels, ecdysteroid metabolic process, and response to stimulus; down-regulated unigenes were related to negative regulation of insulin receptor signaling, germ cell development, oogenesis, spermatid development, and regulation of neuron differentiation. Transcriptome results suggest that the endocrine, reproductive, and neural development of firefly larvae could be impaired by even relatively brief period of ALAN exposure. This report contributes a much-needed molecular perspective to the growing body of research documenting the fitness impacts of ALAN on bioluminescent fireflies.

The Neurotrophic Function of Glucagon-Like Peptide-1 Promotes Human Neuroblastoma Differentiation via the PI3K-AKT Axis.

Simple SummaryThe study demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our results also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.Background: Neurons are terminally-differentiated cells that generally develop from neuronal stem cells stimulated by various neurotrophic factors such as NGF, BDNF, NT3, and NT-4. Neurotrophic factors have multiple functions for neurons, including enabling neuronal development, growth, and protection. Glucagon-like peptide-1 (GLP-1) is an intestinal-secreted incretin that enhances cellular glucose up-take to decrease blood sugar levels. However, many studies suggest that the function of GLP-1 is not limited to the regulation of blood sugar levels. Instead, it may also act as a neurotrophic factor with a role in ensuring neuronal survival and neurite outgrowth, as well as protecting synaptic plasticity and memory formation. Methods: The SH-SY5Y cells were differentiated by sequential treatments of retinoic acid and GLP-1 treatment within polyethylenimine-coated dishes under serum-free Neurobasal medium. PI3K inhibitor (LY294002) and MEK inhibitor (U0126) were used to determine the signaling pathway in regulation of neuronal differentiation. Neuronal marker (TUJ1) and synaptic markers (synapsin 1, synaptophysin, and PSD95) as well as single cell patch-clamp were applied to determine maturity of neurons. Antibodies of AMPA receptor, NMDA receptor subunit 2A, dopamine receptor D1, muscarinic acetylcholine receptor 2, and nicotinic acetylcholine receptor α4 were used to examine the types of differentiated neurons. Results: Our study’s results demonstrated that the treatment with GLP-1 of SH-SY5Y human neuroblastoma cells increased the expression of AMPA receptors, NMDA receptors, dopamine receptors, synaptic proteins-synapsin 1, synaptophysin, and postsynaptic density protein 95, but not muscular and nicotinic acetylcholine receptors. In addition, the biomarker of dividing neuronal cells, vimentin, was decreased after treatment with GLP-1. Tuj1 immunostaining images showed that GLP-1 induced neurite processes and the development of neuronal morphologies. The GLP-1-differentiated neurons were able to be induced to generate action potentials by single cell patch-clamp. Our study also suggested that the PI3K-AKT axis is the dominant signaling pathway promoting the differentiation of SH-SY5Y cells into mature and functional neurons in response to GLP-1 receptor activation. Conclusions: The sequential treatment of retinoic acid and GLP-1 within a serum-free medium is able to trigger the differentiation of SH-SY5Y cells into morphologically and physiologically mature glutamatergic and dopaminergic neurons.

The Requirement of Sox2 for the Spinal Cord Motor Neuron Development of Zebrafish.

Sex-determining region Y box 2 (Sox2), expressed in neural tissues, plays an important role as a transcription factor not only in the pluripotency and proliferation of neuronal cells but also in the opposite function of cell differentiation. Nevertheless, how Sox2 is linked to motor neuron development remains unknown. Here, we showed that Sox2 was localized in the motor neurons of spinal cord by in situ hybridization and cell separation, which acted as a positive regulator of motor neuron development. The deficiency of Sox2 in zebrafish larvae resulted in abnormal PMN development, including truncated but excessively branched CaP axons, loss of MiP, and increase of undifferentiated neuron cells. Importantly, transcriptome analysis showed that Sox2-depleted embryos caused many neurogenesis, axonogenesis, axon guidance, and differentiation-related gene expression changes, which further support the vital function of Sox2 in motor neuron development. Taken together, these data indicate that Sox2 plays a crucial role in the motor neuron development by regulating neuron differentiation and morphology of neuron axons.

A single cell transcriptome atlas of the developing zebrafish hindbrain.

ABSTRACTSegmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at boundaries and segment centres, in part mediated by Fgf20 signalling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation.

A genome-wide multiphenotypic association analysis identified candidate genes and gene ontology shared by four common risky behaviors.

Background: Risky behaviors can lead to huge economic and health losses. However, limited efforts are paid to explore the genetic mechanisms of risky behaviors.Result: MASH analysis identified a group of target genes for risky behaviors, such as APBB2, MAPT and DCC. For GO enrichment analysis, FUMA detected multiple risky behaviors related GO terms and brain related diseases, such as regulation of neuron differentiation (adjusted P value = 2.84×10-5), autism spectrum disorder (adjusted P value =1.81×10-27) and intelligence (adjusted P value =5.89×10-15).Conclusion: We reported multiple candidate genes and GO terms shared by the four risky behaviors, providing novel clues for understanding the genetic mechanism of risky behaviors.Methods: Multivariate Adaptive Shrinkage (MASH) analysis was first applied to the GWAS data of four specific risky behaviors (automobile speeding, drinks per week, ever-smoker, number of sexual partners) to detect the common genetic variants shared by the four risky behaviors. Utilizing genomic functional annotation data of SNPs, the SNPs detected by MASH were then mapped to target genes. Finally, gene set enrichment analysis of the identified candidate genes were conducted by the FUMA platform to obtain risky behaviors related gene ontology (GO) terms as well as diseases and traits, respectively.

Dopamine-Producing Neurons in Rat Ontogeny: Phenotypic Features Underlying Molecular Mechanisms of Secretion and Regulation

Dopamine (DA), which is synthesized in DA-ergic neurons and so-called monoenzymatic neurons, is involved in the regulation of neuronal differentiation during the critical period of morphogenesis. The aim of the present study was to examine phenotypic features of DA-producing neurons in several brain regions, including the striatum, suprachiasmatic nucleus, and arcuate nucleus, during rat ontogeny starting from embryonic day 18 (E18) to postnatal day 60 (P60). The enzymes of DA synthesis tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AAAD), vesicular monoamine transporters (VMAT), and D1 and D2 DA receptors were used as markers for functional activity of DA neurons. Expression of these markers was studied using double immunohistochemical labeling of TH and AAAD or PCR assay of TH, AAAD, VMAT1, VMAT2, and D1 and D2 DA receptors. In the striatum, mRNAs of all mentioned markers were revealed within the whole period of ontogeny studied, bienzymatic TH and AAAD-immunopositive neurons were observed at E18, whereas monoenzymatic TH- or AAAD-containing neurons were observed at P60 only. In the suprachiasmatic nucleus, monoenzymatic AAAD neurons coexist with monoenzymatic TH-immunoreactive fibers, which innervate the ventral part of the nucleus, in the early postnatal period (P10). At P10 in the suprachiasmatic nucleus, the TH, VMAT1, and VMAT2 genes are not expressed; however, transcripts of the D1 and D2 genes were detected. The arcuate nucleus of rat embryos contained only monoenzymatic TH neurons in the ventrolateral area and AAAD neurons in the dorsomedial area in the late prenatal period (E21). At the same time TH, AAAD, VMAT2, D1, and D2 mRNAs were expressed in both parts of the arcuate nucleus. Thus, during rat ontogeny, nondopaminergic neurons containing one of the enzymes, TH or AAAD, are present in the striatum, suprachiasmatic nucleus, and arcuate nucleus, which allows for suggesting the cooperative synthesis of dopamine in these brain structures.

P53 controls genomic stability and temporal differentiation of human neural stem cells and affects neural organization in human brain organoids

In this study, we take advantage of human induced pluripotent stem (iPS) cell-derived neural stem cells and brain organoids to study the role of p53 during human brain development. We knocked down (KD) p53 in human neuroepithelial stem (NES) cells derived from iPS cells. Upon p53KD, NES cells rapidly show centrosome amplification and genomic instability. Furthermore, a reduced proliferation rate, downregulation of genes involved in oxidative phosphorylation (OXPHOS), and an upregulation of glycolytic capacity was apparent upon loss of p53. In addition, p53KD neural stem cells display an increased pace of differentiating into neurons and exhibit a phenotype corresponding to more mature neurons compared to control neurons. Using brain organoids, we modeled more specifically cortical neurogenesis. Here we found that p53 loss resulted in brain organoids with disorganized stem cell layer and reduced cortical progenitor cells and neurons. Similar to NES cells, neural progenitors isolated from brain organoids also show a downregulation in several OXPHOS genes. Taken together, this demonstrates an important role for p53 in controlling genomic stability of neural stem cells and regulation of neuronal differentiation, as well as maintaining structural organization and proper metabolic gene profile of neural progenitors in human brain organoids.

Protein kinases: master regulators of neuritogenesis and therapeutic targets for axon regeneration.

Proper neurite formation is essential for appropriate neuronal morphology to develop and defects at this early foundational stage have serious implications for overall neuronal function. Neuritogenesis is tightly regulated by various signaling mechanisms that control the timing and placement of neurite initiation, as well as the various processes necessary for neurite elongation to occur. Kinases are integral components of these regulatory pathways that control the activation and inactivation of their targets. This review provides a comprehensive summary of the kinases that are notably involved in regulating neurite formation, which is a complex process that involves cytoskeletal rearrangements, addition of plasma membrane to increase neuronal surface area, coupling of cytoskeleton/plasma membrane, metabolic regulation, and regulation of neuronal differentiation. Since kinases are key regulators of these functions during neuromorphogenesis, they have high potential for use as therapeutic targets for axon regeneration after injury or disease where neurite formation is disrupted.

Abstract 4392: Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer

Abstract Background: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with poorly understood drivers of disease progression. We recently characterized five mCRPC phenotypes, including an amphicrine phenotype that co-expresses androgen receptor (AR) and neuroendocrine prostate cancer (NEPC) biomarkers. We determined that loss of RE1-silencing transcription factor (REST), a master regulator of neuronal differentiation, drives the amphicrine phenotype, but is not sufficient for prostate cancer (PC) adenocarcinoma to NEPC conversion. Furthermore, loss of REST through SRRM4-mediated splicing of REST pre-mRNA to REST4 has been suggested to drive adenocarcinoma to NEPC conversion. However, the roles of pre-mRNA splicing factors and REST activity in lineage switching require further investigation. Methods: Transcriptomic (RNASeq) and immunohistochemical/immunofluorescent analysis (IHC and IF) were conducted on amphicrine and NEPC patient metastases, LuCaP patient-derived xenograft (PDX) models and modified CRPC cell lines. The roles of SRRM3 and SRRM4 were examined using overexpression studies in AR-expressing and AR-null CRPC cell lines. Results: RNASeq, IHC and IF of metastatic specimens, LuCaP PDX models and VCaP cells confirmed the existence of the amphicrine phenotype in vitro and in vivo. Interestingly, transcriptome analysis of amphicrine patient specimens and LuCaP 77CR revealed that loss of REST repressor activity occurred without SRRM4 expression in a subset of tumor specimens. Indeed, BaseScope analysis using primers specific to REST4 and SRRM4 verified that amphicrine 77CR tumors were positive for REST4 expression but negative for SRRM4 expression, suggesting an alternative mechanism of REST splicing. Notably, overexpression of SRRM4 in AR-expressing C4-2B and AR-null PC-3 cells did not induce REST splicing. Moreover, RNASeq of SRRM4-overexpressing cells displayed heterogeneous transcriptome profiles inconsistent with canonical amphicrine or NEPC gene expression profiles. Interestingly, SRRM3 transcript was expressed at high levels in amphicrine and NEPC patient and LuCaP PDX biospecimens that lacked SRRM4 expression, suggesting an SRRM3-mediated mechanism of REST splicing. Studies interrogating the roles of SRRM3 in REST splicing and CRPC cellular plasticity are ongoing. Conclusions: Our data highlights an unrecognized mechanism of adenocarcinoma to amphicrine or NEPC conversion that hinges on a SRRM3-REST regulatory axis rather than REST-loss or SRRM4-mediated REST splicing. Identifying the mechanisms that may convert adenocarcinoma to treatment-resistant amphicrine or NEPC phenotypes in mCRPC patients will inform treatment and identify potential molecular pathways for therapeutic intervention. Citation Format: Mark P. Labrecque, Ilsa M. Coleman, Lisha G. Brown, Bryce Lakely, Lori Kollath, Daniel W. Lin, Lawrence D. True, Eva Corey, Peter S. Nelson, Colm Morrissey. Pre-mRNA splicing factors promote cellular plasticity in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4392.