Neural Cell Line Research Articles

Analysis of the interplay between MeCP2 and histone H1 during in vitro differentiation of human ReNCell neural progenitor cells

ABSTRACT An immortalized neural cell line derived from the human ventral mesencephalon, called ReNCell, and its MeCP2 knock out were used. With it, we characterized the chromatin compositional transitions undergone during differentiation, with special emphasis on linker histones. While the WT cells displayed the development of dendrites and axons the KO cells did not, despite undergoing differentiation as monitored by NeuN. ReNCell expressed minimal amounts of histone H1.0 and their linker histone complement consisted mainly of histone H1.2, H1.4 and H1.5. The overall level of histone H1 exhibited a trend to increase during the differentiation of MeCP2 KO cells. The phosphorylation levels of histone H1 proteins decreased dramatically during ReNCell’s cell differentiation independently of the presence of MeCP2. Immunofluorescence analysis showed that MeCP2 exhibits an extensive co-localization with linker histones. Interestingly, the average size of the nucleus decreased during differentiation but in the MeCP2 KO cells, the smaller size of the nuclei at the start of differentiation increased by almost 40% after differentiation by 8 days (8 DIV). In summary, our data provide a compelling perspective on the dynamic changes of H1 histones during neural differentiation, coupled with the intricate interplay between H1 variants and MeCP2. Abbreviations: ACN, acetonitrile; A230, absorbance at 230 nm; bFGF, basic fibroblast growth factor; CM, chicken erythrocyte histone marker; CNS, central nervous system; CRISPR, clustered regulated interspaced short palindromic repeatsDAPI, 4,’6-diaminidino-2-phenylindole; DIV, days in vitro (days after differentiation is induced); DMEM, Dulbecco’s modified Eagle medium; EGF, epidermal growth factor; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic proteinHPLC, high-performance liquid chromatography; IF, immunofluorescence; iPSCs, induced pluripotent stem cells; MAP2, microtubule-associated protein 2; MBD, methyl-binding domain; MeCP2, methyl-CpG binding protein 2; MS, mass spectrometry; NCP, nucleosome core particle; NeuN, neuron nuclear antigen; NPC, neural progenitor cellPAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PFA, paraformaldehyde; PTM, posttranslational modification; RP-HPLC, reversed phase HPLC; ReNCells, ReNCells VM; RPLP0, ribosomal protein lateral stalk subunit P0; RT-qPCR, reverse transcription quantitative polymerase-chain reaction; RTT, Rett Syndrome; SDS, sodium dodecyl sulphate; TAD, topologically associating domain; Triple KO, triple knockout.

CSIG-02. SHH PATHWAY ACTIVATION DRIVES NEURAL CREST CELL SIGNATURE IN A SUBGROUP OF MPNSTS

Abstract INTRODUCTION Neurofibromatosis Type 1 is hereditary tumor predisposition syndrome that results in the development of innumerable nerve sheath tumors that fall within the spectrum of benign, premalignant and malignant lesions. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs. METHODS We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, a SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment. RESULTS We observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6 . We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines. CONCLUSION SHH pathway activation drives dedifferentiation into a neural crest cell-like state in a subset of MPNSTs. We confirm that inhibiting SHH pathway activity in a subset of MPNSTS prevent growth and malignant progression, providing a rational for future clinical trials.

IPSC-derived vascular smooth muscle cell model recapitulates Loeys-Dietz-Syndrome type V aortic phenotype

Abstract Introduction Thoracic aortic aneurysm and dissection (TAAD) is a hallmark of Loeys-Dietz syndrome (LDS) and a prominent cause of morbidity and sudden death. Current drug therapies are only able to slow down but not stop or reverse aneurysm formation. TAAD pathomechanisms remain largely elusive, complicating the development of more effective treatments. Purpose The creation of a disease-relevant in vitro model, that recapitulates the disease phenotype, is incredibly valuable to gain better insights in the pathomechanisms underlying TAAD and to develop new treatments. Methods Validated induced pluripotent stem cells (iPSCs) of two male TAAD patients carrying the same pathogenic p.Asp263His variant in TGFB3 (LDS type V) and one male healthy individual were used in this study. One patient underwent a Bentall procedure at age 66 years because of insufficiency of a bicuspid aortic valve and TAA (50 mm, z-score= 5.2). The second patient presented with a type A dissection at age 31 years, also requiring Bentall surgery. The patients are distantly related, having a common ancestor allele, estimated to be arisen >400 years ago. We created and characterized iPSC-derived neural crest-vascular smooth muscle cell (NC-VSMC) lines from both LDS type V patients and the healthy control. Addition of a chemical thromboxane A2 receptor agonist initiated NC-VSMC contraction which was quantified by the change in impedance using the xCELLigence system and RTCA software. Additionally, the proliferation rate was quantified with the CyQuant Direct Cell Proliferation Assay Kit and inverted DMi8 fluorescence microscope (Leica). With an univariate ANOVA test, we assessed similarity of the variance between groups, after which the corresponding two-way t-test was performed. Results Within 30 minutes after thromboxane A2 receptor agonist addition, a maximal contracted state was reached, which was significantly lower for the first patient (2 clones) as compared to the control (2 cloness) (p=0.03). The time to get to this maximal contracted state was comparable between patient and control, while the surface area coverage over a duration of 1h stimulation was reduced for patient NC-VSMCs (p=0.04). Contraction of the NC-VSMC line (1 clone) from the second LDS patient did not significantly differ from the contraction profile of the control line. After one hour, U46619 was removed resulting in relaxation and full recovery for all NC-VSMCs. Proliferation rate of NC-VSMCs from all patient and control lines was around 1.5, with no significant difference between the groups (p=0.59). Conclusion We created an NC-VSMC model that recapitulates the LDS aortic phenotype with regards to reduced VSMC contractility. Although NC-VSMC lines created from the same individual showed extremely little variability, there is important variability between the lines of different patients which might be due to immaturity of the cells, environmental factors and the presence of genetic modifiers.

Toxic dinoflagellate Karenia mikimotoi induces apoptosis in Neuro-2a cells through an oxidative stress-mediated mitochondrial pathway

The dinoflagellate Karenia mikimotoi is a toxic bloom-forming species that threatens aquaculture and public health worldwide. Previous studies showed that K. mikimotoi induces neurotoxicity; however, the underlying mechanism is poorly understood. In this study, three neural cell lines were used to investigate the potential neurotoxicity of K. mikimotoi. The tested cells were exposed to a ruptured cell solution (RCS) of K. mikimotoi at different concentrations (0.5 × 105, 1.0 × 105, 2.0 × 105, 4.0 × 105, and 6 × 105 cells mL−1) for 24 h, and the RCS decreased cell viabilities and promoted Neuro-2a (N2A) cell apoptosis in a dose-dependent manner. The underlying mechanism was further investigated in N2A cells. At the biochemical level, the RCS stimulated reactive oxygen species (ROS) and malondialdehyde (MDA) formation, decreased SOD activity, and reduced mitochondrial membrane potential (MMP). At the gene level, the moderate RCS treatment (2.0 × 105 cells mL−1) upregulated antioxidant response genes (e.g., nrf-2, HO-1, NQO-1, and cat) to alleviate RCS-induced oxidative stress, while the high RCS treatment (4.0 × 105 cells mL−1) downregulated these genes, thereby aggravating oxidative stress. Meanwhile, apoptosis-related genes (e.g., p53, caspase 3, and bax2) were significantly upregulated and the anti-apoptotic gene bcl2 was suppressed after RCS treatment. Western blotting results for Caspase 3, Bax2 and Bcl2 were consistent with the mRNA trends. These results revealed that K. mikimotoi RCS can induce neural cell apoptosis via the oxidative stress-mediated mitochondrial pathway, providing novel insights into the neurotoxicity of K. mikimotoi.

Pluripotent stem cell-derived neural progenitor cells can be used to model effects of IL-6 on human neurodevelopment

ABSTRACT Maternal immune activation (MIA) increases the risks for neurodevelopmental disorders in offspring through inflammatory cytokines, including interleukin-6 (IL-6). We therefore aimed to establish a human two-dimensional (2D) in vitro neural model to investigate the effects of IL-6 exposure on neurodevelopment. IL-6 signal transduction requires two receptors: interleukin-6 signal transducer (IL6ST) and interleukin-6 receptor (IL6R). Prenatally, neural cells lack IL6R, and hence cannot elicit cis IL-6 signaling, but IL6R can be provided by microglia in trans. We demonstrate here that an immortalized human neural progenitor cell (NPC) line, ReNCell CX, expresses IL6ST and elicits both cis and trans IL-6 signaling, limiting its use as a model of MIA. In contrast, induced pluripotent stem cell (iPSC)-derived NPCs only activate the IL-6 cascade in trans. Activation of the trans IL-6 cascade did not result in increased proliferation of iPSC-derived NPCs or ReNCell CX, as has been demonstrated in animal models. iPSC-derived NPCs upregulated NR2F1 expression in response to IL-6 signaling in line with analogous experiments in organoids. Thus, iPSC-derived NPCs can be used to model gene expression changes in response to MIA in 2D cultures.

Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells

Background• Neural stem/progenitor cells (NSPCs) are multipotent self-renewing cells that can be isolated from the brain or spinal cord. As they need to be isolated from neural tissues, it is difficult to study human NSPCs. To facilitate NSPC research, we attempted to isolate NSPCs from dogs, as dogs share the environment and having many similar diseases with humans. We collected and established primary cultures of ependymal and subependymal cells from the central canal of the cervical spinal cord of adult dogs. To isolate pure NSPCs, we employed the monolayer culture and selective medium culture methods. We further tested the ability of the NSPCs to form neurospheres (using the suspension culture method) and evaluated their differentiation potential.Results• The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested.Conclusion• It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.

PEDOT-Coated PLA Fibers Electrospun from Solutions Incorporating Fe(III)Tosylate in Different Solvents by Vapor-Phase Polymerization for Neural Regeneration.

Therapeutic solutions for injuries in the peripheral nervous system are limited and not existing in the case of the central nervous system. The electrical stimulation of cells through a cell-supporting conductive scaffold may contribute to new therapeutic solutions for nerve regeneration. In this work, biocompatible Polylactic acid (PLA) fibrous scaffolds incorporating Fe(III)Tosylate (FeTos) were produced by electrospinning a mixture of PLA/FeTos solutions towards a rotating cylinder, inducing fiber alignment. Fibers were coated with the conductive polymer Poly(3,4 ethylenedioxythiophene) (PEDOT) formed by vapor-phase polymerization of EDOT at 70 °C for 2 h. Different solvents (ETH, DMF and THF) were used as FeTos solvents to investigate the impact on the scaffold's conductivity. Scaffold conductivity was estimated to be as high as 1.50 × 10-1 S/cm when FeTos was dissolved in DMF. In vitro tests were performed to evaluate possible scaffold cytotoxicity, following ISO 10993-5, revealing no cytotoxic effects. Differentiation and growth of cells from the neural cell line SH-SY5Y seeded on the scaffolds were also assessed, with neuritic extensions observed in cells differentiated in neurons with retinoic acid. These extensions tended to follow the preferential alignment of the scaffold fibers.

A PrP EGFR signaling axis controls neural stem cell senescence through modulating cellular energy pathways

Mis-folding of the prion protein (PrP) is known to cause neurodegenerative disease; however, the native function of this protein remains poorly defined. PrP has been linked with many cellular functions, including cellular proliferation and senescence. It is also known to influence epidermal growth factor receptor (EGFR) signaling, a pathway that is itself linked with both cell growth and senescence. Adult neural stem cells (NSCs) persist at low levels in the brain throughout life and retain the ability to proliferate and differentiate into new neural lineage cells. KO of PrP has previously been shown to reduce NSC proliferative capacity. We used PrP KO and WT NSCs from adult mouse brain to examine the influence of PrP on cellular senescence, EGFR signaling, and the downstream cellular processes. PrP KO NSCs showed decreased cell proliferation and increased senescence in invitro cultures. Expression of EGFR was decreased in PrP KO NSCs compared with WT NSCs and additional supplementation of EGF was sufficient to reduce senescence. RNA-seq analysis confirmed that significant changes were occurring at the mRNA level within the EGFR signaling pathway and these were associated with reduced expression of mitochondrial components and correspondingly reduced mitochondrial function. Metabolomic analysis of cellular energy pathways showed that blockages were occurring at critical sites for production of energy and biomass, including catabolism of pyruvate. We conclude that, in the absence of PrP, NSC growth pathways are downregulated as a consequence of insufficient energy and growth intermediates.

Neuronal conversion from glia to replenish the lost neurons.

Neuronal injury, aging, and cerebrovascular and neurodegenerative diseases such as cerebral infarction, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington's disease are characterized by significant neuronal loss. Unfortunately, the neurons of most mammals including humans do not possess the ability to self-regenerate. Replenishment of lost neurons becomes an appealing therapeutic strategy to reverse the disease phenotype. Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain, but it carries the risk of causing gene mutation, tumorigenesis, severe inflammation, and obstructive hydrocephalus induced by brain edema. Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss, which may overcome the above-mentioned disadvantages of neural stem cell therapy. Thus far, many strategies to transform astrocytes, fibroblasts, microglia, Müller glia, NG2 cells, and other glial cells to mature and functional neurons, or for the conversion between neuronal subtypes have been developed through the regulation of transcription factors, polypyrimidine tract binding protein 1 (PTBP1), and small chemical molecules or are based on a combination of several factors and the location in the central nervous system. However, some recent papers did not obtain expected results, and discrepancies exist. Therefore, in this review, we discuss the history of neuronal transdifferentiation, summarize the strategies for neuronal replenishment and conversion from glia, especially astrocytes, and point out that biosafety, new strategies, and the accurate origin of the truly converted neurons in vivo should be focused upon in future studies. It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transcription factors or down-regulation of PTBP1 or drug interference therapies.

The Foxi3 transcription factor is necessary for the fate restriction of placodal lineages at the neural plate border.

The Foxi3 transcription factor, expressed in the neural plate border at the end of gastrulation, is necessary for the formation of posterior placodes and is thus important for ectodermal patterning. We have created two knock-in mouse lines expressing GFP or a tamoxifen-inducible Cre recombinase to show that Foxi3 is one of the earliest genes to label the border between the neural tube and epidermis, and that Foxi3-expressing neural plate border progenitors contribute primarily to cranial placodes and epidermis from the onset of expression, but not to the neural crest or neural tube lineages. By simultaneously knocking out Foxi3 in neural plate border cells and following their fates, we show that neural plate border cells lacking Foxi3 contribute to all four lineages of the ectoderm - placodes, epidermis, crest and neural tube. We contrast Foxi3 with another neural plate border transcription factor, Zic5, the progenitors of which initially contribute broadly to all germ layers until gastrulation and gradually become restricted to the neural crest lineage and dorsal neural tube cells. Our study demonstrates that Foxi3 uniquely acts early at the neural plate border to restrict progenitors to a placodal and epidermal fate.

P02.08.B ROLE OF THE PROTEIN NETWORK OF THE PARACASPASE MALT1 IN SELF-RENEWAL PROPERTIES OF GLIOBLASTOMA CELLS

Abstract BACKGROUND Glioblastoma is the most common and most aggressive brain tumor in adults. Despite standard treatment combining surgery, radiotherapy and chemotherapy, relapse is inevitable, making this cancer a major public health issue. Within this tumor reside cells with a stem-like character, called GSCs for Glioblastoma Stem-like Cells, which maintain, initiate, and propagate the tumor, making them a potential target to hinder glioblastoma. These cells constitute a reservoir of heterogeneous cells capable of differentiating into neural lineage cell types. In this context, lysosomal homeostasis plays a key role in the maintenance of the GSC reservoir. Furthermore, previous studies demonstrated that the paracaspase MALT1 is a major regulator of lysosomal homeostasis: MALT1 is required for GSC viability as well as for the maintenance of stemness by modulating the abundance of lysosomal proteins. This current study addresses the issue of the balance between self-renewal and viability of GSCs. In addition, this project aims to identify and characterize an actor of self-renewal mechanisms that does not impact the survival-death balance of primary cells from patients. MATERIAL AND METHODS An initial analysis combining DepMap and String Network databases allowed us to select potential relays of the MALT1 activity in GSCs. A functional screening was then set up and performed in GSCs derived from patients to identify among 20 candidates those regulating cell expansion without impacting viability. RESULTS The strategy of functional screening allowed the selection of 14 genes with a global viability effect, while only 2 genes were affecting self-renewal while sparing viability in vitro. We next pursued with one of the identified targets, from the IKK family. The involvement of this serine/threonine protein kinase was then confirmed in multiple patient cells and in CRISPR-mediated knock-out. Future experiments will decipher the involvement of this kinase in the self-renewal properties of GSCs. CONCLUSION This study highlights a putative candidate specifically operating on the self-renewal of glioblastoma cells, that does not affect the life-death balance. Future investigations will permit to characterize and understand the underlying mechanisms.

Two Sides of The Same Coin: Normal and Tumoral Stem Cells, The Relevance of In Vitro Models and Therapeutic Approaches: The Experience with Zika Virus in Nervous System Development and Glioblastoma Treatment.

Neural stem cells (NSCs) were described for the first time more than two decades ago for their ability to differentiate into all neural cell lineages. The isolation of NSCs from adults and embryos was carried out by various laboratories and in different species, from mice to humans. Similarly, no more than two decades ago, cancer stem cells were described. Cancer stem cells, previously identified in hematological malignancies, have now been isolated from several solid tumors (breast, brain, and gastrointestinal compartment). Though the origin of these cells is still unknown, there is a wide consensus about their role in tumor onset, propagation and, in particular, resistance to treatments. Normal and neoplastic neural stem cells share common characteristics, and can thus be considered as two sides of the same coin. This is particularly true in the case of the Zika virus (ZIKV), which has been described as an inhibitor of neural development by specifically targeting NSCs. This understanding prompted us and other groups to evaluate ZIKV action in glioblastoma stem cells (GSCs). The results indicate an oncolytic activity of this virus vs. GSCs, opening potentially new possibilities in glioblastoma treatment.

Down-Regulation of ABCA7 in Human Microglia, Astrocyte and THP-1 Cell Lines by Cholesterol Depletion, IL-1β and TNFα, or PMA.

Adenosine triphosphate-binding cassette transporter subfamily A member 7 (ABCA7) is a major risk factor for Alzheimer's disease. Human neural cell lines were used to investigate the regulation of ABCA7 expression by cholesterol and pro-inflammatory cytokines. Cholesterol was depleted by methyl-β-cyclodextrin, followed by treatment with rosuvastatin to suppress de novo synthesis, while the cells underwent adjustment to low cholesterol. Cholesterol depletion by 50-76% decreased ABCA7 expression by ~40% in C20 microglia and ~21% in A172 astrocytes but had no effect on the protein in SK-N-SH neurons. Cholesterol depletion also suppressed ABCA7 in HMC3 microglia. Previously, cholesterol loss was reported to up-regulate ABCA7 in murine macrophages. ABCA7 was down-regulated during PMA-induced differentiation of human THP-1 monocytes to macrophages. But, cholesterol depletion in THP-1 macrophages by ~71% had no effect on ABCA7. IL-1β and TNFα reduced ABCA7 expression in C20 and HMC3 microglia but not in A172 astrocytes or SK-N-SH neurons. IL-6 did not affect ABCA7 in the neural cells. These findings suggest that ABCA7 is active in regular homeostasis in human neural cells, is regulated by cholesterol in a cell type-dependent manner, i.e., cholesterol depletion down-regulates it in human neuroglia but not neurons, and is incompatible with IL-1β and TNFα inflammatory responses in human microglia.

Deoxyribonucleoside treatment rescues EtBr-induced mtDNA depletion in iPSC-derived neural stem cells with POLG mutations.

Mutations in POLG, the gene encoding the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (Pol-γ), lead to diseases driven by defective mtDNA maintenance. Despite being the most prevalent cause of mitochondrial disease, treatments for POLG-related disorders remain elusive. In this study, we used POLG patient-induced pluripotent stem cell (iPSC)-derived neural stem cells (iNSCs), one homozygous for the POLG mutation c.2243G>C and one compound heterozygous with c.2243G>C and c.1399G>A, and treated these iNSCs with ethidium bromide (EtBr) to study the rate of depletion and repopulation of mtDNA. In addition, we investigated the effect of deoxyribonucleoside (dNs) supplementation on mtDNA maintenance during EtBr treatment and post-treatment repopulation in the same cells. EtBr-induced mtDNA depletion occurred at a similar rate in both patient and control iNSCs, however, restoration of mtDNA levels was significantly delayed in iNSCs carrying the compound heterozygous POLG mutations. In contrast, iNSC with the homozygous POLG mutation recovered their mtDNA at a rate similar to controls. When we treated cells with dNs, we found that this reduced EtBr-induced mtDNA depletion and significantly increased repopulation rates in both patient iNSCs. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation also within intact neural lineage cells and suggest that those with compound heterozygous mutation have a more severe defect of mtDNA synthesis. Our findings further highlight the potential for dNs to improve mtDNA replication in the presence of POLG mutations, suggesting that this may offer a new therapeutic modality for mitochondrial diseases caused by disturbed mtDNA homeostasis.

Protection of high-frequency low-intensity pulsed electric fields and brain-derived neurotrophic factor for SH-SY5Y cells against hydrogen peroxide-induced cell damage.

Neurodegenerative diseases (NDDs) pose a significant global health threat. In particular, Alzheimer disease, the most common type causing dementia, remains an incurable disease. Alzheimer disease is thought to be associated with an imbalance of reactive oxygen species (ROS) in neurons, and scientists considered ROS modulation as a promising strategy for novel remedies. In the study, human neural cell line SH-SY5Y was used in probing the effect of combining noninvasive high-frequency low-intensity pulsed electric field (H-LIPEF) and brain-derived neurotrophic factor (BDNF) in protection against hydrogen peroxide (H2O2)-induced neuron damage. Our result finds that the combination approach has intensified the neuroprotective effect significantly, perhaps due to H-LIPEF and BDNF synergistically increasing the expression level of the phosphorylated epidermal growth factor receptor (p-EGFR), which induces the survival-related mitogen-activated protein kinases (MAPK) proteins. The study confirmed the activation of extracellular signal-regulated kinase (ERK) and the downstream pro-survival and antioxidant proteins as the mechanism underlying neuron protection. These findings highlighted the potential of H-LIPEF combined with BDNF in the treatment of NDDs. Furthermore, BDNF-mimetic drugs combining with noninvasive H-LIPEF to patients is a promising approach worthy of further research. This points to strategies for selecting drugs to cooperate with electric fields in treating neurodegenerative disorders.

Contemporary Antiretroviral Therapy Dysregulates Iron Transport and Augments Mitochondrial Dysfunction in HIV-Infected Human Microglia and Neural-Lineage Cells.

HIV-associated cognitive dysfunction during combination antiretroviral therapy (cART) involves mitochondrial dysfunction, but the impact of contemporary cART on chronic metabolic changes in the brain and in latent HIV infection is unclear. We interrogated mitochondrial function in a human microglia (hμglia) cell line harboring inducible HIV provirus and in SH-SY5Y cells after exposure to individual antiretroviral drugs or cART, using the MitoStress assay. cART-induced changes in protein expression, reactive oxygen species (ROS) production, mitochondrial DNA copy number, and cellular iron were also explored. Finally, we evaluated the ability of ROS scavengers or plasmid-mediated overexpression of the antioxidant iron-binding protein, Fth1, to reverse mitochondrial defects. Contemporary antiretroviral drugs, particularly bictegravir, depressed multiple facets of mitochondrial function by 20-30%, with the most pronounced effects in latently infected HIV+ hμglia and SH-SY5Y cells. Latently HIV-infected hμglia exhibited upregulated glycolysis. Increases in total and/or mitochondrial ROS, mitochondrial DNA copy number, and cellular iron accompanied mitochondrial defects in hμglia and SH-SY5Y cells. In SH-SY5Y cells, cART reduced mitochondrial iron-sulfur-cluster-containing supercomplex and subunit expression and increased Nox2 expression. Fth1 overexpression or pre-treatment with N-acetylcysteine prevented cART-induced mitochondrial dysfunction. Contemporary cART impairs mitochondrial bioenergetics in hμglia and SH-SY5Y cells, partly through cellular iron accumulation; some effects differ by HIV latency.

Generation of a human haploid neural stem cell line for genome-wide genetic screening.

Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens. To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens. Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools. Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the human haNSCs' genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools. This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

PCP4 Promotes Alzheimer's Disease Pathogenesis by Affecting Amyloid-β Protein Precursor Processing.

Down syndrome (DS) is caused by an extra copy of all or part of chromosome 21. The patients with DS develop typical Alzheimer's disease (AD) neuropathology, indicating the role of genes on human chromosome 21 (HSA21) in the pathogenesis of AD. Purkinje cell protein 4 (PCP4), also known as brain-specific protein 19, is a critical gene located on HSA21. However, the role of PCP4 in DS and AD pathogenesis is not clear. To explore the role of PCP4 in amyloid-β protein precursor (AβPP) processing in AD. In this study, we investigated the role of PCP4 in AD progression in vitro and in vivo. In vitro experiments, we overexpressed PCP4 in human Swedish mutant AβPP stable expression or neural cell lines. In vitro experiments, APP23/PS45 double transgenic mice were selected and treated with AAV-PCP4. Multiple topics were detected by western blot, RT-PCR, immunohistochemical and behavioral test. We found that PCP4 expression was altered in AD. PCP4 was overexpressed in APP23/PS45 transgenic mice and PCP4 affected the processing of AβPP. The production of amyloid-β protein (Aβ) was also promoted by PCP4. The upregulation of endogenous AβPP expression and the downregulation of ADAM10 were due to the transcriptional regulation of PCP4. In addition, PCP4 increased Aβ deposition and neural plaque formation in the brain, and exuberated learning and memory impairment in transgenic AD model mice. Our finding reveals that PCP4 contributes to the pathogenesis of AD by affecting AβPP processing and suggests PCP4 as a novel therapeutic target for AD by targeting Aβ pathology.

The human acetylcholinesterase C-terminal T30 peptide activates neuronal growth through alpha 7 nicotinic acetylcholine receptors and the mTOR pathway

Acetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contribute to cellular development and aging. In particular, enzymatic cleavage of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the ⍺7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS). Proteomic analysis of cells exposed to (100 nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and ⍺7 nAChR activation. T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E and S6K1, and a decrease in the autophagy LC3B-II protein. These findings are corroborated in hippocampal neurons and show that T30 promotes dendritic arborization. Taken together, our findings define mTOR as a novel pathway activated by T30 interaction with the nAChR and suggest a role for this process in human disease.

Arrestin domain-containing protein 1-mediated microvesicles (ARMMs) protect against cadmium-induced neurotoxicity

Exposure to environmental heavy metals such as cadmium (Cd) is often linked to neurotoxicity but the underlying mechanisms remain poorly understood. Here we show that Arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMMs)–an important class of extracellular vesicles (EVs) whose biogenesis occurs at the plasma membrane–protect against Cd-induced neurotoxicity. Cd increased the production of EVs, including ARMMs, in a human neural progenitor cell line, ReNcell CX (ReN) cells. ReN cells that lack ARMMs production as a result of CRISPR-mediated ARRDC1 knockout were more susceptible to Cd toxicity as evidenced by increased LDH production as well as elevated level of oxidative stress markers. Importantly, adding ARMMs back to the ARRDC1-knockout ReN cells significantly reduced Cd-induced toxicity. Consistent with this finding, proteomics data showed that anti-oxidative stress proteins are enriched in ARMMs secreted from ReN cells. Together our study reveals a novel protective role of ARMMs in Cd neurotoxicity and suggests that ARMMs may be used therapeutically to reduce neurotoxicity caused by exposure to Cd and potentially other metal toxicants.