Human Primary Neurons Research Articles

Therapeutic potential of Nlrp1 inflammasome, Caspase-1, or Caspase-6 against Alzheimer disease cognitive impairment.

The sequential activation of Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing protein 1 (Nlrp1) inflammasome, Caspase-1 (Casp1), and Caspase-6 (Casp6) is implicated in primary human neuron cultures and Alzheimer Disease (AD) neurodegeneration. To validate the Nlrp1-Casp1-Casp6 pathway in vivo, the APPSwedish/Indiana J20 AD transgenic mouse model was generated on either a Nlrp1, Casp1 or Casp6 null genetic background and mice were studied at 4-5 months of age. Episodic memory deficits assessed with novel object recognition were normalized by genetic ablation of Nlrp1, Casp1, or Casp6 in J20 mice. Spatial learning deficits, assessed with the Barnes Maze, were normalized in genetically ablated J20, whereas memory recall was normalized in J20/Casp1-/- and J20/Casp6-/-, and improved in J20/Nlrp1-/- mice. Hippocampal CA1 dendritic spine density of the mushroom subtype was reduced in J20, and normalized in genetically ablated J20 mice. Reduced J20 hippocampal dentate gyrus and CA3 synaptophysin levels were normalized in genetically ablated J20. Increased Iba1+-microglia in the hippocampus and cortex of J20 brains were normalized by Casp1 and Casp6 ablation and reduced by Nlrp1 ablation. Increased pro-inflammatory cytokines, TNF-α and CXCL1, in the J20 hippocampus were normalized by Nlrp1 or Casp1 genetic ablation. CXCL1was also normalized by Casp6 genetic ablation. IFN-γ was increased and total amyloid β peptide was decreased in genetically ablated Nlrp1, Casp1 or Casp6 J20 hippocampi. We conclude that Nlrp1, Casp1, or Casp6 are implicated in AD-related cognitive impairment, inflammation, and amyloidogenesis. These results indicate that Nlrp1, Casp1, and Casp6 represent rational therapeutic targets against cognitive impairment and inflammation in AD.

Functional Assessment of Stroke-Induced Regulation of miR-20a-3p and Its Role as a Neuroprotectant

MicroRNAs have gained popularity as a potential treatment for many diseases, including stroke. This study identifies and characterizes a specific member of the miR-17–92 cluster, miR-20a-3p, as a possible stroke therapeutic. A comprehensive microRNA screening showed that miR-20a-3p was significantly upregulated in astrocytes of adult female rats, which typically have better stroke outcomes, while it was profoundly downregulated in astrocytes of middle-aged females and adult and middle-aged males, groups that typically have more severe stroke outcomes. Assays using primary human astrocytes and neurons show that miR-20a-3p treatment alters mitochondrial dynamics in both cell types. To assess whether stroke outcomes could be improved by elevating astrocytic miR-20a-3p, we created a tetracycline (Tet)-induced recombinant adeno-associated virus (rAAV) construct where miR-20a-3p was located downstream a glial fibrillary acidic protein promoter. Treatment with doxycycline induced miR-20-3p expression in astrocytes, reducing mortality and modestly improving sensory motor behavior. A second Tet-induced rAAV construct was created in which miR-20a-3p was located downstream of a neuron-specific enolase (NSE) promoter. These experiments demonstrate that neuronal expression of miR-20a-3p is vastly more neuroprotective than astrocytic expression, with animals receiving the miR-20a-3p vector showing reduced infarction and sensory motor improvement. Intravenous injections, which are a therapeutically tractable treatment route, with miR-20a-3p mimic 4 h after middle cerebral artery occlusion (MCAo) significantly improved stroke outcomes including infarct volume and sensory motor performance. Improvement was not observed when miR-20a-3p was given immediately or 24 h after MCAo, identifying a unique delayed therapeutic window. Overall, this study identifies a novel neuroprotective microRNA and characterizes several key pathways by which it can improve stroke outcomes.

MiR-124 synergism with ELAVL3 enhances target gene expression to promote neuronal maturity

Neuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed by repressing antineurogenic genes. How these miRNAs function after the repression of fibroblast genes for neuronal fate remains unclear. Here, we identified targets of miR-9/9*-124 as reprogramming cells activate the neuronal program and reveal the role of miR-124 that directly promotes the expression of its target genes associated with neuronal development and function. The mode of miR-124 as a positive regulator is determined by the binding of both AGO and a neuron-enriched RNA-binding protein, ELAVL3, to target transcripts. Although existing literature indicates that miRNA-ELAVL family protein interaction can result in either target gene up-regulation or down-regulation in a context-dependent manner, we specifically identified neuronal ELAVL3 as the driver for miR-124 target gene up-regulation in neurons. In primary human neurons, repressing miR-124 and ELAVL3 led to the down-regulation of genes involved in neuronal function and process outgrowth and cellular phenotypes of reduced inward currents and neurite outgrowth. Our results highlight the synergistic role between miR-124 and RNA-binding proteins to promote target gene regulation and neuronal function.

MAM-2201, One of the Most Potent-Naphthoyl Indole Derivative-Synthetic Cannabinoids, Exerts Toxic Effects on Human Cell-Based Models of Neurons and Astrocytes.

Considering the consequences on human health, in general population and workplace, associated with the use of new psychoactive substances and their continuous placing on the market, novel in vitro models for neurotoxicology research, applying human-derived CNS cells, may provide a means to understand the mechanistic basis of molecular and cellular alterations in brain. Cytotoxic effects of MAM-2201, a potent-naphthoyl indole derivative-synthetic cannabinoid, have been evaluated applying a panel of human cell-based models of neurons and astrocytes, testing different concentrations (1-30µM) and exposure times (3-24-48h). MAM-2201 induced toxicity in primary neuron-like cells (hNLCs), obtained from transdifferentiation of mesenchymal stem cells derived from human umbilical cord. Effects occurred in a concentration- and time-dependent manner. The lowest concentration affecting cell viability, metabolic function, apoptosis, morphology, and neuronal markers (MAP-2, NSE) was 5μM, and even 1μM induced apoptosis. Effects appeared early (3h) and persisted after 24 and 48h. Similar behavior was evidenced for human D384-astrocytes treated with MAM-2201. Differently, human SH-SY5Y-neurons, both differentiated and undifferentiated, were not sensitive to MAM-2201. On D384, the different altered endpoints were reversed, attenuated, or not antagonized by AM251 indicating that CB1 receptors may partially mediate MAM-2201-induced cytotoxicity. While in hNLCs, all toxic effects caused by MAM-2201 were apparently unrelated to CB-receptors since they were not evidenced by immunofluorescence. The present in vitro findings demonstrate the cytotoxicity of MAM-2201 on human primary neurons (hNLCs) and astrocytes cell line (D384), and support the use of these cellular models as species-specific in vitro tools suitable to clarify the neurotoxicity mechanisms of synthetic cannabinoids.

Papaverine, a Phosphodiesterase 10A Inhibitor, Ameliorates Quinolinic Acid-Induced Synaptotoxicity in Human Cortical Neurons.

Phosphodiesterase-10A (PDE10A) hydrolyse the secondary messengers cGMP and cAMP, two molecules playing important roles in neurodevelopment and brain functions. PDE10A is associated to progression of neurodegenerative diseases like Alzheimer's, Parkinson's, Huntington's diseases, and a critical role in cognitive functions. The present study was undertaken to determine the possible neuroprotective effects and the associated mechanism of papaverine (PAP), a PDE10A isoenzyme inhibitor, against quinolinic acid (QUIN)-induced excitotoxicity using human primary cortical neurons. Cytotoxicity potential of PAP was analysed using MTS assay. Reactive oxygen species (ROS) and mitochondrial membrane potential were measured by DCF-DA and JC10 staining, respectively. Caspase 3/7 and cAMP levels were measured using ELISA kits. Effect of PAP on the CREB, BNDF and synaptic proteins such as SAP-97, synaptophysin, synapsin-I, and PSD-95 expression was analysed by Western blot. Pre-treatment with PAP increased intracellular cAMP and nicotinamide adenine dinucleotide (NAD+) levels, restored mitochondrial membrane potential (ΔΨm), and decreased ROS and caspase 3/7 content in QUIN exposed neurons. PAP up-regulated CREB and BDNF, and synaptic protein expression. In summary, these data indicate that PDE10A is involved in QUIN-mediated synaptotoxicity and its inhibition elicit neuroprotection by reducing the oxidative stress and protecting synaptic proteins via up-regulation of cAMP signalling cascade.

Fitness selection of hyperfusogenic measles virus F proteins associated with neuropathogenic phenotypes

Measles virus (MeV) is resurgent and caused >200,000 deaths in 2019. MeV infection can establish a chronic latent infection of the brain that can recrudesce months to years after recovery from the primary infection. Recrudescent MeV leads to fatal subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MIBE) as the virus spreads across multiple brain regions. Most clinical isolates of SSPE/MIBE strains show mutations in the fusion (F) gene that result in a hyperfusogenic phenotype in vitro and allow for efficient spread in primary human neurons. Wild-type MeV receptor-binding protein is indispensable for manifesting these mutant F phenotypes, even though neurons lack canonical MeV receptors (CD150/SLAMF1 or nectin-4). How such hyperfusogenic F mutants are selected and whether they confer a fitness advantage for efficient neuronal spread is unresolved. To better understand the fitness landscape that allows for the selection of such hyperfusogenic F mutants, we conducted a screen of ≥3.1 × 105 MeV-F point mutants in their genomic context. We rescued and amplified our genomic MeV-F mutant libraries in BSR-T7 cells under conditions in which MeV-F-T461I (a known SSPE mutant), but not wild-type MeV, can spread. We recovered known SSPE mutants but also characterized at least 15 hyperfusogenic F mutations with an SSPE phenotype. Structural mapping of these mutants onto the prefusion MeV-F trimer confirm and extend our understanding of the F regulatory domains in MeV-F. Our list of hyperfusogenic F mutants is a valuable resource for future studies into MeV neuropathogenesis and the regulation of paramyxovirus F.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Neuronal death in pneumococcal meningitis is triggered by pneumolysin and RrgA interactions with β-actin.

Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein β-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal β-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of β-actin filaments, leading to more β-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of β-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against β-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae.

Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Niemann-Pick Type C1.

The lysosomal storage disorders Niemann-Pick disease Type C1 (NPC1) and Type C2 (NPC2) are rare diseases caused by mutations in the NPC1 or NPC2 gene. Both NPC1 and NPC2 are proteins responsible for the exit of cholesterol from late endosomes and lysosomes (LE/LY). Consequently, mutations in one of the two proteins lead to the accumulation of unesterified cholesterol and glycosphingolipids in LE/LY, displaying a disease hallmark. A total of 95% of cases are due to a deficiency of NPC1 and only 5% are caused by NPC2 deficiency. Clinical manifestations include neurological symptoms and systemic symptoms, such as hepatosplenomegaly and pulmonary manifestations, the latter being particularly pronounced in NPC2 patients. NPC1 and NPC2 are rare diseases with the described neurovisceral clinical picture, but studies with human primary patient-derived neurons and hepatocytes are hardly feasible. Obviously, induced pluripotent stem cells (iPSCs) and their derivatives are an excellent alternative for indispensable studies with these affected cell types to study the multisystemic disease NPC1. Here, we present a review focusing on studies that have used iPSCs for disease modeling and drug discovery in NPC1 and draw a comparison to commonly used NPC1 models.

Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons.

The research on human neural progenitor cells holds great potential for the understanding of the molecular programs that control differentiation of cells of glial and neuronal lineages, as well as pathogenetic mechanisms of neurological diseases. Stem cell technologies also provide opportunities for the pharmaceutical industry to develop new approaches for regenerative medicine. Here, we describe the protocol for the isolation and maintenance of neural progenitor cells and cortical neurons using human fetal brain tissue. This protocol can be successfully adapted for the preparation of rodent neural and oligodendrocyte progenitor cells. While several methods for isolating neural and oligodendrocyte progenitors from rodent brain tissue have been described, including techniques utilizing gene transfer and magnetic resonance beads, few methods are specifically focused on deriving human oligodendrocyte progenitor cells. Development of the human cultures provides the most physiologically relevant system for investigating mechanisms which regulate the function of oligodendrocytes, specifically of human origin.

Roflumilast, a cAMP-Specific Phosphodiesterase-4 Inhibitor, Reduces Oxidative Stress and Improves Synapse Functions in Human Cortical Neurons Exposed to the Excitotoxin Quinolinic Acid.

The overexpression of phosphodiesterase 4 (PDE4) enzymes is reported in several neurodegenerative diseases. PDE4 depletes cyclic 3'-5' adenosine monophosphate (cAMP) and, in turn, cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF), the key players in cognitive function. The present study was undertaken to investigate the mechanism behind the protective effects of roflumilast (ROF), a cAMP-specific PDE4 inhibitor, against quinolinic acid (QUIN)-induced neurotoxicity using human primary cortical neurons. Cytotoxicity was analyzed using an MTS assay. Reactive oxygen species (ROS) and mitochondrial membrane potential were measured by DCF-DA and JC-10 staining, respectively. Caspase 3/7 activity was measured using an ApoTox-Glo Triplex assay kit. cAMP was measured using an ELISA kit. The protein expression of CREB, BDNF, SAP-97, synaptophysin, synapsin-I, and PSD-95 was analyzed by the Western blotting technique. QUIN exposure down-regulated CREB, BDNF, and synaptic protein expression in neurons. Pretreatment with ROF increased the intracellular cAMP, mitochondrial membrane potential, and nicotinamide adenine dinucleotide (NAD+) content and decreased the ROS and caspase 3/7 levels in QUIN-exposed neurons. ROF up-regulated the expression of synapse proteins SAP-97, synaptophysin, synapsin-I, PSD-95, and CREB and BDNF, which indicates its potential role in memory. This study suggests for the first time that QUIN causes pre- and postsynaptic protein damage. We further demonstrate the restorative effects of ROF on the mitochondrial membrane potential and antiapoptotic properties in human neurons. These data encourage further investigations to reposition ROF in neurodegenerative diseases and their associated cognitive deficits.

Contemporary circulating enterovirus D68 strains have acquired the capacity for viral entry and replication in human neuronal cells

Background: Enterovirus D68 (EV-D68) is a member of the Picornaviridae family of single-stranded positive-sense RNA viruses. EV-D68 infection has historically been associated with respiratory illnesses in humans. However, in the summers of 2014, 2016, and 2018 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis (AFM) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. Methods and materials: To assess the neurotropic capacity of EV-D68, we explored the use of the neuroblastoma-derived neuronal cell line, SH-SY5Y, as a cell culture model to determine if differential infection is observed for different EV-D68 isolates. Viral replication and infectivity in SH-SY5Y was assessed using four different assays-infectious virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Results: In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to virus binding and entry were at least partly responsible for the differential infectivity phenotype, since transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains which could not directly infect SH-SY5Y. Conclusion: Taken together, these results suggest that EV-D68 has evolved to acquire novel neurotropic characteristics that may contribute to the development of neurological disease in humans. In addition to supporting virus replication and other functional studies, the cell culture model we’ve developed may help confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

Multiple Alu Exonization in 3'UTR of a Primate-Specific Isoform of CYP20A1 Creates a Potential miRNA Sponge.

Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA–miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3′UTR. CYP20A1_Alu-LT, confirmed by 3′RACE, is an outlier in length (9 kb 3′UTR) and widely expressed. Using publically available data sets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15,928 human cortical neurons. miRanda predicts ∼4,700 miRNA recognition elements (MREs) for ∼1,000 miRNAs, primarily originated within these 3′UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. In total, 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared with random sets of differentially expressed genes (P = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA-mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.

Human Immunodeficiency Virus Type 1 and Methamphetamine-Mediated Mitochondrial Damage and Neuronal Degeneration in Human Neurons.

Methamphetamine, a potent psychostimulant, is a highly addictive drug commonly used by persons living with HIV (PLWH), and its use can result in cognitive impairment and memory deficits long after its use is discontinued. Although the mechanism(s) involved with persistent neurological deficits is not fully known, mitochondrial dysfunction is a key component in methamphetamine neuropathology. Specific mitochondrial autophagy (mitophagy) and mitochondrial fusion and fission are protective quality control mechanisms that can be dysregulated in HIV infection, and the use of methamphetamine can further negatively affect these protective cellular mechanisms. Here, we observed that treatment of human primary neurons (HPNs) with methamphetamine and HIV gp120 and Tat increase dynamin-related protein 1 (DRP1)-dependent mitochondrial fragmentation and neuronal degeneration. Methamphetamine and HIV proteins increased microtubule-associated protein 1 light chain 3 beta-II (LC3B-II) lipidation and induced sequestosome 1 (SQSTM1, p62) translocation to damaged mitochondria. Additionally, the combination inhibited autophagic flux, increased reactive oxygen species (ROS) production and mitochondrial damage, and reduced microtubule-associated protein 2 (MAP2) dendrites in human neurons. N-Acetylcysteine (NAC), a strong antioxidant and ROS scavenger, abrogated DRP1-dependent mitochondrial fragmentation and neurite degeneration. Thus, we show that methamphetamine combined with HIV proteins inhibits mitophagy and induces neuronal damage, and NAC reverses these deleterious effects on mitochondrial function.IMPORTANCE Human and animal studies show that HIV infection, combined with the long-term use of psychostimulants, increases neuronal stress and the occurrence of HIV-associated neurocognitive disorders (HAND). On the cellular level, mitochondrial function is critical for neuronal health. In this study, we show that in human primary neurons, the combination of HIV proteins and methamphetamine increases oxidative stress, DRP1-mediated mitochondrial fragmentation, and neuronal injury manifested by a reduction in neuronal network and connectivity. The use of NAC, a potent antioxidant, reversed the neurotoxic effects of HIV and methamphetamine, suggesting a novel approach to ameliorate the effects of HIV- and methamphetamine-associated cognitive deficits.

Enhanced Chemotherapy for Glioblastoma Multiforme Mediated by Functionalized Graphene Quantum Dots

Glioblastoma is the most aggressive and lethal brain cancer. Current treatments involve surgical resection, radiotherapy and chemotherapy. However, the life expectancy of patients with this disease remains short and chemotherapy leads to severe adverse effects. Furthermore, the presence of the blood–brain barrier (BBB) makes it difficult for drugs to effectively reach the brain. A promising strategy lies in the use of graphene quantum dots (GQDs), which are light-responsive graphene nanoparticles that have shown the capability of crossing the BBB. Here we investigate the effect of GQDs on U87 human glioblastoma cells and primary cortical neurons. Non-functionalized GQDs (NF-GQDs) demonstrated high biocompatibility, while dimethylformamide-functionalized GQDs (DMF-GQDs) showed a toxic effect on both cell lines. The combination of GQDs and the chemotherapeutic agent doxorubicin (Dox) was tested. GQDs exerted a synergistic increase in the efficacy of chemotherapy treatment, specifically on U87 cells. The mechanism underlying this synergy was investigated, and it was found that GQDs can alter membrane permeability in a manner dependent on the surface chemistry, facilitating the uptake of Dox inside U87 cells, but not on cortical neurons. Therefore, experimental evidence indicates that GQDs could be used in a combined therapy against brain cancer, strongly increasing the efficacy of chemotherapy and, at the same time, reducing its dose requirement along with its side effects, thereby improving the life quality of patients.

Potential Mechanism of Cellular Uptake of the Excitotoxin Quinolinic Acid in Primary Human Neurons.

In Alzheimer's disease (AD), excessive amounts of quinolinic acid (QUIN) accumulate within the brain parenchyma and dystrophic neurons. QUIN also regulates glutamate uptake into neurons, which may be due to modulation of Na+-dependent excitatory amino acid transporters (EAATs). To determine the biological relationships between QUIN and glutamate dysfunction, we first quantified the functionality and kinetics of [3H]QUIN uptake in primary human neurons using liquid scintillation. We then measured changes in the protein expression of the glutamate transporter EAAT3 and EAAT1b in primary neurons treated with QUIN and the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (2,4-PDC) using western blotting and immunohistochemistry. Immunohistochemistry was further used to elucidate intracellular transport of exogenous QUIN and the lysosomal-associated membrane protein 2 (LAMP2). Structural insights into the binding between QUIN and EAAT3 were further investigated using molecular docking techniques. We report significant temperature-dependent high-affinity transport leading to neuronal uptake of [3H]QUIN with a Km of 42.2μM, and a Vmax of 9.492pmol/2min/mg protein, comparable with the uptake of glutamate. We also found that QUIN increases expression of the EAAT3 monomer while decreasing the functional trimer. QUIN uptake into primary neurons was shown to involve EAAT3 as uptake was significantly attenuated following EAAT inhibition. We also demonstrated that QUIN increases the expression of aberrant EAAT1b protein in neurons further implicating QUIN-induced glutamate dysfunction. Furthermore, we demonstrated that QUIN is metabolised exclusively in lysosomes. The involvement of EAAT3 as a modulator for QUIN uptake was further confirmed using molecular docking. This study is the first to characterise a mechanism for QUIN uptake into primary human neurons involving EAAT3, opening potential targets to attenuate QUIN-induced excitotoxicity in neuroinflammatory diseases.

Protection of MCC950 against Alzheimer's disease via inhibiting neuronal pyroptosis in SAMP8 mice.

Neuronal dysfunction and loss are thought to be one of the causes of cognitive impairment in Alzheimer's disease (AD), but the specific mechanism of neuronal loss in the pathogenesis of AD remains controversial. This study explored the role of NLRP3 inflammasome-induced neuronal pyroptosis in neuronal loss of AD, and pioneered the use of NLRP3 inhibitor MCC950 to intervene in the treatment of senescence-accelerated mouse prone 8 (SAMP8) mice. In vitro, human primary neurons (HPNs) pretreated with MCC950 were stimulated with amyloid-β1-42 (Aβ1-42), and it was found that MCC950 significantly reduced the neurotoxicity of Aβ1-42 by inhibiting neuronal pyroptosis. In vivo, SAMP8 mice were randomly divided into vehicle-treated group and MCC950-treated group, and it was found that MCC950 also played a positive role in treatment. The intervention of MCC950 improved the spatial memory ability and brain histological morphology of SAMP8 mice, and reduced the deposition of amyloid-β in the brain. Furthermore, MCC950 was found to inhibit the overexpressions of NLRP3, caspase-1, and GSDMD, which were the response factors of pyroptosis in SAMP8 mouse neurons, by immunofluorescence staining. In this study, we found that neuronal pyroptosis induced by the NLRP3/caspase-1/GSDMD axis was an important factor in neuronal loss of AD, and revealed that MCC950 might be a potential AD therapeutic agent.

HIV and opiates dysregulate K+- Cl− cotransporter 2 (KCC2) to cause GABAergic dysfunction in primary human neurons and Tat-transgenic mice

Approximately half of people infected with HIV (PWH) exhibit HIV-associated neuropathology (neuroHIV), even when receiving combined antiretroviral therapy. Opiate use is widespread in PWH and exacerbates neuroHIV. While neurons themselves are not infected, they incur sublethal damage and GABAergic disruption is selectively vulnerable to viral and inflammatory factors released by infected/affected glia. Here, we demonstrate diminished K+-Cl− cotransporter 2 (KCC2) levels in primary human neurons after exposure to HIV-1 or HIV-1 proteins ± morphine. Resulting disruption of GABAAR-mediated hyperpolarization/inhibition was shown using genetically-encoded voltage (Archon1) and calcium (GCaMP6f) indicators. The HIV proteins Tat (acting through NMDA receptors) and R5-gp120 (acting via CCR5) but not X4-tropic gp120 (acting via CXCR4), and morphine (acting through μ-opioid receptors) all induced KCC2 loss. We demonstrate that modifying KCC2 levels or function, or antagonizing NMDAR, CCR5 or MOR rescues KCC2 and GABAAR-mediated hyperpolarization/inhibition in HIV, Tat, or gp120 ± morphine-exposed neurons. Using an inducible, Tat-transgenic mouse neuroHIV model, we found that chronic exposure to Tat also reduces KCC2. Our results identify KCC2 as a novel therapeutic target for ameliorating the pathobiology of neuroHIV, including PWH exposed to opiates.

The application of in vitro-derived human neurons in neurodegenerative disease modeling.

The development of safe and effective treatments for age-associated neurodegenerative disorders is an on-going challenge faced by the scientific field. Key to the development of such therapies is the appropriate selection of modeling systems in which to investigate disease mechanisms and to test candidate interventions. There are unique challenges in the development of representative laboratory models of neurodegenerative diseases, including the complexity of the human brain, the cumulative and variable contributions of genetic and environmental factors over the course of a lifetime, inability to culture human primary neurons, and critical central nervous system differences between small animal models and humans. While traditional rodent models have advanced our understanding of neurodegenerative disease mechanisms, key divergences such as the species-specific genetic background can limit the application of animal models in many cases. Here we review in vitro human neuronal systems that employ stem cell and reprogramming technology and their application to a range of neurodegenerative diseases. Specifically, we compare human-induced pluripotent stem cell-derived neurons to directly converted, or transdifferentiated, induced neurons, as both model systems can take advantage of patient-derived human tissue to produce neurons in culture. We present recent technical developments using these two modeling systems, as well as current limitations to these systems, with the aim of advancing investigation of neuropathogenic mechanisms using these models.

N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells.

The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency=0.03282%) and Glu44Gln (allele frequency=0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.