Co-culture Of Neurons Research Articles

Excitatory to inhibitory synaptic ratios are unchanged at presymptomatic stages in multiple models of ALS.

Hyperexcitability of motor neurons and spinal cord motor circuitry has been widely reported in the early stages of Amyotrophic Lateral Sclerosis (ALS). Changes in the relative amount of excitatory to inhibitory inputs onto a neuron (E:I synaptic ratio), possibly through a developmental shift in synapse formation in favour of excitatory transmission, could underlie pathological hyperexcitability. Given that astrocytes play a major role in early synaptogenesis and are implicated in ALS pathogenesis, their potential contribution to disease mechanisms involving synaptic imbalances and subsequent hyperexcitability is also of great interest. In order to assess E:I ratios in ALS, we utilised a novel primary spinal neuron / astrocyte co-culture system, derived from neonatal mice, in which synapses are formed in vitro. Using multiple ALS mouse models we found that no combination of astrocyte or neuron genotype produced alterations in E:I synaptic ratios assessed using pre- and post-synaptic anatomical markers. Similarly, we observed that ephrin-B1, a major contact-dependent astrocytic synaptogenic protein, was not differentially expressed by ALS primary astrocytes. Further to this, analysis of E:I ratios across the entire grey matter of the lumbar spinal cord in young (post-natal day 16-19) ALS mice revealed no differences versus controls. Finally, analysis in co-cultures of human iPSC-derived motor neurons and astrocytes harbouring the pathogenic C9orf72 hexanucleotide repeat expansion showed no evidence of a bias toward excitatory versus inhibitory synapse formation. We therefore conclude, utilising multiple ALS models, that we do not observe significant changes in the relative abundance of excitatory versus inhibitory synapses as would be expected if imbalances in synaptic inputs contribute to early hyperexcitability.

High levels of FAD autofluorescence indicate pathology preceding cell death

Flavin adenine dinucleotide (FAD) autofluorescence from cells reports on the enzymatic activity which involves FAD as a cofactor. Most of the cellular FAD fluorescence comes from complex II of the electron transport chain in mitochondria and can be assessed with inhibitor analysis. The intensity of FAD autofluorescence is not homogeneous and vary between cells in tissue and in cell culture types. Using primary co-culture of neurons and astrocytes, and human skin fibroblasts we have found that very high FAD autofluorescence is a result of an overactivation of the mitochondrial complex II from ETC and from the activity of monoamine oxidases. Cells with high FAD autofluorescence were mostly intact and were not co-labelled with indicators for necrosis or apoptosis. However, cells with high FAD fluorescence showed activation of apoptosis and necrosis within 24 h after initial measurements. Thus, high level of FAD autofluorescence is an indicator of cell pathology and reveals an upcoming apoptosis and necrosis.

CNSC-29. NKCC1 SIGNALING IN GLIOBLASTOMA REGULATES NEURONAL HYPEREXCITABILITY THROUGH GABAERGIC TONE

Abstract Over 90% of patients with glioma experience tumor-associated epilepsy, however first-line treatment fails to control seizures in most patients. Thus, there is a critical need to identify novel drivers of glioblastoma-induced neuronal hyperexcitability. We therefore used high-density electrode arrays to record local field potentials in vivo from gliomas. We then developed cerebral organoid and mouse models using primary glioblastoma cells from patients and neurons derived from induced pluripotent stem cells. Single-cell RNA sequencing (13,730 cells analyzed) in this system identified genes elevated in glioma-intrinsic neurons, including the sodium-potassium-chloride co-transporter 1 (NKCC1), which mediates chloride (Cl-) influx into cells. This elevated NKCC1 expression and subsequent increase of intracellular Cl- sets the reversal potential of GABAAR-mediated currents promoting depolarizing excitatory GABAergic responses. Electrophysiological analysis of glioma-neuron co-cultures using multi-electrode array and live-cell calcium imaging demonstrated increased network burst synchrony in the presence of NKCC1-high expressing tumor cells. Single-nucleus sequencing (54,000 cells analyzed) of embryonic neurons in co-culture with GBM cells further confirmed the elevated expression of circuit assembly genes including neuronal NKCC1. Live-cell imaging of iPSC-induced neuron organoids co-cultured with NKCC1-high-and low-GBM cells revealed increased migration and integration of NKCC1-elevated tumor cells. Mechanistic and functional studies validating therapeutic vulnerabilities to NKCC1 silencing by shRNA knockdown and by the FDA-approved diuretic drug, bumetanide was performed in vitro. The reduction in neuronal synchrony induced by NKCC1 downregulation correlated with a decrease in extracellular GABA and glutamate, suggesting a strong association between excitatory GABAergic and glutamate signaling with neuronal hypersynchrony in glioblastoma. Patient-derived xenograft in vivo experiments evaluated the effect of NKCC1 on tumor growth and progression and NKCC1-high GBM cells exhibited greater tumor burden compared to control mice. Collectively, these findings reveal a novel mechanism driving neuronal hyperactivity in glioblastoma and identifies potential therapeutic vulnerabilities for the treatment of glioma-associated epilepsy.

Investigating epithelial-neuronal signaling contribution in visceral pain through colon organoid-dorsal root ganglion neuron co-cultures.

Investigating epithelial-neuronal signaling contribution in visceral pain through colon organoid-dorsal root ganglion neuron co-cultures.

A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.

Communication and contact between neurons and astrocytes is important for proper brain physiology. How neuron/astrocyte crosstalk is affected by intraneuronal tau aggregation in neurodegenerative tauopathies is largely elusive. Human induced pluripotent stem cell (iPSC)-derived neurons provide the opportunity to model tau pathology in a translationally relevant in vitro context. However, current iPSC models inefficiently develop tau aggregates, and co-culture models of tau pathology have thus far utilized rodent astrocytes. In this article, we describe the co-culture of human iPSC-derived neurons with primary human astrocytes in a 96-well format compatible with high-content microscopy. By lentiviral overexpression of different mutated tau variants, this protocol can be flexibly adapted for the efficient induction of seeded or spontaneous tau aggregation. We used this novel co-culture model to identify cell type-specific disease mechanisms and to provide proof of concept for intervention by antisense therapy. These results show that this human co-culture model provides a highly translational tool for target discovery and drug development for human tauopathies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Human neuron/astrocyte co-culture for seeded and spontaneous intraneuronal tau aggregation Support Protocol 1: Human induced pluripotent stem cell culture Support Protocol 2: Human primary astrocyte culture.

Rasd1 is involved in white matter injury through neuron-oligodendrocyte communication after subarachnoid hemorrhage.

Rasd1 has been reported to be correlated with neurotoxicity, metabolism, and rhythm, but its effect in case of subarachnoid hemorrhage (SAH) remained unclear. White matter injury (WMI) and ferroptosis participate in the early brain injury (EBI) after SAH. In this work, we have investigated whether Rasd1 can cause ferroptosis and contribute to SAH-induced WMI. Lentivirus for Rasd1 knockdown/overexpression was administrated by intracerebroventricular (i.c.v) injection at 7 days before SAH induction. SAH grade, brain water content, short- and long-term neurobehavior, Western blot, real-time PCR, ELISA, biochemical estimation, immunofluorescence, diffusion tensor imaging (DTI), and transmission electron microscopy (TEM) were systematically performed. Additionally, genipin, a selective uncoupling protein 2(UCP2) inhibitor, was used in primary neuron and oligodendrocyte co-cultures for further in vitro mechanistic studies. Rasd1 knockdown has improved the neurobehavior, glia polarization, oxidative stress, neuroinflammation, ferroptosis, and demyelination. Conversely, Rasd1 overexpression aggravated these changes by elevating the levels of reactive oxygen species (ROS), inflammatory cytokines, MDA, free iron, and NCOA4, as well as contributing to the decrease of the levels of UCP2, GPX4, ferritin, and GSH mechanistically. According to the in vitro study, Rasd1 can induce oligodendrocyte ferroptosis through inhibiting UCP2, increasing reactive oxygen species (ROS), and activating NCOA4-mediated ferritinophagy. It can be concluded that Rasd1 exerts a modulated role in oligodendrocytes ferroptosis in WMI following SAH.

SRRM4-mediated REST to REST4 dysregulation promotes tumor growth and neural adaptation in breast cancer leading to brain metastasis.

Effective control of brain metastasis remains an urgent clinical need due a limited understanding of the mechanisms driving it. Although the gain of neuro-adaptive attributes in breast-to-brain metastases (BBMs) has been described, the mechanisms that govern this neural acclimation and the resulting brain metastasis competency are poorly understood. Herein, we define the role of neural-specific splicing factor Serine/Arginine Repetitive Matrix Protein 4 (SRRM4) in regulating microenvironmental adaptation and brain metastasis colonization in breast cancer cells. Utilizing pure neuronal cultures and brain-naive and patient-derived BM tumor cells, along with in vivo tumor modeling, we surveyed the early induction of mediators of neural acclimation in tumor cells. When SRRM4 is overexpressed in systemic breast cancer cells, there is enhanced BBM leading to poorer overall survival in vivo. Concomitantly, SRRM4 knockdown expression does not provide any advantage in central nervous system metastasis. In addition, reducing SRRM4 expression in breast cancer cells slows down proliferation and increases resistance to chemotherapy. Conversely, when SRRM4/REST4 levels are elevated, tumor cell growth is maintained even in nutrient-deprived conditions. In neuronal coculture, decreasing SRRM4 expression in breast cancer cells impairs their ability to adapt to the brain microenvironment, while increasing SRRM4/RE-1 Silencing Transcription Factor (REST4) levels leads to greater expression of neurotransmitter and synaptic signaling mediators and a significant colonization advantage. Collectively, our findings identify SRRM4 as a regulator of brain metastasis colonization, and a potential therapeutic target in breast cancer.

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell-neuron cocultures.

Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris.

First person – Clara Mutschler

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Clara Mutschler is first author on ‘Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell–neuron cocultures’, published in JCS. Clara is a PhD student in the lab of Peter Arthur-Farraj at the John Van Geest Centre for Brain Repair, University of Cambridge, UK, investigating axon–Schwann cell interactions after peripheral nervous system injury.

P16-52: Modulation of GABA activity: Investigations in hiPSC-derived neuronal co-cultures and human ion channel assays

P16-52: Modulation of GABA activity: Investigations in hiPSC-derived neuronal co-cultures and human ion channel assays

Cell type–specific labeling of newly synthesized proteins by puromycin inactivation

Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here we show how a widely used mammalian selection marker, puromycin N-acetyltransferase (PAT), can be re-purposed for cell-specific metabolic labelling. This approach, which we named Puromycin Inactivation for Cell-Selective proteome Labelling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing PAT and detection of translation in other cell types. Using co-cultures of neurons and glial cells from the rat brain cortex, we show the application of PICSL for puromycin immunostaining, western blot and mass-spectrometric identification of nascent proteins. By combining PICSL and OPP-ID proteomics, cell-type enriched proteins can be identified based on reduced OPP labelling in the cell type of interest.

New human in vitro co-culture model of keratinocytes and sensory neurons like cells releasing substance P with an evaluation of the expression of ZIKV entry receptors: A potent opportunity to test Zika virus entry and to study Zika virus' infection in neurons?

During the course of acute ZIKV infection, pruritus is a cardinal symptom widely documented in the literature. Its frequent association with dysesthesia and several dysautonomic manifestations, suggests a pathophysiological mechanism involving the peripheral nervous system. The aim of this study was to develop a functional human model to potentially able to be infected by ZIKV: by demonstrating the functionality on a new human model of co-culture of keratinocyte and sensory neuron derived from induced pluripotent stem cells using a classical method of capsaicin induction and SP release, and verify the presence of ZIKV entry receptor in these cells. Depending of cellular type, receptors of the TAMs family, TIMs (TIM1, TIM3 and TIM4) and DC-SIGN and RIG1 were present/detected. The cells incubations with capsaicin resulted in an increase of the substance P. Hence, this study demonstrated the possibility to obtain co-cultures of human keratinocytes and human sensory neurons that release substance P in the same way than previously published in animal models which can be used as a model of neurogenic skin inflammation. The demonstration of the expression of ZIKV entry receptors in these cells allows to considerate the potent possibility that ZIKV is able to infect cells.

Human Induced Pluripotent Stem Cell-Derived Pericytes as Scalable and Editable Source to Study Direct Lineage Reprogramming Into Induced Neurons.

Studying human somatic cell-to-neuron conversion using primary brain-derived cells as starting cell source is hampered by limitations and variations in human biopsy material. Thus, delineating the molecular variables that allow changing the identity of somatic cells, permit adoption of neuronal phenotypes, and foster maturation of induced neurons (iNs) is challenging. Based on our previous results that pericytes derived from the adult human cerebral cortex can be directly converted into iNs (Karow et al., 2018; Karow et al., 2012), we here introduce human induced pluripotent stem cell (hiPSC)-derived pericytes (hiPSC-pericytes) as a versatile and more uniform tool to study the pericyte-to-neuron conversion process. This strategy enables us to derive scalable cell numbers and allows for engineering of the starting cell population such as introducing reporter tools before differentiation into hiPSC-pericytes and subsequent iN conversion. Harvesting the potential of this approach, we established hiPSC-derived human-human neuronal cocultures that not only allow for independent manipulation of each coculture partner but also resulted in morphologically more mature iNs. In summary, we exploit hiPSC-based methods to facilitate the analysis of human somatic cell-to-neuron conversion.

Functional attachment of primary neurons and glia on radiopaque implantable biomaterials for nerve repair

Repairing peripheral nerve injuries remains a challenge, even with use of auxiliary implantable biomaterial conduits. After implantation the location or function of polymeric devices cannot be assessed via clinical imaging modalities. Adding nanoparticle contrast agents into polymers can introduce radiopacity enabling imaging using computed tomography. Radiopacity must be balanced with changes in material properties impacting device function. In this study radiopaque composites were made from polycaprolactone and poly(lactide-co-glycolide) 50:50 and 85:15 with 0–40 wt% tantalum oxide (TaOx) nanoparticles. To achieve radiopacity, ≥5 wt% TaOx was required, with ≥20 wt% TaOx reducing mechanical properties and causing nanoscale surface roughness. Composite films facilitated nerve regeneration in an in vitro co-culture of adult glia and neurons, measured by markers for myelination. The ability of radiopaque films to support regeneration was driven by the properties of the polymer, with 5–20 wt% TaOx balancing imaging functionality with biological response and proving that in situ monitoring is feasible.

Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

Studying NF-κB signaling during neuroinflammation in Alzheimer’s Diseases in vitrousing hiPSC-derived brain models

Abstract Neuroinflammation is a key pathological driver of various neurological diseases, including Alzheimer’s disease (AD). NF-κB signaling pathway is widely recognized as a hallmark of inflammation and cellular senescence in which aged microglia may undergo cellular senescence partly influenced by NF-κB. Our previous data indicated that ex vivo microglia comprised of two distinct subpopulations distinguished by morphology and motility. Most microglia cells formed a tight cell cluster, termed “clustered microglia”. The rest was a subpopulation of microglia with smaller cell size and high motility, termed “free-roaming microglia”. In microglia from aged animals, the composition of clustered versus free-roaming subsets was shifted toward a higher prevalence of free-roaming microglia where c-Rel is expressed and the canonical NF-κB signaling is more sustained. To study NF-κB signaling in the context of neuroinflammation, we are developing 3D models, using hiPSC neurons derived from healthy donors of various age groups and co-culturing them with primary microglia from knock-in mice in which their endogenous c-Rel was labeled with a fluorescent protein. Moreover, individuals with APOE-e4 genotype have an increased risk of developing AD. To develop 3D brain models for AD, we will use co-culture of microglia and neurons derived from hiPSCs of APOE-e4 carriers. Live cell imaging and biomarker assessment of microglia and neurons activated by Amyloid β (1–42) will uncover important molecular mechanisms associated with age-dependent interactions between neurons from healthy versus AD-risk individuals, and resident macrophages of the young and aged brains.

339 Microfluidic device supporting the coculture of iPSC-derived sensitive neurons and epidermis

339 Microfluidic device supporting the coculture of iPSC-derived sensitive neurons and epidermis

302 Super Mitochondria, the Next Super Therapy to Reverse Ischemic Damage in Stroke?

INTRODUCTION: Stroke represents a massive healthcare emergency, ranking in the top 5 causes of death and disability and impacting nearly 800,000 Americans annually. Current therapies are limited. A deeper analysis of the mechanisms of cell death after ischemic brain insult may usher novel safe and effective treatments. Mitochondrial damage serves as a key modulator of ischemic cell death, which may be reversible with transfer of healthy mitochondria from mesenchymal stem cells (MSCs) to ischemic neurons. METHODS: MSCs were grown under ambient condition (normal MSCs) (nMSCs) or metabolic switching paradigm involving exchanging galactose for glucose in the cell culture medium (switched MSCs) (sMSCs) then assayed for oxygen consumption rates using the Seahorse technology. Subsequently, primary neurons were subjected to OGD then co-cultured with either nMSCs or sMSCs, and assessed for cell viability, cell metabolism, mitochondrial reactive oxidative species mRNA, and mitochondrial ATP mRNA. RESULTS: Compared to nMSCs, sMSCs displayed robust mitochondrial respiration as evidenced by higher basal energy production, larger spare respiratory capacity, greater ATP production, and decreased proton (H+) leak. Co-culture of OGD-exposed neurons with sMSCs conferred increased cell viability and enhanced cell metabolism, coupled with reduced mitochondrial reactive oxidative species mRNA and elevated mitochondrial ATP mRNA, which were significantly better than those afforded by nMSCs. CONCLUSIONS: These findings suggest sMSCs generated “super” mitochondria. Metabolic switching produces healthy mitochondria that may enhance the mitochondrial transfer between MSCs and ischemic cells warranting consideration as a stroke therapeutic.

Sodium houttuyfonate exerts its neuroprotection effect by inhibiting the M1 microglia polarization in a TLR4/NF-κB signal pathway

Neuroinflammation plays an important role in secondary injury after spinal cord injury (SCI) and may aggravate neurological dysfunction. Several studies have indicated that sodium houttuyfonate (SH) can significantly inhibit macrophage- mediated inflammation; however, its effects on SCI still needs to be elucidated. We found that SH improved Basso, Beattie, and Bresnahan scores and performance in the inclined plane test of SCI model rats. The injured spinal cord exhibited less neuronal loss, cell apoptosis, and M1 microglial polarization after SH treatment. In vitro, SH reduced TLR4/NF-κB expression in cultured primary microglia and decreased M1 microglial polarization and cell apoptosis in a lipopolysaccharide (LPS)-pretreated microglia and neuron coculture system. These results indicated that SH may exert a neuroprotective effect by inhibiting M1 microglial polarization after SCI via the TLR4/NF-κB signalling pathway.

Phosphodiesterase (PDE) 4 inhibition boosts Schwann cell myelination in a 3D regeneration model

Phosphodiesterase 4 (PDE4) inhibitors have been extensively researched for their anti-inflammatory and neuroregenerative properties. Despite the known neuroplastic and myelin regenerative properties of nonselective PDE4 inhibitors on the central nervous system, the direct impact on peripheral remyelination and subsequent neuroregeneration has not yet been investigated. Therefore, to examine the possible therapeutic effect of PDE4 inhibition on peripheral glia, we assessed the differentiation of primary rat Schwann cells exposed in vitro to the PDE4 inhibitor roflumilast. To further investigate the differentiation promoting effects of roflumilast, we developed a 3D model of rat Schwann cell myelination that closely resembles the in vivo situation. Using these in vitro models, we demonstrated that pan-PDE4 inhibition using roflumilast significantly promoted differentiation of Schwann cells towards a myelinating phenotype, as indicated by the upregulation of myelin proteins, including MBP and MAG. Additionally, we created a unique regenerative model comprised of a 3D co-culture of rat Schwann cells and human iPSC-derived neurons. Schwann cells treated with roflumilast enhanced axonal outgrowth of iPSC-derived nociceptive neurons, which was accompanied by an accelerated myelination speed, thereby showing not only phenotypic but also functional changes of roflumilast-treated Schwann cells. Taken together, the PDE4 inhibitor roflumilast possesses a therapeutic benefit to stimulate Schwann cell differentiation and, subsequently myelination, as demonstrated in the biologically relevant in vitro platform used in this study. These results can aid in the development of novel PDE4 inhibition-based therapies in the advancement of peripheral regenerative medicine.