Pure Neuronal Cultures Research Articles

Alzheimer’s disease risk gene BIN1 modulates neural network activity through the regulation of L‐type calcium channel expression in human induced neurons

AbstractBackgroundBridging Integrator 1 (BIN1) is the second most important Alzheimer’s disease (AD) risk gene after APOE, but its physiological roles and contribution to brain pathology are largely elusive. In this work, we tackled the short‐ and long‐term effects of BIN1 deletion in human induced neurons (hiNs).MethodWe generated isogenic BIN1 WT, KO and HET human induced pluripotent stem cells (hiPSCs), which were differentiated into hiNs grown in 2D and 3D (cerebral organoids) cultures. We also developed a novel protocol for the generation of highly pure glutamatergic neuron cultures, allowing the study of cell‐autonomous roles of BIN1. Using these three different systems, we probed neuronal electrical activity using MEA electrophysiology and gene expression alterations in individual cell types using snRNA‐seq.ResultWe show that BIN1 depletion leads to specific transcriptional alterations in glutamatergic neurons involving mainly genes associated with calcium homeostasis, ion transport and synapse function. We also show that BIN1 regulates calcium transients and neuronal electrical activity through interaction with the L‐type voltage‐gated calcium channel Cav1.2 and regulation of activity‐dependent internalization of this channel. Treatment with the Cav1.2 antagonist nifedipine partly rescues neuronal electrical and gene expression alterations in BIN1 knockout hiNs.ConclusionOur results indicate that BIN1 loss‐of‐function impairs calcium homeostasis at least in glutamatergic neurons, potentially contributing to the transcriptional changes and neural network dysfunctions observed in AD.

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.

A functional network of highly pure enteric neurons in a dish.

The enteric nervous system (ENS) is the intrinsic nervous system that innervates the entire digestive tract and regulates major digestive functions. Recent evidence has shown that functions of the ENS critically rely on enteric neuronal connectivity; however, experimental models to decipher the underlying mechanisms are limited. Compared to the central nervous system, for which pure neuronal cultures have been developed for decades and are recognized as a reference in the field of neuroscience, an equivalent model for enteric neurons is lacking. In this study, we developed a novel model of highly pure rat embryonic enteric neurons with dense and functional synaptic networks. The methodology is simple and relatively fast. We characterized enteric neurons using immunohistochemical, morphological, and electrophysiological approaches. In particular, we demonstrated the applicability of this culture model to multi-electrode array technology as a new approach for monitoring enteric neuronal network activity. This in vitro model of highly pure enteric neurons represents a valuable new tool for better understanding the mechanisms involved in the establishment and maintenance of enteric neuron synaptic connectivity and functional networks.

Co-culturing Immune Cells and Mouse-Derived Mixed Cortical Cultures with Oxygen-Glucose Deprivation to In Vitro Simulate Neuroinflammatory Interactions After Stroke.

Studying interactions between neural cells and glial cells in vitro remains an essential tool for scientists worldwide, and with the addition of oxygen-glucose deprivation (OGD) can be particularly useful for identifying mechanisms related to ischemic stroke-related injury and repair. In developing these protocols in the lab, however, we discovered the limitation of co-culturing immune cells with pure neuronal cultures as the standard media for immune cells impair neuronal growth and vice versa. Thus, we optimized a mixed cortical cell culture system that does not require the use of glial-conditioned media to support the viability and growth of neurons but can nonetheless be used to quantify neuronal survival and dendritic arborization. The following methods provide a guide as to how to culture mixed cortical cells from mouse pups (postnatal day 0-2). Additionally, we demonstrate how to co-culture mixed cortical cells with immune cells (e.g., B cells) to study neuro-immune interactions in vitro.

MAPT genotype-dependent mitochondrial aberration and ROS production trigger dysfunction and death in cortical neurons of patients with hereditary FTLD

Tauopathies are a major type of proteinopathies underlying neurodegenerative diseases. Mutations in the tau-encoding MAPT-gene lead to hereditary cases of frontotemporal lobar degeneration (FTLD)-tau, which span a wide phenotypic and pathological spectrum. Some of these mutations, such as the N279K mutation, result in a shift of the physiological 3R/4R ratio towards the more aggregation prone 4R isoform. Other mutations such as V337M cause a decrease in the in vitro affinity of tau to microtubules and a reduced ability to promote microtubule assembly. Whether both mutations address similar downstream signalling cascades remains unclear but is important for potential rescue strategies. Here, we developed a novel and optimised forward programming protocol for the rapid and highly efficient production of pure cultures of glutamatergic cortical neurons from hiPSCs. We apply this protocol to delineate mechanisms of neurodegeneration in an FTLD-tau hiPSC-model consisting of MAPTN279K- or MAPTV337M-mutants and wild-type or isogenic controls. The resulting cortical neurons express MAPT-genotype-dependent dominant proteome clusters regulating apoptosis, ROS homeostasis and mitochondrial function. Related pathways are significantly upregulated in MAPTN279K neurons but not in MAPTV337M neurons or controls. Live cell imaging demonstrates that both MAPT mutations affect excitability of membranes as reflected in spontaneous and stimulus evoked calcium signals when compared to controls, albeit more pronounced in MAPTN279K neurons. These spontaneous calcium oscillations in MAPTN279K neurons triggered mitochondrial hyperpolarisation and fission leading to mitochondrial ROS production, but also ROS production through NOX2 acting together to induce cell death. Importantly, we found that these mechanisms are MAPT mutation-specific and were observed in MAPTN279K neurons, but not in MAPTV337M neurons, supporting a pathological role of the 4R tau isoform in redox disbalance and highlighting MAPT-mutation specific clinicopathological-genetic correlations, which may inform rescue strategies in different MAPT mutations.

Validation of Mitochondria‐Targeted AD Therapeutics in Human Neuronal Cells

AbstractBackgroundNeurons and astrocytes were differentiated separately from human induced pluripotent stem cells (HiPSC) derived from Late‐Onset Alzheimer Disease (LOAD) female patients carrying Apolipoprotein E4 (ApoE4) allele. Cells from age and sex matched healthy subject carrying Apolipoprotein E3 allele were used as control. These neuronal, astrocytic, and their co‐cultures were assessed to investigate cell‐specific AD phenotypes (Figure 1). We recently demonstrated that application of a small molecule partial mitochondrial complex I inhibitor CP2 blocked neurodegeneration and cognitive decline in multiple mouse models of AD. However, the demonstration that this novel therapeutic approach is efficacious in human brain cells has not been done. Here, we aimed to validate neuroprotective mechanisms induced by CP2 using patient‐derived neuronal cells form LOAD patient. Outcomes will further support the development of these compounds for clinical application.MethodAxonal trafficking of MitoTracker Red stained mitochondria in neuron‐astrocyte co‐cultures was captured using live confocal imaging. The Seahorse XFe96 flux analyzer was used to evaluate mitochondrial bioenergetics in co‐cultures and pure neuronal or astrocytic cultures. Cell Viability Assays were used to perform H2O2 challenge. Quantitative polymerase chain reaction (qPCR), immunohistochemistry, and western blot were carried out to assess alterations in the expression of relevant genes and protein products.ResultWe have found that LOAD cells have a significant reduction in the percentage of motile mitochondria in axons, increased accumulation of neuronal phosphorylated Tau, and altered bioenergetics in neurons and astrocytes compared to the control cells. qPCR revealed elevated expression of inflammation related genes in LOAD co‐cultures. CP2 treatment at concentrations found therapeutically relevant in vivo in APP/PS1 and 3xTgAD mice improved cellular bioenergetics, rescued mitochondrial motility, and provided neuroprotection against H2O2 induced oxidative stress.ConclusionLOAD HiPSC‐derived neuronal co‐cultures and individual brain cells recapitulate mechanisms associated with LOAD allowing validation of a novel mitochondria‐targeted therapeutics. Our results support previous observation in AD mice and the hypothesis that partial inhibition of mitochondrial complex I activates neuroprotective compensatory mechanisms improving cellular energetics in LOAD human neurons even in the presence of mitochondria with abnormal dynamics and energetics. Further efforts are focused on the determination of a cell‐specific mechanisms of neuroprotection.

Neuronal exposure induces neurotransmitter signaling and synaptic mediators in tumors early in brain metastasis.

Brain metastases (BM) are responsible for neurological decline and poor overall survival. Although the pro-metastatic roles of glial cells, and the acquisition of neuronal attributes in established BM tumors have been described, there are no studies that investigate the initial interplay between neurons and brain-seeking tumor cells. The aim of this study was to characterize early tumor-neuron interactions and the induced CNS-adaptive changes in tumor cells prior to macro-colonization. Utilizing pure neuronal cultures and brain-naïve and patient-derived BM tumor cells, we surveyed the early induction of mediators of neurotransmitter (NT) and synaptic signaling in breast and lung tumor cells. Reliance on microenvironmental GABA in breast-to-brain metastatic cells (BBMs) was assessed in vitro and in vivo. Coculture with neurons induces early expression of classical NT receptor genes (HTR4, GRIA2, GRIN2B, GRM4, GRM8, DRD1) and neuronal synaptic mediators (CNR1, EGR2, ARC, NGFR, NRXN1) in breast and lung cancer cells. NT-dependent classification of tumor cells within the neuronal niche shows breast cancer cells become GABAergic responsive brain metastases (GRBMs) and transition from relying on autocrine GABA, to paracrine GABA from adjacent neurons; while autocrine Dopaminergic breast and lung tumor cells persist. In vivo studies confirm reliance on paracrine GABA is an early CNS-acclimation strategy in breast cancer. Moreover, neuronal contact induces early resurgence in Reelin expression in tumor cells through epigenetic activation, facilitating CNS adaptation. Tumor-neuron interactions allow for CNS adaptation early in the course of brain metastasis.

The cAMP Inducers Modify N-Acetylaspartate Metabolism in Wistar Rat Brain.

Neuronal N-acetylaspartate production appears in the presence of aspartate N-acetyltransferase (NAT8L) and binds acetyl groups from acetyl-CoA with aspartic acid. Further N-acetylaspartate pathways are still being elucidated, although they seem to involve neuron-glia crosstalk. Together with N-acetylaspartate, NAT8L takes part in oligoglia and astroglia cell maturation, myelin production, and dopamine-dependent brain signaling. Therefore, understanding N-acetylaspartate metabolism is an emergent task in neurobiology. This project used in in vitro and in vivo approaches in order to establish the impact of maturation factors and glial cells on N-acetylaspartate metabolism. Embryonic rat neural stem cells and primary neurons were maturated with either nerve growth factor, trans-retinoic acid or activators of cAMP-dependent protein kinase A (dibutyryl-cAMP, forskolin, theophylline). For in vivo, adult male Wistar rats were injected with theophylline (20 mg/kg b.w.) daily for two or eight weeks. Our studies showed that the N-acetylaspartate metabolism differs between primary neurons and neural stem cell cultures. The presence of glia cells protected N-acetylaspartate metabolism from dramatic changes within the maturation processes, which was impossible in the case of pure primary neuron cultures. In the case of differentiation processes, our data points to dibutyryl-cAMP as the most prominent regulator of N-acetylaspartate metabolism.

Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica.

Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3–5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16–18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.

Primary neuron and astrocyte cultures from postnatal Callithrix jacchus: a non-human primate in vitro model for research in neuroscience, nervous system aging, and neurological diseases of aging.

The ability to generate in vitro cultures of neuronal cells has been instrumental in advancing our understanding of the nervous system. Rodent models have been the principal source of brain cells used in primary cultures for over a century, providing insights that are widely applicable to human diseases. However, therapeutic agents that showed benefit in rodent models, particularly those pertaining to aging and age-associated dementias, have frequently failed in clinical trials. This discrepancy established a potential "translational gap" between human and rodent studies that may at least partially be explained by the phylogenetic distance between rodent and primate species. Several non-human primate (NHP) species, including the common marmoset (Callithrix jacchus), have been used extensively in neuroscience research, but in contrast to rodent models, practical approaches to the generation of primary cell culture systems amenable to molecular studies that can inform in vivo studies are lacking. Marmosets are a powerful model in biomedical research and particularly in studies of aging and age-associated diseases because they exhibit an aging phenotype similar to humans. Here, we report a practical method to culture primary marmoset neurons and astrocytes from brains of medically euthanized postnatal day 0 (P0) marmoset newborns that yield highly pure primary neuron and astrocyte cultures. Primary marmoset neuron and astrocyte cultures can be generated reliably to provide a powerful NHP in vitro model in neuroscience research that may enable mechanistic studies of nervous system aging and of age-related neurodegenerative disorders. Because neuron and astrocyte cultures can be used in combination with in vivo approaches in marmosets, primary marmoset neuron and astrocyte cultures may help bridge the current translational gap between basic and clinical studies in nervous system aging and age-associated neurological diseases.

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis.

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine β-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

Isolation and Culture of Chick Ciliary Ganglion Neurons.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Pathological pain processing in mouse models of multiple sclerosis and spinal cord injury: contribution of plasma membrane calcium ATPase 2 (PMCA2)

BackgroundNeuropathic pain is often observed in individuals with multiple sclerosis (MS) and spinal cord injury (SCI) and is not adequately alleviated by current pharmacotherapies. A better understanding of underlying mechanisms could facilitate the discovery of novel targets for therapeutic interventions. We previously reported that decreased plasma membrane calcium ATPase 2 (PMCA2) expression in the dorsal horn (DH) of healthy PMCA2+/− mice is paralleled by increased sensitivity to evoked nociceptive pain. These studies suggested that PMCA2, a calcium extrusion pump expressed in spinal cord neurons, plays a role in pain mechanisms. However, the contribution of PMCA2 to neuropathic pain processing remains undefined. The present studies investigated the role of PMCA2 in neuropathic pain processing in the DH of wild-type mice affected by experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and following SCI.MethodsEAE was induced in female and male C57Bl/6N mice via inoculation with myelin oligodendrocyte glycoprotein fragment 35–55 (MOG35–55) emulsified in Complete Freund’s Adjuvant (CFA). CFA-inoculated mice were used as controls. A severe SC contusion injury was induced at thoracic (T8) level in female C57Bl/6N mice. Pain was evaluated by the Hargreaves and von Frey filament tests. PMCA2 levels in the lumbar DH were analyzed by Western blotting. The effectors that decrease PMCA2 expression were identified in SC neuronal cultures.ResultsIncreased pain in EAE and SCI was paralleled by a significant decrease in PMCA2 levels in the DH. In contrast, PMCA2 levels remained unaltered in the DH of mice with EAE that manifested motor deficits but not increased pain. Interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and IL-6 expression were robustly increased in the DH of mice with EAE manifesting pain, whereas these cytokines showed a modest increase or no change in mice with EAE in the absence of pain. Only IL-1β decreased PMCA2 levels in pure SC neuronal cultures through direct actions.ConclusionsPMCA2 is a contributor to neuropathic pain mechanisms in the DH. A decrease in PMCA2 in DH neurons is paralleled by increased pain sensitivity, most likely through perturbations in calcium signaling. Interleukin-1β is one of the effectors that downregulates PMCA2 by acting directly on neurons.

Neuronal apolipoprotein E4 increases cell death and phosphorylated tau release in alzheimer disease.

The apolipoprotein E (APOE) E4 isoform is the strongest genetic risk factor for sporadic Alzheimer disease (AD). Although APOE is predominantly expressed by astrocytes in the central nervous system, neuronal expression of APOE is of increasing interest in age-related cognitive impairment, neurological injury, and neurodegeneration. Here, we show that endogenous expression of E4 in stem-cell-derived neurons predisposes them to injury and promotes the release of phosphorylated tau. Induced pluripotent stem cells from 2 unrelated AD patients carrying the E4 allele were corrected to the E3/E3 genotype with the CRISPR/Cas9 system and differentiated into pure cultures of forebrain excitatory neurons without contamination from other cells types. Compared to unedited E4 neurons, E3 neurons were less susceptible to ionomycin-induced cytotoxicity. Biochemically, E4 cells exhibited increased tau phosphorylation and ERK1/2 phosphoactivation. Moreover, E4 neurons released increased amounts of phosphorylated tau extracellularly in an isoform-dependent manner by a heparin sulfate proteoglycan-dependent mechanism. Our results demonstrate that endogenous expression of E4 by stem-cell-derived forebrain excitatory neurons predisposes neurons to calcium dysregulation and ultimately cell death. This change is associated with increased cellular tau phosphorylation and markedly enhanced release of phosphorylated tau. Importantly, these effects are independent of glial APOE. These findings suggest that E4 accelerates spreading of tau pathology and neuron death in part by neuron-specific, glia-independent mechanisms. Ann Neurol 2019;85:726-739.

Ceramide Induces the Death of Retina Photoreceptors Through Activation of Parthanatos.

Ceramide (Cer) has a key role inducing cell death and has been proposed as a messenger in photoreceptor cell death in the retina. Here, we explored the pathways induced by C2-acetylsphingosine (C2-Cer), a cell-permeable Cer, to elicit photoreceptor death. Treating pure retina neuronal cultures with 10μM C2-Cer for 6h selectively induced photoreceptor death, decreasing mitochondrial membrane potential and increasing the formation of reactive oxygen species (ROS). In contrast, amacrine neurons preserved their viability. Noteworthy, the amount of TUNEL-labeled cells and photoreceptors expressing cleaved caspase-3 remained constant and pretreatment with a pan-caspase inhibitor did not prevent C2-Cer-induced death. C2-Cer provoked polyADP ribosyl polymerase-1 (PARP-1) overactivation. Inhibiting PARP-1 decreased C2-Cer-induced photoreceptor death; C2-Cer increased polyADP ribose polymer (PAR) levels and induced the translocation of apoptosis inducing factor (AIF) from mitochondria to photoreceptor nuclei, which was prevented by PARP-1 inhibition. Pretreatment with a calpain and cathepsin inhibitor and with a calpain inhibitor reduced photoreceptor death, whereas selective cathepsin inhibitors granted no protection. Combined pretreatment with a PARP-1 and a calpain inhibitor evidenced the same protection as each inhibitor by itself. Neither autophagy nor necroptosis was involved in C2-Cer-elicited death; no increase in LDH release was observed upon C2-Cer treatment and pretreatment with inhibitors of necroptosis and autophagy did not rescue photoreceptors. These results suggest that C2-Cer induced photoreceptor death by a novel, caspase-independent mechanism, involving activation of PARP-1, decline of mitochondrial membrane potential, calpain activation, and AIF translocation, all of which are biochemical features of parthanatos.

Complement-dependent bystander injury to neurons in AQP4-IgG seropositive neuromyelitis optica

BackgroundAquaporin-4-immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune disease of the central nervous system in which AQP4-IgG binding to AQP4 on astrocytes results in complement-dependent astrocyte injury and secondary inflammation, demyelination, and neuron loss. We previously reported evidence for a complement bystander mechanism for early oligodendrocyte injury in NMO. Herein, we tested the hypothesis that complement bystander injury, which involves diffusion to nearby cells of activated soluble complement components from complement-injured astrocytes, is a general phenomenon that may contribute to neuronal injury in NMO.MethodsPrimary cocultures of rat astrocytes and cortical neurons were established to study complement-dependent cell death after exposure to AQP4-IgG and complement. In animal experiments, AQP4-IgG was delivered to adult rats by intracerebral injection. Cell cultures and rat brain were studied by immunofluorescence.ResultsIn primary astrocyte-neuron cocultures, addition of AQP4-IgG and complement resulted in death of neurons nearby astrocytes. Deposition of complement membrane attack complex C5b-9 was seen on neurons nearby astrocytes, whereas C1q, the initiating protein in the complement pathway, was seen only on astrocytes. Neuron death was not seen with a complement inhibitor, with C1q- or C6-depleted complement, in pure neuron cultures exposed to AQP4-IgG and complement or in cocultures exposed to an astrocyte toxin. Intracerebral injection in rats of AQP4-IgG and a fixable dead cell fluorescent marker produced death of neurons near astrocytes, with C5b-9 deposition. Neuron death was not seen in rats receiving a complement inhibitor or in AQP4-IgG-injected AQP4 knockout rats.ConclusionThese results support a novel mechanism for early neuron injury in NMO and provide evidence that complement bystander injury may be a general phenomenon for brain cell injury following AQP4-IgG-targeted astrocyte death.

Interleukin-1β Protects Neurons against Oxidant-Induced Injury via the Promotion of Astrocyte Glutathione Production.

Interleukin-1β (IL-1β), a key cytokine that drives neuroinflammation in the Central Nervous System (CNS), is enhanced in many neurological diseases/disorders. Although IL-1β contributes to and/or sustains pathophysiological processes in the CNS, we recently demonstrated that IL-1β can protect cortical astrocytes from oxidant injury in a glutathione (GSH)-dependent manner. To test whether IL-1β could similarly protect neurons against oxidant stress, near pure neuronal cultures or mixed cortical cell cultures containing neurons and astrocytes were exposed to the organic peroxide, tert-butyl hydroperoxide (t-BOOH), following treatment with IL-1β or its vehicle. Neurons and astrocytes in mixed cultures, but not pure neurons, were significantly protected from the toxicity of t-BOOH following treatment with IL-1β in association with enhanced GSH production/release. IL-1β failed to increase the GSH levels or to provide protection against t-BOOH toxicity in chimeric mixed cultures consisting of IL-1R1+/+ neurons plated on top of IL-1R1−/− astrocytes. The attenuation of GSH release via block of multidrug resistance-associated protein 1 (MRP1) transport also abrogated the protective effect of IL-1β. These protective effects were not strictly an in vitro phenomenon as we found an increased striatal vulnerability to 3-nitropropionic acid-mediated oxidative stress in IL-1R1 null mice. Overall, our data indicate that IL-1β protects neurons against oxidant injury and that this likely occurs in a non-cell-autonomous manner that relies on an increase in astrocyte GSH production and release.

A role for astrocyte-derived amyloid β peptides in the degeneration of neurons in an animal model of temporal lobe epilepsy.

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid-β (Aβ) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aβ peptides. Thus, we seek to establish what role Aβ plays in kainic acid-induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aβ-related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aβ in kainic acid-induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aβ production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co-cultures than in pure neuronal culture, and this is attenuated by precluding Aβ production. Collectively, these results indicate that increased production/secretion of Aβ-related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid-treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aβ peptides derived from astrocytes may have a role in TLE pathogenesis.

NEURONAL APOE MODULATES A SPORADIC ALZHEIMER’S DISEASE PHENOTYPE IN PATIENT-DERIVED INDUCED NEURONS

The APOE-ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (sAD) and may be enriched in other clinically relevant populations. While APOE is predominantly expressed by astrocytes in the central nervous system, neuronal expression of APOE is of increasing interest in age-related cognitive impairment, neurological injury, and neurodegeneration. The human and mouse APOE promoter sequences are dissimilar, and contain distinct regions that control expression in a species specific, cell-type specific manner. APOE mRNA and protein is detectable in primary human neuronal cultures in a manner suggestive of de novo protein synthesis. However, neuronal expression of APOE has not been linked to neurotoxicity in a human system. We reprogrammed induced pluripotent stem cells (iPSCs) from three unrelated sAD patients with the ε3/ε4 genotype, used CRISPR/Cas9 gene editing to generate ε3/ε3 isogenic lines, and differentiated them into pure cultures of glutamatergic neurons with no detectable glia. APOE is synthesized and secreted by human induced neurons. Genetic correction of ε4 to ε3 reduces tau phosphorylation and phosphoactivation of a specific kinase. Moreover, ε4 is associated with altered amyloid secretion, and exacerbates the neurotoxic effects of a calcium ionophore. APOE is expressed in human glia-free neuronal culture system, and APOE isoform alters sAD-associated molecular pathways. More detailed mechanistic studies are required to better understand this phenomenon. However, the net effect is that endogenous expression of APOE-ε4 weakens neurons and contributes to the sAD cellular phenotype independently of, but likely cooperatively with, glial APOE.

Micro electrode arrays to investigate neuron-glia crosstalk in neuropathic pain in-vitro models

Micro electrode arrays to investigate neuron-glia crosstalk in neuropathic pain in-vitro models