Neuronal-glial Co-cultures Research Articles

Sustained fentanyl exposure inhibits neuronal activity in dissociated striatal neuronal-glial cocultures through actions independent of opioid receptors.

Besides having high potency and efficacy at the µ-opioid (MOR) and other opioid receptor types, fentanyl has some affinity for some adrenergic receptor types, which may underlie its unique pathophysiological differences from typical opioids. To better understand the unique actions of fentanyl, we assessed the extent to which fentanyl alters striatal medium spiny neuron (MSN) activity via opioid receptors or α1-adrenoceptors in dopamine type 1 or type 2 receptor (D1 or D2)-expressing MSNs. In neuronal and mixed-glial cocultures from the striatum, acute fentanyl (100 nM) exposure decreased the frequency of spontaneous action potentials. Overnight exposure of cocultures to 100 nM fentanyl severely reduced the proportion of MSNs with spontaneous action potentials, which was unaffected by coexposure to the opioid receptor antagonist naloxone (10 µM) but fully negated by coadministering the pan-α1-adrenoceptor inverse agonist prazosin (100 nM) and partially reversed by the selective α1A-adrenoceptor antagonist RS 100329 (300 nM). Acute fentanyl (100 nM) exposure modestly reduced the frequency of action potentials and caused firing rate adaptations in D2, but not D1, MSNs. Prolonged (2-5 h) fentanyl (100 nM) application dramatically attenuated firing rates in both D1 and D2 MSNs. To identify possible cellular sites of α1-adrenoceptor action, α1-adrenoceptors were localized in subpopulations of striatal astroglia and neurons by immunocytochemistry and Adra1a mRNA by in situ hybridization in astrocytes. Thus, sustained fentanyl exposure can inhibit striatal MSN activity via a nonopioid receptor-dependent pathway, which may be modulated via complex actions in α1-adrenoceptor-expressing striatal neurons and/or glia.NEW & NOTEWORTHY Acute fentanyl exposure attenuated the activity of striatal medium spiny neurons (MSNs) in vitro and in dopamine D2, but not D1, receptor-expressing MSNs in ex vivo slices. By contrast, sustained fentanyl exposure suppressed the spontaneous activity of MSNs cocultured with glia through a nonopioid receptor-dependent mechanism modulated, in part, by α1-adrenoceptors. Fentanyl exposure can affect striatal function via a nonopioid receptor mechanism of action that appears mediated by α1-adrenoreceptor-expressing striatal neurons and/or astroglia.

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging.

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y6 receptor (P2Y6 R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y6 R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y6 R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal-glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y6 R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y6 R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y6 R has the potential to prevent age-associated memory impairment.

Discovery of Small Molecule KCC2 Potentiators Which Attenuate In Vitro Seizure-Like Activity in Cultured Neurons.

KCC2 is a K+-Cl− cotransporter that is expressed in neurons throughout the central nervous system. Deficits in KCC2 activity have been implicated in a variety of neurological disorders, including epilepsy, chronic pain, autism spectrum disorders, and Rett syndrome. Therefore, it has been hypothesized that pharmacological potentiation of KCC2 activity could provide a treatment for these disorders. To evaluate the therapeutic potential of pharmacological KCC2 potentiation, drug-like, selective KCC2 potentiators are required. Unfortunately, the lack of such tools has greatly hampered the investigation of the KCC2 potentiation hypothesis. Herein, we describe the discovery and characterization of a new class of small-molecule KCC2 potentiator. This newly discovered class exhibits KCC2-dependent activity and a unique mechanistic profile relative to previously reported small molecules. Furthermore, we demonstrate that KCC2 potentiation by this new class of KCC2 potentiator attenuates seizure-like activity in neuronal-glial co-cultures. Together, our results provide evidence that pharmacological KCC2 potentiation, by itself, is sufficient to attenuate neuronal excitability in an in vitro model that is sensitive to anti-epileptic drugs. Our findings and chemical tools are important for evaluating the promise of KCC2 as a therapeutic target and could lay a foundation for the development of KCC2-directed therapeutics for multiple neurological disorders.

The epileptic landscape of IDH mutant gliomas.

2064 Background: Tumor-associated epilepsy (TAE) is a frequent complication of diffusely infiltrative gliomas. TAE not only impairs quality-of-life, but it can even be life-threatening. Furthermore, glioma cells have been shown to proliferate and migrate faster when exposed to firing neurons. We previously showed that isocitrate dehydrogenase 1 mutant (IDHmut) gliomas were more likely to cause preoperative seizures than IDH wildtype (IDHwt) gliomas, and that the chemical product of IDHmut, D2HG, increased synchronized network bursts in cultured neurons (PMID: 28404805). But the mechanism whereby this occurs, whether IDHmut inhibitors can block this, and whether seizure risk also varies by IDHmut status postoperatively, are unknown. Methods: Methods are embedded in Results. Results: We discovered that exogenous 3 mM D2HG had a 150% greater effect on the firing rate of cultured mouse cortical neurons when nonneoplastic glia cells were present versus when they were absent ( P=0.002). Although a recently published study suggested that D2HG causes seizures through mTOR activation (PMID: 34994387), we found that D2HG reduced neuronal mTOR activity in neuronal-glial cocultures by 54% ( P=0.0004). Patch clamp analyses showed that, while D2HG does not directly activate glutamate receptors, it does act as a glutamate transport substrate, thus potentially interfering with the ability of glial cells to take up glutamate released into the synaptic cleft. Coculture with patient-derived IDHmut glioma cells increased the firing rate of human cortical neuron/astrocyte spheroids by up to 272%, and an IDHmut inhibitor currently being tested in clinical trials, AG881, reduced the excitatory effect of IDHmut glioma cells on spheroids by 79% ( P=0.0008). Using a novel in vivo model of TAE, wherein EEG recordings were taken of immunocompetent mice engrafted with an isogenic pair of Sleeping Beauty transposase-engineered mouse glioma lines (NRAS/ATRX/TP53mut ± IDHmut), we found that IDHmut gliomas produced 7.6-fold more epileptiform spikes than IDHwt gliomas ( P=0.004). RNA-Seq analysis of the peritumoral mouse brain tissue showed that this increase in spikes was associated with significantly increased expression of key genes known to be upregulated in epilepsy, including SLC12A5, THSB1, VEGFA, FOSL2, and SYNPO. Treatment with 5 mg/kg AG881 by daily oral gavage reduced those spikes in IDHmut-engrafted mice by 51% within three days ( P=0.027), whereas vehicle control had no effect ( P=0.33). Among 247 patients with grade 2–4 adult-type diffuse gliomas, multivariable time-to-event analysis showed that postoperative seizure risk increased with preoperative seizures, subtotal resection, and IDHmut astrocytoma. Conclusions: Together, these data show that (i) the D2HG product of IDHmut gliomas increases neuronal excitation in a glial-dependent manner; (ii) IDHmut also affects postoperative seizure risk; (iii) IDHmut inhibitors may improve TAE control.

Triiodothyronine or Antioxidants Block the Inhibitory Effects of BDE-47 and BDE-49 on Axonal Growth in Rat Hippocampal Neuron-Glia Co-Cultures.

We previously demonstrated that polybrominated diphenyl ethers (PBDEs) inhibit the growth of axons in primary rat hippocampal neurons. Here, we test the hypothesis that PBDE effects on axonal morphogenesis are mediated by thyroid hormone and/or reactive oxygen species (ROS)-dependent mechanisms. Axonal growth and ROS were quantified in primary neuronal-glial co-cultures dissociated from neonatal rat hippocampi exposed to nM concentrations of BDE-47 or BDE-49 in the absence or presence of triiodothyronine (T3; 3–30 nM), N-acetyl-cysteine (NAC; 100 µM), or α-tocopherol (100 µM). Co-exposure to T3 or either antioxidant prevented inhibition of axonal growth in hippocampal cultures exposed to BDE-47 or BDE-49. T3 supplementation in cultures not exposed to PBDEs did not alter axonal growth. T3 did, however, prevent PBDE-induced ROS generation and alterations in mitochondrial metabolism. Collectively, our data indicate that PBDEs inhibit axonal growth via ROS-dependent mechanisms, and that T3 protects axonal growth by inhibiting PBDE-induced ROS. These observations suggest that co-exposure to endocrine disruptors that decrease TH signaling in the brain may increase vulnerability to the adverse effects of developmental PBDE exposure on axonal morphogenesis.

25-Hydroxycholesterol modulates tau-mediated neurodegeneration and microglial chemotaxis and phagocytosis.

Immune activation is an important component of Alzheimer disease (AD) pathology, with microglia playing a critical role in driving ApoE-dependent tau-mediated neurodegeneration. The oxysterol 25-hydroxycholesterol (25-HC) is an established potent regulator of peripheral innate immune response, though less is known regarding its role in CNS neuroimmune pathobiology. The enzyme that synthesizes 25-HC, cholesterol 25-hydroxylase (CH25H), is exclusively expressed by microglia in the CNS and is significantly upregulated in human AD brain and in transgenic AD mouse models. We have recently reported pro-inflammatory effects of 25-HC in ApoE4-expressing mouse microglia, including increased microglial IL-1β secretion and inflammasome activation. In this study we explored the role of 25-HC in ApoE-dependent tau-mediated neurodegeneration. PS19 tauopathy mice previously crossed with ApoE targeted replacement (TR) mice were subsequently crossed with CH25H KO mice. Hippocampal (HC), entorhinal and pyriform cortical (EC/PC) and ventricular volumes were measured at 9 months of age. In vitro primary cell cultures containing neurons, mixed glia or microglia were utilized to evaluate effects of 25-HC or 7α,25-dihydroxycholesterol (7α,25-diHC), on neurotoxicity, microglial cytokine secretion, chemotaxis and phagocytic activity. We found that in female tau mice expressing human ApoE4, genetic deletion of CH25H was protective (increased EC/PC volumes and reduced ventricular volumes). Similar trends were observed in male mice though these were not statistically significant. In a neuronal-glial co-culture model of ApoE4-mediated neurotoxicity, 25-HC protected against neurite loss and neuronal death at low concentrations but was potently neurotoxic at higher concentrations. In primary microglial cultures, 25-HC modulated secretion of a number of chemokines and provoked a potent microglial chemotaxic response that was most robust in microglia derived from CH25H KO, ApoE4-TR or ApoE4-TR-CH25H KO mice. 7α,25-diHC also stimulated chemotaxis but less robustly than 25-HC. Assays testing the effect of 25-HC and 7α,25-diHC on phagocytosis in microglia were also performed, and these data will be presented. 25-HC appears to modulate tau-mediated neurodegeneration in an ApoE-dependent manner, especially in female mice, possibly through effects on critical microglial cellular functions. CH25H and 25-HC may be viable therapeutic targets for AD-related neuroimmune activation.

Marine and Anthropogenic Bromopyrroles Alter Cellular Ca2+ Dynamics of Murine Cortical Neuronal Networks by Targeting the Ryanodine Receptor and Sarco/Endoplasmic Reticulum Ca2+-ATPase.

Bromopyrroles (BrPyr) are synthesized naturally by marine sponge symbionts and produced anthropogenically as byproducts of wastewater treatment. BrPyr interact with ryanodine receptors (RYRs) and sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA). Influences of BrPyr on the neuronal network activity remain uncharted. BrPyr analogues with differing spectra of RYR/SERCA activities were tested using RYR-null or RYR1-expressing HEK293 and murine cortical neuronal/glial cocultures (NGCs) loaded with Fluo-4 to elucidate their mechanisms altering Ca2+ dynamics. The NGC electrical spike activity (ESA) was measured from NGCs plated on multielectrode arrays. Nanomolar tetrabromopyrrole (TBP, 1) potentiated caffeine-triggered Ca2+ release independent of extracellular [Ca2+] in RYR1-HEK293, whereas higher concentrations produce slow and sustained rise in cytoplasmic [Ca2+] independent of RYR1 expression. TBP, 2,3,5-tribromopyrrole (2), pyrrole (3), 2,3,4-tribromopyrrole (4), and ethyl 4-bromopyrrole-2-carboxylate (5) added acutely to NGC showed differential potency; rank order TBP (IC50 ≈ 220 nM) > 2 ≫ 5, whereas 3 and 4 were inactive at 10 μM. TBP >2 μM elicited sustained elevation of cytoplasmic [Ca2+] and loss of neuronal viability. TBP did not alter network ESA. BrPyr from marine and anthropogenic sources are ecological signaling molecules and emerging anthropogenic pollutants of concern to environmental and human health that potently alter ER Ca2+ dynamics and warrant further investigation in vivo.

Extracellular tau induces microglial phagocytosis of living neurons in cell cultures.

Tau is a microtubule-associated protein, found at high levels in neurons, and itsaggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.

Activated microglia desialylate their surface, stimulating complement receptor 3‐mediated phagocytosis of neurons

The glycoproteins and glycolipids of the cell surface have sugar chains that normally terminate in a sialic acid residue, but inflammatory activation of myeloid cells can cause sialidase enzymes to remove these residues, resulting in desialylation and altered activity of surface receptors, such as the phagocytic complement receptor 3 (CR3). We found that activation of microglia with lipopolysaccharide (LPS), fibrillar amyloid beta (Aβ), Tau or phorbol myristate acetate resulted in increased surface sialidase activity and desialylation of the microglial surface. Desialylation of microglia by adding sialidase, stimulated microglial phagocytosis of beads, but this was prevented by siRNA knockdown of CD11b or a blocking antibody to CD11b (a component of CR3). Desialylation of microglia by a sialyl‐transferase inhibitor (3FAx‐peracetyl‐Neu5Ac) also stimulated microglial phagocytosis of beads. Desialylation of primary glial‐neuronal co‐cultures by adding sialidase or the sialyl‐transferase inhibitor resulted in neuronal loss that was prevented by inhibiting phagocytosis with cytochalasin D or the blocking antibody to CD11b. Adding desialylated microglia to glial‐neuronal cultures, in the absence of neuronal desialylation, also caused neuronal loss prevented by CD11b blocking antibody. Adding LPS or Aβ to primary glial‐neuronal co‐cultures caused neuronal loss, and this was prevented by inhibiting endogenous sialidase activity with N‐acetyl‐2,3‐dehydro‐2‐deoxyneuraminic acid or blockage of CD11b. Thus, activated microglia release a sialidase activity that desialylates the cell surface, stimulating CR3‐mediated phagocytosis of neurons, making extracellular sialidase and CR3 potential treatment targets to prevent inflammatory loss of neurons.

Hydrogel scaffolds promote neural gene expression and structural reorganization in human astrocyte cultures

Biomaterial scaffolds have the potential to enhance neuronal development and regeneration. Understanding the genetic responses of astrocytes and neurons to biomaterials could facilitate the development of synthetic environments that enable the specification of neural tissue organization with engineered scaffolds. In this study, we used high throughput transcriptomic and imaging methods to determine the impact of a hydrogel, PuraMatrix™, on human glial cells in vitro. Parallel studies were undertaken with cells grown in a monolayer environment on tissue culture polystyrene. When the Normal Human Astrocyte (NHA) cell line is grown in a hydrogel matrix environment, the glial cells adopt a structural organization that resembles that of neuronal-glial cocultures, where neurons form clusters that are distinct from the surrounding glia. Statistical analysis of next generation RNA sequencing data uncovered a set of genes that are differentially expressed in the monolayer and matrix hydrogel environments. Functional analysis demonstrated that hydrogel-upregulated genes can be grouped into three broad categories: neuronal differentiation and/or neural plasticity, response to neural insult, and sensory perception. Our results demonstrate that hydrogel biomaterials have the potential to transform human glial cell identity, and may have applications in the repair of damaged brain tissue.

From the Cover: BDE-47 and BDE-49 Inhibit Axonal Growth in Primary Rat Hippocampal Neuron-Glia Co-Cultures via Ryanodine Receptor-Dependent Mechanisms.

Polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with adverse neurodevelopmental outcomes in children and preclinical models; however, the mechanisms by which PBDEs cause developmental neurotoxicity remain speculative. The structural similarity between PBDEs and nondioxin-like (NDL) polychlorinated biphenyls (PCBs) suggests shared toxicological properties. Consistent with this, both NDL PCBs and PBDEs have been shown to stabilize ryanodine receptors (RyRs) in the open configuration. NDL PCB effects on RyR activity are causally linked to increased dendritic arborization, but whether PBDEs similarly enhance dendritic growth is not known. In this study, we quantified the effects of individual PBDE congeners on not only dendritic but also axonal growth since both are regulated by RyR-dependent mechanisms, and both are critical determinants of neuronal connectivity. Neuronal-glial co-cultures dissociated from the neonatal rat hippocampus were exposed to BDE-47 or BDE-49 in the culture medium. At concentrations ranging from 20 pM to 2 µM, neither PBDE congener altered dendritic arborization. In contrast, at concentrations ≥ 200 pM, both congeners delayed neuronal polarization resulting in significant inhibition of axonal outgrowth during the first few days in vitro. The axon inhibitory effects of these PBDE congeners occurred independent of cytotoxicity, and were blocked by pharmacological antagonism of RyR or siRNA knockdown of RyR2. These results demonstrate that the molecular and cellular mechanisms by which PBDEs interfere with neurodevelopment overlap with but are distinct from those of NDL PCBs, and suggest that altered patterns of neuronal connectivity may contribute to the developmental neurotoxicity of PBDEs.

Glia and glial polyamines. Role in brain function in health and disease

The roles of glia and polyamines (PA) in brain function and dysfunction are highlighted in this review. We emphasize that PA accumulation preferentially in glia, but not in neurons, is clearly evolutionarily determined; it is found throughout the brain, retina, peripheral nervous system, and in glial-neuronal co-cultures of multiple species, including man. This phenomenon raises key questions: (i) What are the mechanisms that underlie such uneven distribution, accumulation and release from glia? (ii) What are the consequences of PA fluxes within the brain on neuronal function? (iii) What are the roles of PAs in brain disorders and diseases? This review includes suggestions on the roles of PAs, such as putrescine (PT), spermidine (SPD), spermine (SPM) and their derivatives as novel glio-transmitters in brain since PA affect many neuronal and glial receptors, channels and transporters. Polyamines hitherto have been neglected, although it is evident that these molecules are key elements for normal brain function and their metabolic disorders, apparently, cause the development of many pathological syndromes and diseases. The study of endogenous PA allows one to put forward the basic principles of scientific research on glio-neuronal interactions and clinical therapies, which are based on the exclusivity of glial cells in terms of accumulation of PA and PA-dependent functions.

5α-reduced progestogens ameliorate mood-related behavioral pathology, neurotoxicity, and microgliosis associated with exposure to HIV-1 Tat

Human immunodeficiency virus (HIV) is associated with motor and mood disorders, likely influenced by reactive microgliosis and subsequent neural damage. We have recapitulated aspects of this pathology in mice that conditionally express the neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat). Progestogens may attenuate Tat-related behavioral impairments and reduce neurotoxicity in vitro, perhaps via progesterone’s 5α-reductase-dependent metabolism to the neuroprotective steroid, allopregnanolone. To test this, ovariectomized female mice that conditionally expressed (or did not express) central HIV-1 Tat were administered vehicle or progesterone (4mg/kg), with or without pretreatment of a 5α-reductase inhibitor (finasteride, 50mg/kg). Tat induction significantly increased anxiety-like behavior in an open field, elevated plus maze and a marble burying task concomitant with elevated protein oxidation in striatum. Progesterone administration attenuated anxiety-like effects in the open field and elevated plus maze, but not in conjunction with finasteride pretreatment. Progesterone also attenuated Tat-promoted protein oxidation in striatum, independent of finasteride pretreatment. Concurrent experiments in vitro revealed Tat (50nM)-mediated reductions in neuronal cell survival over 60h, as well as increased neuronal and microglial intracellular calcium, as assessed via fura-2 AM fluorescence. Co-treatment with allopregnanolone (100nM) attenuated neuronal death in time-lapse imaging and blocked the Tat-induced exacerbation of intracellular calcium in neurons and microglia. Lastly, neuronal-glial co-cultures were labeled for Iba-1 to reveal that Tat increased microglial numbers in vitro and co-treatment with allopregnanolone attenuated this effect. Together, these data support the notion that 5α-reduced pregnane steroids exert protection over the neurotoxic effects of HIV-1 Tat.

Increased BDNF protein expression after ischemic or PKC epsilon preconditioning promotes electrophysiologic changes that lead to neuroprotection.

Ischemic preconditioning (IPC) via protein kinase C epsilon (PKCɛ) activation induces neuroprotection against lethal ischemia. Brain-derived neurotrophic factor (BDNF) is a pro-survival signaling molecule that modulates synaptic plasticity and neurogenesis. Interestingly, BDNF mRNA expression increases after IPC. In this study, we investigated whether IPC or pharmacological preconditioning (PKCɛ activation) promoted BDNF-induced neuroprotection, if neuroprotection by IPC or PKCɛ activation altered neuronal excitability, and whether these changes were BDNF-mediated. We used both in vitro (hippocampal organotypic cultures and cortical neuronal-glial cocultures) and in vivo (acute hippocampal slices 48 hours after preconditioning) models of IPC or PKCɛ activation. BDNF protein expression increased 24 to 48 hours after preconditioning, where inhibition of the BDNF Trk receptors abolished neuroprotection against oxygen and glucose deprivation (OGD) in vitro. In addition, there was a significant decrease in neuronal firing frequency and increase in threshold potential 48 hours after preconditioning in vivo, where this threshold modulation was dependent on BDNF activation of Trk receptors in excitatory cortical neurons. In addition, 48 hours after PKCɛ activation in vivo, the onset of anoxic depolarization during OGD was significantly delayed in hippocampal slices. Overall, these results suggest that after IPC or PKCɛ activation, there are BDNF-dependent electrophysiologic modifications that lead to neuroprotection.

Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis.

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, expressed in many brain pathologies and associated with neuronal loss. We show here that addition of TNF-α to neuronal-glial co-cultures increases microglial proliferation and phagocytosis, and results in neuronal loss that is prevented by eliminating microglia. Blocking microglial phagocytosis by inhibiting phagocytic vitronectin and P2Y6 receptors, or genetically removing opsonin MFG-E8, prevented TNF-α induced loss of live neurons. Thus TNF-α appears to induce neuronal loss via microglial activation and phagocytosis of neurons, causing neuronal death by phagoptosis.

Molecule of the month: miRNA and proteins in Alzheimer's disease

The analysis of Alzheimer's disease is an important topic of research because the results and findings help elucidate problems associated with aging as well as other diseases including Downs' syndrome, Parkinson's disease, dementia with Lewy bodies, Multiple Sclerosis, Amyotrophic Lateral Sclerosis,and infectious disease including NeuroAIDS. Here we review just a few points from an ever growing and productive literature. A recent article dealt with oxidation effects and neurodegeneration [1]. The hypothesis addressed was that oxidation of RNA affects miRNA and protein expression. In human as well as in rodent brain, neurodegeneration (neuronal) that is age-associated was demonstrated that is consequent to oxidation of nucleic acids and proteins. Such neuronal degenerative factors have also been analyzed in dementia with Lewy bodies, Amyotrophic Lateral Sclerosis, Parkinson disease, and Alzheimer disease. These findings occurred in cellular as well as animal models. Coding RNA as well as non-coding RNA were affected and deleterious effects on cell fate were analyzed. Such studies are needed for developing strategies of therapy. A recent study also analyzed miRNAs in Alzheimer's disease pathogenesis [2]. The analysis of extracellular fluid from neocortex associated with temporal lobe from patients who had Alzheimer's disease indicated increased miRNAs. These miRNAs included miRNA-155 and miRNA-146a that are proinflammatory. Moreover, when cell culture medium was used with these miRNA added, Alzheimer's disease-like gene expression occurred. This profile was associated with downregulation of complement factor H (CFH), a repressor of the innate immune response. Anti-miRNA approaches reduced the pro-inflammatory effects. Additional miRNAs, miRNA-9 and miRNA-125bwere involved as well [3]. These miRNAs were regulated by NF-кB andbound independently to the mRNA 3'- untranslated region to reduce CFH translation. The authors of an article in 2011 [4] stated that transcription factor, TAp73, drove expression of miRNA-34a (but not miRNA-34b or miRNA-34c). This was associated with decreased expression of synaptic proteins synaptotagmin-1and syntaxin-1A. These controlled molecular reactions generally occurred in retinoid-stimulated differentiation of neuroblastoma cells and rodents. The synaptotagmin-1 protein expression changes also occurred in Alzheimer's disease brain tissue. These observations were extended to non-classical infectious disease related to prion infections. Several neurotrophic RNA and DNA viruses induced miRNA-146a in human brain cells. In addition, oxidative stress, neurotoxic metals,pro-inflammatory cytokines,as well as Aβ42 peptideupregulated miRNA-146a as wellin primary cell human glial-neuronal cocultures. These findings were in Alzheimer's disease and murine scrapie studies. miRNA-146a quantitieswere related to disease severity and inflammatory neuropathology in Alzheimer's disease. Moreover, findings were similar in Gerstmann-Straussler- Scheinker syndrome and sporadicCreutzfeldt-Jakob disease [5]. In addition, Aβ oligopeptide down regulated miRNA-9 and miRNA-181c in earlier studies. This occurred in hippocampal cultures. In addition, APP23 transgenicmice and human Alzheimer's disease tissue showed deposition of Aβ. Moreover, APP is a target of miRNA regulation and additional 3'- untranslated regions were identified from several mRNAs. These are down regulatedbymiR-9 and -181c, separately and contemporaneously and includedTGFBI, TRIM2, SIRT1 and BTBD3 [6]. NF-kB has a very large well-known number of protein-protein interactions and is not included here. Based on the literature, the protein Tap73 is more often recognized as TP73. Tap73 was absent in the GNCPro database and so the term TP73 was used. The figure illustrates various gene interactions among the proteins mentioned above:complement factor H (CFH), synaptotagmin-1 (SYT1), syntaxin-1A (STX1A), tumor protein p73 transcription factor (TP73), transforming growth factor (TGFBI), tripartite motif containing 2(TRIM2),sirtuin 1(SIRT1), andBTB (POZ) domain containing 3(BTBD3). It is left as a puzzle for the interested reader to identify the various genes and their functions in the Figure 1 [7–9]. Figure 1 Network of input proteins (complement factor H, synaptotagmin-1, syntaxin-1A, TP73, TGFBI, TRIM2, SIRT1, andBTBD3) and the input neighbors of these proteins. In this figure, line-colors and various interactions with other genes are red Down-regulation, ...

RONS in inflammatory neurodegeneration

Inflammatory neurodegeneration is neuronal degeneration due to inflammation, and is thought to contribute to neuronal loss in infectious, ischemic, traumatic and neurodegenerative brain pathologies. We have identified three mechanisms by which inflamed glia kill neurons: iNOS, PHOX and phagocytosis. Inflamed microglia (brain macrophages) and astrocytes express inducible nitric oxide synthase (iNOS), producing high levels of NO, which inhibits mitochondrial respiration at cytochrome oxidase in competition with oxygen, sensitising neurons to hypoxic death. In addition, NO-derivatives peroxynitrite and S-nitrosothiols inactivate mitochondrial complex I, stimulating oxidant production by mitochondria. The phagocyte NADPH oxidase (PHOX) is constitutively expressed by microglia, and we find that H2O2 from PHOX activation is required for inflammatory activation of microglia. Alternatively, superoxide from PHOX can react with NO from iNOS to produce peroxynitrite that induces reversible phosphatidyserine (PS) exposure on neurons, which induces microglia to phagocytose viable neurons. We found inflammatory activation of neuronal-glial co-cultures with LPS, LTA, TNF-αor α-amyloid, or in vivo with LPS results, in progressive loss of neurons, accompanied by microglial phagocytosis of neurons, and is prevented by blocking phagocytosis or knockout of PS-binding adaptor protein MFG-E8. Cell death by primary phagocytosis may constitute a novel form of cell death: ‘phagoptosis’. References M. Fricker, J.J. Neher, J.W. Zhao, C. Thery, A.M. Tolkovsky, G.C. Brown, MFG-E8 mediates primary phagocytosis of viable neurons during neuroinflammation. J Neurosci. 32 (2012) 2657–2666. J.J. Neher, U. Neniskyte, Z.W. Zhao, A. Bal-Price, A.M. Tolkovsky, G.C. Brown, Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J. Immunol. 186 (2011) 4973–4983. G.C. Brown, J.J. Neher, Inflammatory neurodegeneration and mechanisms of microglial killing of neurons. Mol. Neurobiol. 41 (2010) 242–247. G.C. Brown, Nitric oxide and neuronal death. Nitric Oxide 23 (2010) 153–165.

An aligned 3D neuronal-glial co-culture model for peripheral nerve studies

In this study we report on the development of a 3D in vitro peripheral nerve model using aligned electrospun polycaprolactone fibre scaffolds manufactured under tightly controlled and reproducible conditions with uniform diameters of 1 μm, 5 μm and 8 μm. Fibres were characterized by SEM for diameter, density and alignment properties and formed in to scaffolds for 3D in vitro culture. Three different approaches were adopted using: i) neuronal or primary Schwann cell cultures alone; ii) neuronal and primary Schwann cells in co-culture and iii) isolated dorsal root ganglion cultures, containing both neuronal and Schwann cells, with immunohistochemical and 3D confocal microscopy analysis. Neurite guidance was evident on all fibres diameters with the longest neurites detected on 8 μm fibres when cultured alone. However, co-culture with primary Schwann cells was found to enable neurite formation on all scaffolds. Dorsal root ganglion explants when grown on scaffolds showed both organised aligned neurite guidance and notably the co-localization of Schwann cells with neurites. Neurite lengths of up to 2.50 mm were routinely observed using 1 μm diameter fibres after 10 days and all cultures demonstrated a migrating Schwann cell ‘front’ of up to 2.70 mm (1 μm diameter fibres). Thus, a direct relationship was found between fibre diameter, neurite outgrowth and Schwann cell morphology. This work therefore supports the use of aligned electrospun PCL microfibres for the development of 3D peripheral nerve models in vitro. We envisage such models having future value in a number of areas including developmental biology, disease studies and the design of devices and scaffolds for peripheral nerve repair.

An in vitro model for studying the effects of continuous ethanol exposure on N-methyl- d-aspartate receptor function

Long-term ethanol exposure has deleterious effects on both glial and neuronal function. We assessed alterations in both astrocytic and neuronal viability, and alterations in N-methyl- d-aspartate receptor (NMDAR) function, in cocultures of rat cerebellar granule cells (CGCs) and astrocytes after continuous ethanol exposure (CEE). Treatment of cells with 100 mM EtOH once every 24 h for 4 days resulted in a mean ethanol concentration of 57.3 ± 2.1 mM. Comparisons between control and post–ethanol-treated cells were made 4 days after the last ethanol treatment. CEE did not alter glial cell viability, as indicated by the absence of either changes in astrocytic morphology, actin depolymerization, or disruption of astrocytic intracellular mitochondrial distribution at any day postethanol treatment. The CGCs were healthy and viable after CEE, as indicated by phase-contrast microscopy and the trypan-blue exclusion method. Whole-cell patch-clamp experiments indicated that NMDA-induced currents (I NMDA) were altered by CEE treatment. Similar to previous results obtained during the withdrawal phase from chronic ethanol exposure, I NMDA from CEE-treated cells were significantly larger than I NMDA from NMDARs in control CGCs, but returned to control values by the fourth day post-CEE. However, after the last ethanol dosing and during a time when ethanol concentrations remained high, I NMDA were significantly smaller than control values. Identical results were observed in CGCs expressing the NR2A or NR2B subunit. In summary, both neurons and astrocytes remained healthy following exposure to CEE with no signs of neurotoxicity at the cellular level, and modulation of NMDAR function is consistent with findings from prior experiments. Thus, we conclude that the CEE paradigm in glial–neuronal cocultures readily lends itself to long-term in vitro studies of ethanol effects that include glial–neuronal interactions and the ability to study ethanol withdrawal-induced neurotoxicity.

Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR‐155 and miR‐155*

Astrocytes, together with microglia and macrophages, participate in innate inflammatory responses in the CNS. Although inflammatory mediators such as interferons generated by astrocytes may be critical in the defense of the CNS, sustained unopposed cytokine signaling could result in harmful consequences. Interferon regulatory factor 3 (IRF3) is a transcription factor required for IFNβ production and antiviral immunity. Most cells express low levels of IRF3 protein, and the transcriptional mechanism that upregulates IRF3 expression is not known. In this study, we explored the consequence of adenovirus-mediated IRF3 gene transfer (Ad-IRF3) in primary human astrocytes. We show that IRF3 transgene expression suppresses proinflammatory cytokine gene expression upon challenge with IL-1/IFNγ and alters astrocyte activation phenotype from a proinflammatory to an anti-inflammatory one, akin to an M1-M2 switch in macrophages. This was accompanied by the rescue of neurons from cytokine-induced death in glial-neuronal co-cultures. Furthermore, Ad-IRF3 suppressed the expression of microRNA-155 and its star-form partner miR-155*, immunoregulatory miRNAs highly expressed in multiple sclerosis lesions. Astrocyte miR-155/miR155* were induced by cytokines and TLR ligands with a distinct hierarchy and involved in proinflammatory cytokine gene induction by targeting suppressor of cytokine signaling 1, a negative regulator of cytokine signaling and potentially other factors. Our results demonstrate a novel proinflammatory role for miR-155/miR-155* in human astrocytes and suggest that IRF3 can suppress neuroinflammation through regulating immunomodulatory miRNA expression. © 2011 Wiley-Liss, Inc.