Primary Cultures Of Murine Neurons Research Articles

IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasite Toxoplasma gondii

Dogma holds that Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites, even with IFN-γ stimulation. As several recent studies questioned this idea, here we use primary murine neuronal cultures from wild type and transgenic mice in combination with IFN-γ stimulation and parental and transgenic parasites to reassess IFN-γ dependent neuronal clearance of intracellular parasites. We find that neurons respond to IFN-γ and that a subset of neurons clear intracellular parasites via immunity regulated GTPases. Whole neuron reconstructions from mice infected with parasites that trigger neuron GFP expression only after full invasion reveal that ~50% of these T. gondii-invaded neurons no longer harbor parasites. Finally, IFN-γ stimulated human pluripotent stem cell derived neurons show an ~50% decrease in parasite infection rate when compared to unstimulated cultures. This work highlights the capability of human and murine neurons to mount cytokine-dependent anti-T. gondii defense mechanisms in vitro and in vivo.

Identification of First-in-Class Inhibitors of Kallikrein-Related Peptidase 6 That Promote Oligodendrocyte Differentiation.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that causes severe motor, sensory, and cognitive impairments. Kallikrein-related peptidase (KLK)6 is the most abundant serine protease secreted in the CNS, mainly by oligodendrocytes, the myelin-producing cells of the CNS, and KLK6 is assumed to be a robust biomarker of MS, since it is highly increased in the cerebrospinal fluid (CSF) of MS patients. Here, we report the design and biological evaluation of KLK6's low-molecular-weight inhibitors, para-aminobenzyl derivatives. Interestingly, selected hit compounds were selective of the KLK6 proteolytic network encompassing KLK1 and plasmin that also participate in the development of MS physiopathology. Moreover, hits were found noncytotoxic on primary cultures of murine neurons and oligodendrocyte precursor cells (OPCs). Among them, two compounds (32 and 42) were shown to promote the differentiation of OPCs into mature oligodendrocytes in vitro constituting thus emerging leads for the development of regenerative therapies.

Metabotropic glutamate receptor 5 (mGlu5 )-positive allosteric modulators differentially induce or potentiate desensitization of mGlu5 signaling in recombinant cells and neurons.

Allosteric modulators of metabotropic glutamate receptor 5 (mGlu5 ) are a promising therapeutic strategy for a number of neurological disorders. Multiple mGlu5 -positive allosteric modulator (PAM) chemotypes have been discovered that act as either pure PAMs or as PAM-agonists in recombinant and native cells. While these compounds have been tested in paradigms of receptor activation, their effects on receptor regulatory processes are largely unknown. In this study, acute desensitization of mGlu5 mediated intracellular calcium mobilization by structurally diverse mGlu5 orthosteric and allosteric ligands was assessed in human embryonic kidney 293 cells and primary murine neuronal cultures from both striatum and cortex. We aimed to determine the intrinsic efficacy and modulatory capacity of diverse mGlu5 PAMs [(R)-5-((3-fluorophenyl)ethynyl)-N-(3-hydroxy-3-methylbutan-2-yl)picolinamide (VU0424465), N-cyclobutyl-6-((3-fluorophenyl)ethynyl)picolinamide (VU0360172), 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone(DPFE), ((4-fluorophenyl) (2-(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)methanone) (VU0409551), 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide(CDPPB)] on receptor desensitization and whether cellular context influences receptor regulatory processes. Only VU0424465 and VU0409551 induced desensitization alone in human embryonic kidney 293-mGlu5 cells, while all PAMs enhanced (S)-3,5-dihydroxyphenylglycine (DHPG)-induced desensitization. All mGlu5 PAMs induced receptor desensitization alone and enhanced DHPG-induced desensitization in striatal neurons. VU0424465 and VU0360172 were the only PAMs that induced desensitization alone in cortical neurons. With the exception of (CDPPB), PAMs enhanced DHPG-induced desensitization in cortical neurons. Moreover, differential apparent affinities, efficacies, and cooperativities with DHPG were observed for VU0360172, VU0409551, and VU0424465 when comparing receptor activation and desensitization in a cell type-dependent manner. These data indicate that biased mGlu5 allosteric modulator pharmacology extends to receptor regulatory processes in a tissue dependent manner, adding yet another layer of complexity to rational mGlu5 drug discovery.

Primary murine neurons as in vitro model for studying neuroinfections caused by human adenoviruses.

Adenoviral infections of the central nervous system are rare, but they are characteristic for their high mortality rate. People with impaired immunity and children are particularly vulnerable. A few reports of neuroinfections caused by adenoviruses are found in literature. In this study the tropism of the human adenoviruses type 4, 5, 7 to primary cultures of murine neurons and the influence of infection on the neuron morphology and actin cytoskeleton was examined. The A549 non-small-cell lung cancer cell line was used as a reference line. Viral effects upon the cell culture were evaluated by direct immunofluorescence. The levels of adenovirus replication in the above-mentioned cell cultures were determined by real-time PCR. In the current study we demonstrated for the first time that human adenovirus (HAdV) type 4, 5 and 7 exhibits tropism for neurons cultured in vitro followed by the extensive replication of all serotypes in the primary culture of murine neurons. Immunofluorescent labelling and confocal microscopy revealed the changes in cell morphology, destruction of actin cytoskeleton and cell lysis as the final stage of infection. According to the obtained results we can assume that productive cycle of HAdV in the studied cell cultures occurred. We also observed accumulation of nuclear actin within nuclei of infected cells which may indicate that it plays role in adenovirus infection and replication in neurons and A549 cells.

GPR40 activation leads to CREB and ERK phosphorylation in primary cultures of neurons from the mouse CNS and in human neuroblastoma cells

GPR40, the free fatty acid receptor 1, is expressed strongly in the primate pancreas and brain. While the role of pancreatic GPR40 in glucose homeostasis has been extensively studied, the absence of this G-protein-coupled receptor from the brain of rodents has hampered studies into its role in the central nervous system. However, we found intense GPR40 mRNA expression by in situ hybridization in mouse hippocampal and motor cortex neurons. Furthermore, in a neuroblastoma cell GPR40 was activated by docosahexaenoic acid and selective agonists, yet not by palmitic acid. Significantly, the activation of GPR40 provoked the phosphorylation of the cAMP response element-binding protein, CREB. The receptor was also functional in primary cultures of murine neurons, in which its activation by a selective agonist produced the phosphorylation of CREB and of extracellular signal-regulated kinases, ERK1/2. These results suggest that mice represent a suitable model for elucidating the role of GPR40 in brain function.

Neuronal MHC-I display in T-cell mediated neurodegeneration

Parkinson’s disease (PD) and other disorders feature the degeneration of ventral midbrain (VM) catecholamine neurons. Recent data suggest that neuroinflammatory mechanisms contribute to a cascade of events leading to chronic neuronal degeneration. In primary murine neuronal cultures, substantia nigra (SN) and locus coeruleus (LC) neurons are induced to express the major histocompatibility class I complex (MHC-I) by the proinflammatory cytokine, γ-interferon, L-DOPA, or conditioned medium from microglia exposed to α-synuclein or NM. SN DA neurons, moreover, process the foreign protein ovalbumin to an antigenic peptide that is presented by their MHC-I and triggers their specific destruction by CD8+ killer T-cells. In human postmortem samples, we find by immunolabel, mRNA profiling, and proteomic analysis that neuromelanin (NM)-containing catecholamine SN and LC neurons in adult human control and PD brains express MHC-I, often in proximity to CD8+ T-cells. These data reveal a novel inflammatory T-cell mediated neurodegenerative processes that could underlie neuronal death.

Hemopexin decreases hemin accumulation and catabolism by neural cells

Hemopexin is a serum, CSF, and neuronal protein that is protective after experimental stroke. Its efficacy in the latter has been linked to increased expression and activity of heme oxygenase (HO)-1, suggesting that it facilitates heme degradation and subsequent release of cytoprotective biliverdin and carbon monoxide. In this study, the effect of hemopexin on the rate of hemin breakdown by CNS cells was investigated in established in vitro models. Equimolar hemopexin decreased hemin breakdown, as assessed by gas chromatography, by 60–75% in primary cultures of murine neurons and glia. Extracellular hemopexin reduced cell accumulation of 55Fe-hemin by over 90%, while increasing hemin export or extraction from membranes by fourfold. This was associated with significant reduction in HO-1 expression and neuroprotection. In a cell-free system, hemin breakdown by recombinant HO-1 was reduced over 80% by hemopexin; in contrast, albumin and two other heme-binding proteins had no effect. Although hemopexin was detected on immunoblots of cortical lysates from adult mice, hemopexin knockout per se did not alter HO activity in cortical cells treated with hemin. These results demonstrate that hemopexin decreases the accumulation and catabolism of exogenous hemin by neural cells. Its beneficial effect in stroke models is unlikely to be mediated by increased production of cytoprotective heme breakdown products.

Equine herpesvirus type 1 (EHV-1) replication in primary murine neurons culture

Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Bańbura et al. (Bańbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.

Role for Metallothioneins-I/II in Isoflurane Preconditioning of Primary Murine Neuronal Cultures

Pretreatment with inhaled anesthetics, including isoflurane, can induce long-lasting cellular protection against ischemia-derived toxicity in multiple tissues, including brain tissue. Metal-regulatory genes, metallothioneins-I/II (MT-I/II), have been shown to protect against oxidative damage in multiple tissues. Furthermore, MT have been found to be differentially regulated in response to isoflurane and ischemic preconditioning. In this study, we assess the role of MT-I/II in mediating isoflurane preconditioning in primary neuronal-glial cultures. Primary mouse neuronal-glial cultures were preconditioned with isoflurane (3 h, 1.5%) 24-96 h before 3-h oxygen-glucose deprivation (OGD, ischemic model). After OGD, isoflurane protection and responsiveness of MT-I/II knockdown and knockout cultures to preconditioning were assessed by lactate dehydrogenase release. Immunoassays for microtubule associated protein 2 and glial fibrillary acidic protein determined neuronal-glial sensitivity to preconditioning. MT-I/II messenger RNA was assessed by quantitative reverse transcriptase-polymerase chain reaction. Cultures transfected with exogenous MT-I/II were analyzed for protection against OGD toxicity. Isoflurane preconditioning reduced OGD-mediated toxicity by 11.6 +/- 7.9% at 24 h, with protection increasing to 37.5 +/- 2.5% at 72 h after preconditioning. Immunolabeling showed that neurons were more sensitive to OGD and more responsive to isoflurane preconditioning compared to glia. Quantitative reverse transcriptase-polymerase chain reaction showed MT-I/II messenger RNA were upregulated (approximately 2.5-fold) by isoflurane treatments. Also MT-I/II protein transfection significantly decreased OGD-mediated toxicity. Finally, knockdown and knockout of MT-I/II diminished and abolished isoflurane-mediated protection, respectively. MT-I/II play an important role in isoflurane-mediated delayed preconditioning against OGD toxicity of neuronal and glial cells in vitro.

Hypoxic injury of isolated axons is independent of ionotropic glutamate receptors

Axonal injury in white matter is an important consequence of many acute neurological diseases including ischemia. A role for glutamate-mediated excitotoxicity is suggested by observations from in vitro and in situ models that AMPA/kainate blockers can reduce axonal injury. We assessed axonal vulnerability in primary murine neuronal cultures, with axons isolated from their cell bodies using a compartmented chamber design. Transient removal of oxygen and glucose in the axon compartment resulted in irreversible loss of axon length and neurofilament labeling. This injury was not prevented by addition of ionotropic glutamate receptor blockers and could not be reproduced by glutamate receptor agonists. However, hypoxic injury was prevented by blockade of voltage-gated sodium channels, inhibition of calpain and removal of extracellular calcium. These results suggest that isolated, unmyelinated axons are vulnerable to hypoxic injury which is mediated by influx of sodium and calcium but is independent of glutamate receptor activation.

Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly- l-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved.

Dexmedetomidine, a potent and highly specific alpha 2 agonist, evokes cytosolic calcium surge in astrocytes but not in neurons

Dexmedetomidine is an extremely potent alpha 2 adrenoceptor agonist which can reduce anaesthetic requirements by up to 90%. However, the precise cellular mechanism of action of this agent is not known. Primary cultures of murine neurons and astrocytes were grown to test the hypothesis that changes in free intracellular calcium may trigger cellular responses to this drug. In astrocyte cultures, experiments revealed a pronounced increase in cytosolic calcium concentration when 100 nM dexmedetomidine was administered. However, in cultured cerebellar granule cell neurons there was no calcium response observed with similar or even higher concentrations of the drug. Results suggest dexmedetomidine may exert its primary effect on the central nervous system via astrocytes.