Minocycline and Risperidone Prevent Microglia Activation and Rescue Behavioral Deficits Induced by Neonatal Intrahippocampal Injection of Lipopolysaccharide in Rats

Background

Various signs of activation of microglia have been reported in schizophrenia, and it is hypothesized that microglia activation is closely associated with the neuropathology of schizophrenia.

Methods

Neonatal intrahippocampal injection of lipopolysaccharide (LPS), an activator of microglia, was performed in rats at postnatal day 7 (P7), and they were separately given saline, risperidone (0.5 mg/kg), minocycline (40 mg/kg) or a combination of both of them at P42 for consecutive 14 days. Behavioral changes (locomotion activity, social interaction, novel object recognition and prepulse inhibition) were examined and the number of microglia was assessed by using immunohistochemistry in adulthood.

Results

The adult rats in LPS-injected group showed obvious behavioral alteration (e. g. deficits in social interaction, novel object recognition and prepulse inhibition) and a dramatic increase of number of activated microglial cells in the hippocampus and other brain regions such as cerebral cortex and thalamus compared to those in saline-injected group. Interestingly, application of either minocycline, risperidone or both of them significantly rescued behavioral deficits and attenuated microglia activation.

Conclusion

Our results suggest that inhibition of microglia activation may be one of mechanisms underlying the antipsychotic effect of minocycline and risperidone.     

Regulation of progranulin expression in human microglia and proteolysis of progranulin by matrix metalloproteinase-12 (MMP-12)

Background

The essential role of progranulin (PGRN) as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia.

Goal

In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12), as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI), in human CNS cells.

Methods

Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC), Th1 cytokines (IL-1/IFNγ), or Th2 cytokines (IL-4, IL-13). Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined.

Results

Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNγ, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation, with its effect on TNFα being the most conspicuous.

Conclusions

Our study is the first detailed examination of PGRN in human microglia. Our results establish microglia as a significant source of PGRN, and MMP-12 and SLPI as modulators of PGRN proteolysis. Negative and positive regulation of microglial PGRN release by the proinflammatory/Th1 and the Th2 stimuli, respectively, suggests a fundamentally different aspect of PGRN regulation compared to other known microglial activation products. Microglial PGRN appears to function as an endogenous modulator of innate immune responses.     

Prenatal activation of microglia induces delayed impairment of glutamatergic synaptic function

Background

Epidemiological studies have linked maternal infection during pregnancy to later development of neuropsychiatric disorders in the offspring. In mice, experimental inflammation during embryonic development impairs behavioral and cognitive performances in adulthood. Synaptic dysfunctions may be at the origin of cognitive impairments, however the link between prenatal inflammation and synaptic defects remains to be established.

Methodology/Principal Findings

In this study, we show that prenatal alteration of microglial function, including inflammation, induces delayed synaptic dysfunction in the adult. DAP12 is a microglial signaling protein expressed around birth, mutations of which in the human induces the Nasu-Hakola disease, characterized by early dementia. We presently report that synaptic excitatory currents in mice bearing a loss-of-function mutation in the DAP12 gene (DAP12^KI mice) display enhanced relative contribution of AMPA. Furthermore, neurons from DAP12^KI P0 pups cultured without microglia develop similar synaptic alterations, suggesting that a prenatal dysfunction of microglia may impact synaptic function in the adult. As we observed that DAP12^KI microglia overexpress genes for IL1β, IL6 and NOS2, which are inflammatory proteins, we analyzed the impact of a pharmacologically-induced prenatal inflammation on synaptic function. Maternal injection of lipopolysaccharides induced activation of microglia at birth and alteration of glutamatergic synapses in the adult offspring. Finally, neurons cultured from neonates born to inflamed mothers and cultured without microglia also displayed altered neuronal activity.

Conclusion/Significance

Our results demonstrate that prenatal inflammation is sufficient to induce synaptic alterations with delay. We propose that these alterations triggered by prenatal activation of microglia provide a cellular basis for the neuropsychiatric defects induced by prenatal inflammation.     

Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

Rod and Cone Function in Patients with KCNV2 Retinopathy

Background

To investigate rod and cone function and disease mechanisms in patients with KCNV2 retinopathy.

Methodology/Principal Findings

Psychophysical examinations as well as detailed electrophysiological examinations with Ganzfeld and multifocal electroretinogram (ERG) were performed to study response dynamics. Additionally, fundus photography, autofluorescence imaging and spectral domain OCTs were carried out for morphological characterization. Molecular genetic analysis revealed compound heterozygosity in five patients and homozygosity for the KCNV2 gene in one patient. The mutations resulted in complete absence of Kv8.2 subunits in three patients (no protein group, NOP), while the other three patients expressed mutant Kv8.2 subunits resulting in altered Kv2.1/Kv8.2 heteromeric or residual Kv2.1 homomeric potassium channel function (altered protein group, ALP). Although more advanced morphological changes were visible in the NOP group, a clear functional difference between the two groups could not be observed. All patients showed characteristic dynamics of the b-wave intensity-response function, however, scotopic b-wave response amplitudes were within normal limits. We also observed severely reduced oscillatory potentials.

Conclusions/Significance

A specific genotype-phenotype correlation in retinal function could not be demonstrated. KCNV2 mutations cause a unique form of retinal disorder illustrating the importance of K⁺-channels for the resting potential, activation and deactivation of photoreceptors, while phototransduction remains unchanged. The reduced oscillatory potentials further suggest an altered function of the inner retina. Besides the characteristically steep amplitude-versus-intensity relationship, flicker responses at intermediate frequencies (5–15 Hz) are significantly reduced and shifted in phase.     

Microglial morphology and dynamic behavior is regulated by ionotropic glutamatergic and GABAergic neurotransmission

Purpose

Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior.

Methods

We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia.

Results

Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels.

Conclusions

Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity.     

The Acrylamide (S)-2 As a Positive and Negative Modulator of Kv7 Channels Expressed in Xenopus laevis Oocytes

Background

Activation of voltage-gated potassium channels of the Kv7 (KCNQ) family reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. Retigabine, which opens Kv7.2-5, is now in clinical trial phase III for the treatment of partial onset seizures. One of the main obstacles in developing Kv7 channel active drugs has been to identify compounds that can discriminate between the neuronal subtypes, a feature that could help diminish side effects and increase the potential of drugs for particular indications.

Methodology/Principal Findings

In the present study we have made a thorough investigation of the Bristol-Myers Squibb compound (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1], [4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide [(S)-2] on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. We found that the compound was a weak inhibitor of Kv7.1. In contrast, (S)-2 efficiently opened Kv7.2-5 by producing hyperpolarizing shifts in the voltage-dependence of activation and enhancing the maximal current amplitude. Further, it reduced inactivation, accelerated activation kinetics and slowed deactivation kinetics. The mechanisms of action varied between the subtypes. The enhancing effects of (S)-2 were critically dependent on a tryptophan residue in S5 also known to be crucial for the effects of retigabine, (S)-1 and BMS-204352. However, while (S)-2 did not at all affect a mutant Kv7.4 with a leucine in this position (Kv7.4-W242L), a Kv7.2 with the same mutation (Kv7.2-W236L) was inhibited by the compound, showing that (S)-2 displays a subtype-selective interaction with in the Kv7 family.

Conclusions/Significance

These results offer further insight into pharmacological activation of Kv7 channels, add to the understanding of small molecule interactions with the channels and may contribute to the design of subtype selective modulators.     

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Background

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer''s disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Methods and Findings

In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer''s disease mouse model.

Conclusions

Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.     

Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K⁺ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.     

Amyloid β peptide-mediated neurotoxicity is attenuated by the proliferating microglia more potently than by the quiescent phenotype

Background

The specific role of microglia on Aβ-mediated neurotoxicity is difficult to assign in vivo due to their complicated environment in the brain. Therefore, most of the current microglia-related studies employed the isolated microglia. However, the previous in vitro studies have suggested either beneficial or destructive function in microglia. Therefore, to investigate the phenotypes of the isolated microglia which exert activity of neuroprotective or destructive is required.

Results

The present study investigates the phenotypes of isolated microglia on protecting neuron against Aβ-mediated neurotoxicity. Primary microglia were isolated from the mixed glia culture, and were further cultured to distinct phenotypes, designated as proliferating amoeboid microglia (PAM) and differentiated process-bearing microglia (DPM). Their inflammatory phenotypes, response to amyloid β (Aβ), and the beneficial or destructive effects on neurons were investigated. DPM may induce both direct neurotoxicity without exogenous stimulation and indirect neurotoxicity after Aβ activation. On the other hand, PAM attenuates Aβ-mediated neurotoxicity through Aβ phagocytosis and/or Aβ degradation.

Conclusions

Our results suggest that the proliferating microglia, but not the differentiated microglia, protect neurons against Aβ-mediated neurotoxicity. This discovery may be helpful on the therapeutic investigation of Alzheimer’s disease.     

Cudrania cochinchinensis attenuates amyloid β protein-mediated microglial activation and promotes glia-related clearance of amyloid β protein

Background

Microglial inflammation may significantly contribute to the pathology of Alzheimer’s disease. To examine the potential of Cudrania cochinchinensis to ameliorate amyloid β protein (Aβ)-induced microglia activation, BV-2 microglial cell line, and the ramified microglia in the primary glial mixed cultured were employed.

Results

Lipopolysaccharide (LPS), Interferon-γ (IFN-γ), fibrillary Aβ (fAβ), or oligomeric Aβ (oAβ) were used to activate microglia. LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ. On the other hand, oAβ effectively activated the ramified microglia in mixed glial culture by observing the morphological alteration of the microglia from ramified to amoeboid. CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia. Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance. Results are expressed as mean ± S.D. and were analyzed by ANOVA with post-hoc multiple comparisons with a Bonferroni test.

Conclusions

The components of Cudrania cochinchinensis including CC-EW and CCB are potential for novel therapeutic intervention for Alzheimer’s disease.     

A two-way communication between microglial cells and angiogenic sprouts regulates angiogenesis in aortic ring cultures

Background

Myeloid cells have been associated with physiological and pathological angiogenesis, but their exact functions in these processes remain poorly defined. Monocyte-derived tissue macrophages of the CNS, or microglial cells, invade the mammalian retina before it becomes vascularized. Recent studies correlate the presence of microglia in the developing CNS with vascular network formation, but it is not clear whether the effect is directly caused by microglia and their contact with the endothelium.

Methodology/Principal Findings

We combined in vivo studies of the developing mouse retina with in vitro studies using the aortic ring model to address the role of microglia in developmental angiogenesis. Our in vivo analyses are consistent with previous findings that microglia are present at sites of endothelial tip-cell anastomosis, and genetic ablation of microglia caused a sparser vascular network associated with reduced number of filopodia-bearing sprouts. Addition of microglia in the aortic ring model was sufficient to stimulate vessel sprouting. The effect was independent of physical contact between microglia and endothelial cells, and could be partly mimicked using microglial cell-conditioned medium. Addition of VEGF-A promoted angiogenic sprouts of different morphology in comparison with the microglial cells, and inhibition of VEGF-A did not affect the microglia-induced angiogenic response, arguing that the proangiogenic factor(s) released by microglia is distinct from VEGF-A. Finally, microglia exhibited oriented migration towards the vessels in the aortic ring cultures.

Conclusions/Significance

Microglia stimulate vessel sprouting in the aortic ring cultures via a soluble microglial-derived product(s), rather than direct contact with endothelial cells. The observed migration of microglia towards the growing sprouts suggests that their position near endothelial tip-cells could result from attractive cues secreted by the vessels. Our data reveals a two-way communication between microglia and vessels that depends on soluble factors and should extend the understanding of how microglia promote vascular network formation.     

Prostaglandin E2 reverses aberrant production of an inflammatory chemokine by microglia from Sandhoff disease model mice through the cAMP-PKA pathway

Background

Sandhoff disease (SD) is a neurodegenerative lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including terminal GlcNAc-oligosaccharides and GM2 ganglioside. Microglia-mediated neuroinflammation contributes to the pathogenesis and progression of SD. Our previous study demonstrated that MIP-1α, a putative pathogenic factor for SD, is up-regulated in microglial cells derived from SD model mice (SD-Mg) through activation of Akt and JNK.

Methodology/Principal Findings

In this study, we first demonstrated that prostaglandin E2 (PGE2), which is one of the lipid mediators derived from arachidonic acid and is known to suppress activation of microglia, reduced the aberrant MIP-1α production by SD-Mg to the same level as by WT-Mg. PGE2 also attenuated the activation of Akt and JNK. The inhibition of MIP-1α production and the activation of Akt and JNK occurred through the EP2 and 4/cAMP/PKA signaling pathway in the murine microglia derived from SD model mice.

Conclusions/Significance

We propose that PGE2 plays a role as a negative regulator of MIP-1α production in the pathogenesis of SD, and that PGE2-EP2 and 4/cAMP/PKA signaling could be a target pathway for therapy for SD.     

Chronic apocynin treatment attenuates beta amyloid plaque size and microglial number in hAPP(751)(SL) mice

Background

NADPH oxidase is implicated in neurotoxic microglial activation and the progressive nature of Alzheimer''s Disease (AD). Here, we test the ability of two NADPH oxidase inhibitors, apocynin and dextromethorphan (DM), to reduce learning deficits and neuropathology in transgenic mice overexpressing human amyloid precursor protein with the Swedish and London mutations (hAPP(751)_SL).

Methods

Four month old hAPP(751)_SL mice were treated daily with saline, 15 mg/kg DM, 7.5 mg/kg DM, or 10 mg/kg apocynin by gavage for four months.

Results

Only hAPP(751)_SL mice treated with apocynin showed reduced plaque size and a reduction in the number of cortical microglia, when compared to the saline treated group. Analysis of whole brain homogenates from all treatments tested (saline, DM, and apocynin) demonstrated low levels of TNFα, protein nitration, lipid peroxidation, and NADPH oxidase activation, indicating a low level of neuroinflammation and oxidative stress in hAPP(751)_SL mice at 8 months of age that was not significantly affected by any drug treatment. Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751)_SL mice. To discern how apocynin was affecting plaque levels (plaque load) and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ) phagocytosis, microglial proliferation, or microglial survival.

Conclusions

Together, this study suggests that while hAPP(751)_SL mice show increases in microglial number and plaque load, they fail to exhibit elevated markers of neuroinflammation consistent with AD at 8 months of age, which may be a limitation of this animal model. Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional therapeutic effects independent of anti-inflammatory characteristics.     

NLRP3 inflammasome: key mediator of neuroinflammation in murine Japanese encephalitis

Background

Japanese Encephalitis virus (JEV) is a common cause of acute and epidemic viral encephalitis. JEV infection is associated with microglial activation resulting in the production of pro-inflammatory cytokines including Interleukin-1 β (IL-1β) and Interleukin-18 (IL-18). The Pattern Recognition Receptors (PRRs) and the underlying mechanism by which microglia identify the viral particle leading to the production of these cytokines is unknown.

Methodology/Principal Findings

For our studies, we have used murine model of JEV infection as well as BV-2 mouse microglia cell line. In this study, we have identified a signalling pathway which leads to the activation of caspase-1 as the key enzyme responsible for the maturation of both IL-1β and IL-18 in NACHT, LRR and PYD domains-containing protein-3 (NLRP3) dependent manner. Depletion of NLRP3 results in the reduction of caspase-1 activity and subsequent production of these cytokines.

Conclusion/Significance

Our results identify a mechanism mediated by Reactive Oxygen Species (ROS) production and potassium efflux as the two danger signals that link JEV infection to caspase-1 activation resulting in subsequent IL-1β and IL-18 maturation.     

Hepatitis C Virus Core Protein Induces Neuroimmune Activation and Potentiates Human Immunodeficiency Virus-1 Neurotoxicity

Background

Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection.

Methodology

Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed.

Principal Findings

HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05).

Conclusions

HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection.     

Long-chain acylcarnitines regulate the HERG channel

Background and purpose

In some pathological conditions carnitine concentration is high while in othersitis low.In bothcases,cardiac arrhythmiascan occur and lead to sudden cardiac death. It has been proposed that in ischaemia, acylcarnitine (acyl-CAR), but not carnitine, is involved in arrhythmiasthrough modulation of ionic currents. We studied the effects of acyl-CARs on hERG, K_IR2.1 and K_v7.1/minKchannels (channels responsible for I_KR, I_K1 and I_KS respectively).

Experimental approach

HEK293 cells stably expressing hERG, K_IR2.1 or Kv7.1/minK were studied using the patch clamp technique. Free carnitine (CAR) and acyl-CAR derivatives from medium- (C8 and C10) and long-chain (C16 and C18∶1) fatty acids were applied intra- and extracellularly at different concentrations. Forstudies onhERG, C16 and C18∶1 free fatty acid were also used.

Key results

Extracellular long-chain (LCAC), but not medium-chain, acyl-CAR,induced an increase of I_hERG amplitude associated with a dose-dependent speeding of deactivation kinetics. They had no effect on K_IR2.1 or Kv7.1/minK currents.Computer simulations of these effects wereconsistent with changes in action potential profile.

Conclusions and applications

Extracellular LCAC tonically regulates I_hERG amplitude and kineticsunder physiological conditions. This modulation maycontribute tothe changes in action potential duration thatprecede cardiac arrhythmias in ischaemia, diabetes and primary systemic carnitine deficiency.