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Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

ABSTRACT: It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of beta-amyloid protein (Abeta) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Abeta and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Abeta, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-alpha (TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Abeta-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-alpha release induced by Abeta stimulation. These results provide a direct link between Abeta-induced microglial activation and CLIC1 functional expression.

SUBMITTER: Novarino G

PROVIDER: S-EPMC6729296 | biostudies-literature | 2004 Jun

REPOSITORIES: biostudies-literature

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Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

Novarino Gaia G Fabrizi Cinzia C Tonini Raffaella R Denti Michela A MA Malchiodi-Albedi Fiorella F Lauro Giuliana M GM Sacchetti Benedetto B Paradisi Silvia S Ferroni Arnaldo A Curmi Paul M PM Breit Samuel N SN Mazzanti Michele M

The Journal of neuroscience : the official journal of the Society for Neuroscience 20040601 23

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of beta-amyloid protein (Abeta) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Abeta and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell prolife ...[more]

PMID: 15190104

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Project description:Background: Extracellular vesicles, including exosomes, are secreted by a variety of cell types in the central nervous system. Exosomes play a role in removing intracellular materials from the endosomal system. Alzheimer's disease (AD) is caused by an overproduction or reduced amyloid-beta (Aβ) peptide clearance. Increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway. Therapeutic ultrasound stimulation demonstrated its potential for promoting Aβ degradation efficiency in clinical trials. However, the underlying mechanism of ultrasound stimulation is still unclear. Methods: In this study, astrocytes, the most abundant glial cells in the brain, were used for exosome production. Post insonation, exosomes from ultrasound-stimulated HA cells (US-HA-Exo) were collected, nanoparticle tracking analysis and protein analysis were used to measure and characterize exosomes. Neuroprotective effect of US-HA-Exo in oligomeric Aβ42 toxicated SH-SY5Y cells was tested. Cellular uptake and distribution of exosomes were observed by flow cytometry and confocal laser scanning microscopy. Focused ultrasound (FUS) with microbubbles was employed for blood-brain-barrier opening to achieve brain-targeted exosome delivery. After US-HA-Exo/FUS treatment, amyloid-β plaque in APP/PS1 mice were evaluated by Aβ immunostaining and thioflavin-S staining. Results: We showed that ultrasound resulted in an almost 5-fold increase in the exosome release from human astrocytes. Exosomes were rapidly internalized in SH-SY5Y cells, and colocalized with FITC-Aβ42, causing a decreased uptake of FITC-Aβ42. CCk-8 test results showed that US-HA-Exo could mitigate Aβ toxicity to neurons in vitro. The therapeutic potential of US-HA-Exo/FUS delivery was demonstrated by a decrease in thioflavin-S-positive amyloid plaques and Aβ immuno-staining, a therapeutic target for AD in APP/PS1 transgenic mice. The iTRAQ-based proteomic quantification was performed to gain mechanistic insight into the ultrasound effect on astrocyte-derived exosomes and their ability to alleviate Aβ neurotoxicity. Conclusion: Our results imply that US-HA-Exo have the potential to provide neuroprotective effects to reverse oligomeric amyloid-β-induced cytotoxicity in vitro and, when combined with FUS-induced BBB opening, enable the clearance of amyloid-β plaques in vivo.

Project description:Small-conductance Ca2+ activated K+ channels are expressed in the CNS, where KCNN2/SK2/KCa2.2 and KCNN3/SK3/KCa2.3 help shape the electrical activity of some neurons. The SK3 channel is considered a potential therapeutic target for diseases and disorders involving neuron hyper-excitability but little is known about its expression and roles in non-neuronal cells in either the healthy or damaged CNS. The purpose of this study was to examine expression of KCNN3/SK3 in CNS microglia in vivo and in vitro, and to use an established in vitro model to determine if this channel contributes to the neurotoxic capacity of activated microglia.KCNN3 mRNA (real-time RT-PCR) and SK3 immunoreactivity were examined in rat microglia. Lipopolysaccharide was then used to activate microglia (monitored by iNOS, nitric oxide, activation of NF-kappaB and p38 MAPK) and transform them to a neurotoxic state. Microglia-mediated neuron damage (TUNEL, activated caspase 3) and nitrotyrosine levels were quantified using a two-chamber system that allowed microglia to be treated with channel blockers, washed and then added to neuron/astrocyte cultures. Contributions of SK3 to these processes were discriminated using a subtractive pharmacological approach with apamin and tamapin. ANOVA and post-hoc tests were used to assess the statistical significance of differences between treatment groups. SK3 immunoreactivity was then compared in the normal and damaged adult rat striatum, by injecting collagenase (a hemorrhagic stroke) or endothelin-1 (a transient ischemic stroke).KCNN3 mRNA was prevalent in cultured microglia and increased after lipopolysaccharide-induced activation; SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons. SK3 immunoreactivity was prevalent in cultured microglia and throughout the adult rat striatum (except white matter tracts). After strokes, SK3 was highly expressed in activated microglia/macrophages within the lesions, but reduced in other cells.SK3 is expressed in microglia in both the healthy and damaged adult striatum, and mechanistic in vitro studies show it contributes to transformation of microglia to an activated neurotoxic phenotype. Thus, SK3 might be a therapeutic target for reducing inflammation-mediated acute CNS damage. Moreover, its roles in microglia must be considered when targeting this channel for CNS diseases, disorders and reducing neuron hyper-excitability.

Project description:UnlabelledMicroglial dysfunction is increasingly recognized as a key contributor to the pathogenesis of Alzheimer's disease (AD). Environmental enrichment (EE) is well documented to enhance neuronal form and function, but almost nothing is known about whether and how it alters the brain's innate immune system. Here we found that prolonged exposure of naive wild-type mice to EE significantly altered microglial density and branching complexity in the dentate gyrus of hippocampus. In wild-type mice injected intraventricularly with soluble Aβ oligomers (oAβ) from hAPP-expressing cultured cells, EE prevented several morphological features of microglial inflammation and consistently prevented oAβ-mediated mRNA changes in multiple inflammatory genes both in vivo and in primary microglia cultured from the mice. Microdialysis in behaving mice confirmed that EE normalized increases in the extracellular levels of the key cytokines (CCL3, CCL4, TNFα) identified by the mRNA analysis. Moreover, EE prevented the changes in microglial gene expression caused by ventricular injection of oAβ extracted directly from AD cerebral cortex. We conclude that EE potently alters the form and function of microglia in a way that prevents their inflammatory response to human oAβ, suggesting that prolonged environmental enrichment could protect against AD by modulating the brain's innate immune system.Significance statementEnvironmental enrichment (EE) is a potential therapy to delay Alzheimer's disease (AD). Microglial inflammation is associated with the progression of AD, but the influence of EE on microglial inflammation is unclear. Here we systematically applied in vivo methods to show that EE alters microglia in the dentate gyrus under physiological conditions and robustly prevents microglial inflammation induced by human Aβ oligomers, as shown by neutralized microglial inflammatory morphology, mRNA changes, and brain interstitial fluid cytokine levels. Our findings suggest that EE alters the innate immune system and could serve as a therapeutic approach to AD and provide new targets for drug discovery. Further, we propose that the therapeutic benefits of EE could extend to other neurodegenerative diseases involving microglial inflammation.

Dataset Information

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

Publications

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

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