Blockade of chloride intracellular ion channel 1 stimulates Abeta phagocytosis

In amyloid-\u3b2 (A\u3b2)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-\u3b1 and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce A\u3b2 phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on A\u3b2-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, A\u3b225-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of A\u3b2 phagocytosis, we treated BV-2 cells with biotinylated A\u3b21-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased A\u3b2 phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested A\u3b21-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed A\u3b21-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (iNOS), stimulated by A\u3b2, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates A\u3b2 phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD

Comparison of different methods to extract RNA from cardiac tissue for miRNA profiling by qRT-PCR

Despite the growing interest in cardiac miRNA expression profiling, having high quality and yield in RNA extraction from cardiac tissue is still challenging. We compared different methods of tissue homogenization and total RNA extraction from pig cardiac tissue aimed at miRNAs expression profiling. Small biopsies of right atrial appendages were obtained from pig hearts and treated according to four different protocols: no homogenization (P1) and homogenization by manual (P2) or automatic (P3 and P4) methods, followed by Proteinase K digestion (PKD) except in P4. Total RNA was extracted using miRNeasy mini kit, assessing RNA yield and quality by Nanodrop. cDNA synthesis and qRT-PCR were performed using TaqMan MicroRNA Assay. Homogenization was crucial to obtain high yield of pure total RNA. Automatic methods displayed higher yield (0.27 μg RNA/mg tissue in P3) than manual (0.06 μg RNA/mg tissue in P2), with better performance without PKD step (0.38μg RNA/mg tissue in P4). RNA from P4 was suitable for miRNA expression profiling, as demonstrated by qRT-PCR on miRNA 21 and 29. These results suggest the efficacy of an automatic homogenization to extract RNA suitable for miRNA expression profiling

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

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 \u3b2-amyloid protein (A\u3b2) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although A\u3b2 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 A\u3b2 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 A\u3b2, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-\u3b1 (TNF-\u3b1). 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 A\u3b2-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-\u3b1 release induced by A\u3b2 stimulation. These results provide a direct link between A\u3b2-induced microglial activation and CLIC1 functional expression

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

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 β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ 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 Aβ 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 Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). 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 Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression

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

It is widely believed that the inflammatory events mediated by microglialactivation contribute to several neurodegenerative processes. Alzheimer'sdisease, for example, is characterized by an accumulation of beta-amyloidprotein (Abeta) in neuritic plaques that are infiltrated by reactive microgliaand astrocytes. Although Abeta and its fragment 25-35 exert a direct toxiceffect on neurons, they also activate microglia. Microglial activation isaccompanied by morphological changes, cell proliferation, and release of variouscytokines and growth factors. A number of scientific reports suggest that theincreased proliferation of microglial cells is dependent on ionic membranecurrents and in particular on chloride conductances. An unusual chloride ionchannel known to be associated with macrophage activation is the chlorideintracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatalrat microglia specifically leads to the increase in CLIC1 protein and to thefunctional expression of CLIC1 chloride conductance, both barely detectable onthe plasma membrane of quiescent cells. CLIC1 protein expression in microgliaincreases after 24 hr of incubation with Abeta, simultaneously with theproduction of reactive nitrogen intermediates and of tumor necrosis factor-alpha(TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by aspecific 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 withAbeta-treated microglia. Furthermore, we show that small interfering RNAs usedto knock down CLIC1 expression prevent TNF-alpha release induced by Abetastimulation. These results provide a direct link between Abeta-inducedmicroglial activation and CLIC1 functional expression

miR-205 Expression levels in nonsmall cell lung cancer do not always distinguish adenocarcinomas from squamous cell carcinomas

Accurate classification of nonsmall cell lung cancers is of paramount clinical relevance, as novel chemotherapeutic agents show different efficacy in adenocarcinomas (ADCs) compared with squamous cell carcinomas (SQCCs). Cyto and histomorphology may sometimes be insufficient for this distinction and immunohistochemistry may improve diagnostic accuracy. The measurement of miR-205 may be another tool for the distinction between ADC and SQCC. The aim of our study was to compare morphologic and immunohistochemical classification with the relative quantification of miR-205 and miR-21 in surgically resected and well-characterized lung tumors (25 ADCs, 24 SQCCs, 1 adenosquamous). The miR-21 relative levels were similar in SQCC and ADC, whereas the miR-205 relative levels were lower in ADC (P < 0.0001). The miR-205 sample score value, determined according to Lebanony et al, was higher in ADC (range, 2.8 to 9.08) compared with SQCC (range, -4.17 to 2.445) (P < 0.0001). Accordingly, 22 tumors were classified as ADC and 28 tumors as SQCC, although 8 cases (2 SQCCs and 6 ADCs) were in the range of "near cutoff values." Four cases classified as SQCC (according to the sample score method) corresponded to cases classified as ADC on the basis of morphoimmunohistochemical evaluation. In conclusion, the relative quantification of miR-205 and miR-21 seems to be a promising diagnostic tool. However, the molecular approach is still not completely satisfactory as it may misclassify a non-negligible percentage of cases. Therefore, it cannot be used as a substitute of accurate morphologic and immunophenotypical characterization of tumors, but could be used as an adjunctive diagnostic criterion in selected cases