Cinzia Fabrizi

Activation of microglial cells by PrP and β-amyloid fragments raises intracellular calcium through L-type voltage sensitive calcium channels

The prion protein (PrP) and the amyloid beta (Abeta) precursor protein (APP) are two normal proteins constitutively synthesised in human brain. An altered form of PrP accumulates in Creutzfeldt-Jakob disease, while Abeta is involved in the pathogenesis of Alzheimers disease. Synthetic fragments of both proteins, PrP106-126 and beta25-35 (beta25-35), have been demonstrated to induce neurodegeneration and microglia activation. This study was undertaken to compare PrP106-126 and beta25-35 capability of activating human resting microglial cells. Our results show that both peptides are able to induce microglial activation and to elicit an increase in [Ca2+]i levels in cells loaded with calcium-green 1. Inhibitors of L-type voltage-sensitive calcium channels (verapamil, nifedipine and diltiazem) prevented the increase in [Ca2+]i concentration as observed after treatment with PrP106-126 and beta25-35, thus indicating a transmembrane calcium influx through these channels. In addition, verapamil abolished the proliferative effect of both PrP106-126 and beta25-35.

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. Alzheimers 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.

S100B protects LAN‐5 neuroblastoma cells against Aβ amyloid‐induced neurotoxicity via RAGE engagement at low doses but increases Aβ amyloid neurotoxicity at high doses

At the concentrations normally found in the brain extracellular space the glial‐derived protein, S100B, protects neurons against neurotoxic agents by interacting with the receptor for advanced glycation end products (RAGE). It is known that at relatively high concentrations S100B is neurotoxic causing neuronal death via excessive stimulation of RAGE. S100B is detected within senile plaques in Alzheimers disease, where its role is unknown. The present study was undertaken to evaluate a putative neuroprotective role of S100B against Aβ amyloid‐induced neurotoxicity. We treated LAN‐5 neuroblastoma cultures with toxic amounts of Aβ25‐35 amyloid peptide. Our results show that at nanomolar concentrations S100B protects cells against Aβ‐mediated cytotoxicity, as assessed by 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase‐mediated dUTP‐fluorescein isothiocyanate nick end‐labeling (TUNEL) experiments, by countering the Aβ‐mediated decrease in the expression of the anti‐apoptotic factor Bcl‐2. This effect depends on S100B binding to RAGE because S100B is unable to contrast Aβ‐mediated neurotoxicity in neurons overexpressing a signaling‐deficient RAGE mutant lacking the cytosolic and transducing domain. Our data suggest that at nanomolar doses S100B counteracts Aβ peptide neurotoxicity in a RAGE‐mediated manner. However, at micromolar doses S100B is toxic to LAN‐5 cells and its toxicity adds to that of the Aβ peptide, suggesting that additional molecular mechanisms may be involved in the neurotoxic process.

Expression of phosphoinositide‐specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide

Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide‐specific phospholipase C (PI‐PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI‐PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI‐PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL‐6. PI‐PLCs expression in LPS treated neonatal rat astrocytes performed by using RT‐PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI‐PLC beta1, beta4 and gamma1 in all intervals analysed; PI‐PLC delta1 at 6, 18 and 24 h; PI‐PLC delta3 at 6 h after treatment. PI‐PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub‐cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI‐PLCs expression and distribution may play a role in ongoing inflammation process of CNS. J. Cell. Biochem. 109: 1006–1012, 2010.

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes.

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT‐PCR and immunocytochemistry experiments. While in both cell models the most represented PI‐PLC isoforms were β4, γ1, δ4, and ε, isoforms PI‐PLC β2 and δ3 were not detected. Moreover, in primary astrocyte cultures PI‐PLC δ3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms β3 and β4) resulted mainly nuclear, others (isoforms δ4 and ε) were preferentially localized at cytoplasmic and plasma membrane level. J. Cell. Biochem. 100: 952–959, 2007.

Blockade of chloride intracellular ion channel 1 stimulates Aβ phagocytosis

In amyloid‐β (Aβ)‐stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor‐α and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimers disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce Aβ phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on Aβ‐stimulated mononuclear phagocytosis. In the microglial cell line BV‐2, Aβ25–35 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 Aβ phagocytosis, we treated BV‐2 cells with biotinylated Aβ1–42 and measured intracellular amyloid by morphometric analysis. IAA‐94‐treated cells showed an increased Aβ phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested Aβ1–42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed Aβ1–42 in the presence of IAA‐94. However, the increased expression of inducible NO synthase (iNOS), stimulated by Aβ, was reverted by IAA‐94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates Aβ phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD.

Partial biochemical characterization of nitric oxide synthase in the caudal spinal cord of the teleost Oreochromis niloticus

The present study demonstrates that a NADPH/Ca2+-dependent nitric oxide synthase (NOS) activity is present in the soluble and in the particulate fractions of fish caudal spinal cord homogenates, both activities being inhibited by calmodulin inhibitors (W7 and/or TFP) and by the NOS inhibitor L-NAME. Moreover, Western blot analysis using either anti-NOS I or anti-NOS III antibodies shows that the soluble enzyme corresponds to the brain NOS (NOS-I) of mammals, whereas the particulate one is likely attributable to NOS I and/or NOS III (ecNOS) enzymes. To confirm the nitric oxide (NO) production by the caudal spinal cord homogenates, the NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored spectroscopically. The present results are consistent with a constitutive, Ca2+-calmodulin-dependent, NO production by the caudal neurosecretory system.

Interferon gamma up-regulates α2 macroglobulin expression in human astrocytoma cells

An established human astrocytoma cell line (T67) was shown to constitutively produce the proteinase inhibitor alpha 2 macroglobulin (alpha 2M). Interferon gamma (IFN gamma), a potent immunoregulatory lymphokine, was able to increase the synthesis of alpha 2M by these cells, as measured by ELISA on cell supernatants. The alpha 2M induction was also observed in other human glioma cell lines (T70 and ADF) and in human fetal astrocyte cultures following IFN gamma treatment. In T67 cells this effect was dose-dependent and the maximum (2.7-fold increase) was obtained with 2000 U/ml of IFN gamma. A corresponding enhanced alpha 2M mRNA accumulation was demonstrated by PCR and Northern blot techniques. Our results suggest an important role of alpha 2M during inflammatory and immune processes in the CNS. An increased release of alpha 2M following IFN gamma stimulation may allow the removal of the bulk of proteases released at the site of inflammation, strengthening at the same time the antigen presentation processes.

Protease-Activated Receptor–1 Expression in Rat Microglia after Trimethyltin Treatment

In the nervous system, protease-activated receptors (PARs), which are activated by thrombin and other extracellular proteases, are expressed widely at both neuronal and glial levels and have been shown to be involved in several brain pathologies. As far as the glial receptors are concerned, previous experiments performed in rat hippocampus showed that expression of PAR-1, the prototypic member of the PAR family, increased in astrocytes both in vivo and in vitro following treatment with trimethyltin (TMT). TMT is an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. In the present experiments, the authors extended their investigation to microglial cells. In particular, by 7 days following TMT intoxication in vivo, confocal immunofluorescence revealed an evident PAR-1-related specific immunoreactivity in OX-42-positive microglial cells of the CA3 and hilus hippocampal regions. In line with the in vivo results, when primary rat microglial cells were treated in vitro with TMT, a strong upregulation of PAR-1 was observed by immunocytochemistry and Western blot analysis. These data provide further evidence that PAR-1 may be involved in microglial response to brain damage.

14‐3‐3ε marks the amyloid‐stimulated microglia long‐term activation

Microglia‐mediated inflammation in the central nervous system is a hallmark of the pathogenesis of several neurodegenerative diseases including Alzheimers disease. Microglial cells activation follows the deposition of amyloid β fibrils and it is generally considered a triggering factor in the early steps of the onset of Alzheimers disease. Although the initial engagement of microglia seems to play a neuroprotective role, many lines of evidence indicate that a persistent activation with the production of proinflammatory molecules contributes to dismantle neuronal activity and to induce neuronal loss occurring in neurodegenerative diseases. To date, limited proteomic data are available on activated microglial cells in response to extracellular amyloidogenic peptides. In this study, murine microglial cells have been employed to investigate the effects of amyloid β peptides in triggering microglial activation. The response was monitored at the proteome level through a two‐dimensional gel electrophoresis‐based approach. Results show only a limited number of differentially expressed proteins, among these a more acidic species of the cytosolic actin, and the 14‐3‐3ε protein, found significantly upregulated in Aβ‐activated cells. 14‐3‐3ε belongs to a regulatory protein family involved in important cellular processes, including those leading to neurodegenerative diseases, and thus its increased expression suggests a role of this protein in tuning microglia activation.