Biotinylation Assay to Determine LFA-1 Recycling in Motile T-Lymphocytes.

The cycles of internalization of the cell surface ß2 integrin receptor lymphocyte function-associated antigen 1 (LFA-1) and its re-exposure on the plasma membrane are important for T-cell trafficking. Biotinylation of cells enables to measure surface expression of receptors, and after reducing surface biotin with reducing buffer, enables to measure the internalization of receptors. Here, by using biotin in combination with reducing buffer and recombinant intercellular adhesion molecule-1 (rICAM-1)-coated dishes and subsequent Western immunoblot analysis, we describe how to measure internalization of the LFA-1 receptor and its re-expression back to the cell surface in motile T-lymphocytes.

Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes.

To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow cytometry-based assay that allows the quantification of LFA-1 internalization and its re-expression on the cell surface in T lymphocytes. An advantage of using flow cytometry-based assay over biochemical methods is the low number of needed cells. This protocol can be also used to measure recycling of other receptors.

Integrin Dependent RhoB Activation Assay Using Leukocytes.

To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including RhoB. Here we describe an activation assay of RhoB in leukocytes migrating on ICAM-1Fc coated dishes using commercially available Rhoteikin coated agarose beads. Although this is a specific protocol for LFA-1 induced RhoB activation, it can also be used for RhoB activation induced by other soluble and insoluble factors.

RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes.

The regulation of cell adhesion and motility is complex and requires the intracellular trafficking of integrins to and from sites of cell adhesion, especially in fast-moving cells such as leukocytes. The Rab family of guanosine triphosphatases (GTPases) is essential for vesicle transport, and vesicles mediate intracellular integrin trafficking. We showed that RhoB regulates the vesicular transport of the integrin LFA-1 along the microtubule network in migrating T lymphocytes. Impairment in RhoB function resulted in the accumulation of both LFA-1 and the recycling endosomal marker Rab11 at the rear of migrating T lymphocytes and decreased the association between these molecules. T lymphocytes lacking functional RhoB exhibited impaired recycling and subsequently decreased surface amounts of LFA-1, leading to reduced T cell adhesion and migration mediated by the cell adhesion molecule ICAM-1 (intercellular adhesion molecule-1). We propose that vesicle-associated RhoB is a regulator of the Rab11-mediated recycling of LFA-1 to the cell surface, an event that is necessary for T lymphocyte motility.

Superresolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated T cell adhesion with autoimmunity.

Integrins are heterodimeric transmembrane proteins that play a fundamental role in the migration of leukocytes to sites of infection or injury. We found that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) inhibits signaling by the integrin lymphocyte function-associated antigen-1 (LFA-1) in effector T cells. PTPN22 colocalized with its substrates at the leading edge of cells migrating on surfaces coated with the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1). Knockout or knockdown of PTPN22 or expression of the autoimmune disease-associated PTPN22-R620W variant resulted in the enhanced phosphorylation of signaling molecules downstream of integrins. Superresolution imaging revealed that PTPN22-R620 (wild-type PTPN22) was present as large clusters in unstimulated T cells and that these disaggregated upon stimulation of LFA-1, enabling increased association of PTPN22 with its binding partners at the leading edge. The failure of PTPN22-R620W molecules to be retained at the leading edge led to increased LFA-1 clustering and integrin-mediated cell adhesion. Our data define a previously uncharacterized mechanism for fine-tuning integrin signaling in T cells, as well as a paradigm of autoimmunity in humans in which disease susceptibility is underpinned by inherited phosphatase mutations that perturb integrin function.

Anchored FRET sensors detect local caspase activation prior to neuronal degeneration.

BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-ß peptide as a causative agent in AD. RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-ß peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed. CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-ß peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

The CCAAT/enhancer binding protein (C/EBP) δ is differently regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-ß peptide.

BACKGROUND: The transcription factors CCAAT/enhancer binding proteins (C/EBP) α, ß and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB). In general, C/EBPα is down-regulated, whereas both C/EBPß and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD) one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-ß (Aß) deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aß deposits with the aim of defining new therapeutic targets. METHODS: Here we have investigated the effects of Aß on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aß deposits (tg-ArcSwe) by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay. RESULTS: We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aß inhibit up-regulation of C/EBPδ expression in interleukin-1ß-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPß was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aß deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1ß-induced C/EBPδ DNA binding activity to both C/EBP and κB sites is abolished after exposure to Aß. CONCLUSIONS: These data suggest that both expression and function of C/EBPδ are dysregulated in Alzheimer's disease. C/EBPδ seems to be differently regulated in response to different conformations of Aß. We propose that Aß induces an imbalance between NF-κB and C/EBP transcription factors that may result in abnormal responses to inflammatory stimuli.

Alzheimer amyloid-beta peptides block the activation of C/EBPbeta and C/EBPdelta in glial cells.

Members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors have been reported to be up-regulated in Alzheimer's disease. In the present study, we have investigated the effects of amyloid-beta (Abeta) peptides on C/EBPbeta and C/EBPdelta, previously shown to be induced by inflammatory stimuli in glial cells. Surprisingly, electrophoretic mobility shift assay showed that both Abeta(1-42) and Abeta(25-35) blocked C/EBP activation induced by the inflammatory cytokine interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS) in mixed primary glial cell cultures from rat. Abeta also blocked IL-1beta- or LPS-induced C/EBP protein levels. The most prominent effects were observed on DNA binding activity and protein levels of C/EBPdelta. Our results demonstrate a dysregulation of C/EBP when glial cells are activated in the presence of Alzheimer Abeta peptides.

Targeting cytokine expression in glial cells by cellular delivery of an NF-kappaB decoy.

Inhibition of nuclear factor (NF)-kappaB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer's disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-kappaB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer beta-amyloid peptide in the presence of the inflammatory cytokine interleukin (IL)-1beta. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-kappaB binding activity and IL-6 mRNA expression, respectively.

beta-Amyloid and interleukin-1beta induce persistent NF-kappaB activation in rat primary glial cells.

An increasing body of evidence suggests that beta-amyloid (Abeta) and activated glial cells play a crucial part in the pathogenesis of Alzheimer's disease (AD). Activated glial cells surrounding the senile plaques, formed by Abeta peptides, have been proposed to promote neurodegeneration by producing putatively toxic factors, including the inflammatory cytokine interleukin-1beta (IL-1beta). Elevated levels of both IL-1beta and activated nuclear factor kappaB (NF-kappaB), a key transcription factor regulating a wide variety of inflammatory genes, have been found in the brains of AD patients. In this study, we have investigated the ability of the Abeta(25-35) peptide and IL-1beta, either alone or together, in activating NF-kappaB in glial cells. Mixed primary glial cells from rat were treated with IL-1beta and/or Abeta(25-35), and NF-kappaB binding activity was analyzed by electophoretic mobility shift assay. We observed that the induction of NF-kappaB binding activity induced by either IL-1beta or Abeta(25-35) showed a peak at 30 min, and significantly declined after 2 h. The induced NF-kappaB activation persisted after 24 h and even seemed to increase in cells treated with Abeta(25-35). The activation of NF-kappaB by Abeta(25-35) was shown to be dose-dependent. In addition, Abeta(25-35) potentiated the effect of IL-1beta in a dose-dependent manner when co-stimulating the cells. The potentiating effect of Abeta(25-35) on IL-1beta-induced NF-kappaB binding activity was observed after 30 min, 2 h and 24 h, and did not significantly differ over time. A possible explanation is that when glial cells are stimulated by inflammatory factors in the presence of Abeta peptides or senile plaques, the NF-kappaB negative feedback regulation is no longer functional.