p75 Neurotrophin Receptor Cleavage by α- and γ-Secretases Is Required for Neurotrophin-mediated Proliferation of Brain Tumor-initiating Cells

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.     

Both Functional LTβ Receptor and TNF Receptor 2 Are Required for the Development of Experimental Cerebral Malaria

Background

TNF-related lymphotoxin α (LTα) is essential for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The pathway involved has been attributed to TNFR2. Here we show a second arm of LTα-signaling essential for ECM development through LTβ-R, receptor of LTα1β2 heterotrimer.

Methodology/Principal Findings

LTβR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTαβ deficient mice. Resistance of LTαβ or LTβR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin⁺ CD8⁺ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTβR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM.

Conclusions/Significance

LTβR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTβR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.     

Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α₁-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α₁-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α₁-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca²⁺ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α₁-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α₁-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.     

A Membrane-proximal,C-terminal α-Helix Is Required for Plasma Membrane Localization and Function of the G Protein-coupled Receptor (GPCR) TGR5

The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β₂ adrenergic receptor (β₂AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β₂AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β₂AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.     

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

Background

Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.

Methodology/Principal Finding

We demonstrate that stable cell lines inducibly expressing S1P-adapted α₁-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α₁-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α₁-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α₁-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.

Conclusions/Significance

Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.     

γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling

The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.     

Toll-like Receptor 2 (TLR2), Transforming Growth Factor-β, Hyaluronan (HA), and Receptor for HA-mediated Motility (RHAMM) Are Required for Surfactant Protein A-stimulated Macrophage Chemotaxis

The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA- mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFβ. In turn, TGFβ1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2^−/− mice failed to migrate in response to SPA but responded normally to TGFβ1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44^−/− mice had similar responses to SPA, whereas those from RHAMM^−/− mice had decreased chemotaxis to SPA, TGFβ1, and HA. In primary macrophages, SPA-stimulated TGFβ production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFβ production. TGFβ1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.     

Integrin α5β1 Expression Is Required for Inhibition of Keratinocyte Migration by Ganglioside GT1b

Polysialoganglioside GT1b, a keratinocyte membrane glycosphingolipid, inhibits normal keratinocyte adhesion and migration on a fibronectin matrix. The specificity of the inhibition for cells plated on a fibronectin matrix and competition of GT1b inhibition with peptide RGDS suggest that GT1b abrogates the α₅β₁/fibronectin interaction. We examined the effects of GT1b on the adhesion and migration of keratinocyte-derived cell lines and correlated GT1b responsiveness and α₅β₁integrin expression. GT1b (5 nM) significantly inhibited migration of normal human keratinocytes, immortalized keratinocytes, and squamous cell carcinoma SCC12F2 cells on fibronectin, but not on collagen I. Concentrations as high as 5 μM had no effect on SCC13 or HaCaT cells. Likewise, GT1b inhibited fibronectin-dependent cell adhesion of normal human keratinocytes, immortalized keratinocytes, and SCC12F2 cells, but had no effect on SCC13 or HaCaT cells. Flow cytometric and Western immunoblot analysis of integrin expression showed significantly decreased α₅and β₁integrin expression in SCC13 and HaCaT cells compared to normal keratinocytes, immortalized keratinocytes, and SCC12F2 cells. Incubation with TGF-β1 increased α₅β₁integrin expression and induced responsiveness to GT1b in HaCaT cells. These data imply that GT1b “response” requires sufficient expression of α₅β₁and further suggest that the mechanism of the inhibitory effect of GT1b involves GT1b/α₅β₁interaction.     

Class I α-Mannosidases Are Required for N-Glycan Processing and Root Development in Arabidopsis thaliana

In eukaryotes, class I α-mannosidases are involved in early N-glycan processing reactions and in N-glycan–dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type α-mannosidase I (MNS3) and the two Golgi-α-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected α1,2-mannose residue from Man₉GlcNAc₂, MNS1/2 readily removed three α1,2-mannose residues from Man₈GlcNAc₂. Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man₈GlcNAc₂ accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man₉GlcNAc₂, demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I α-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I α-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.N-glycosylation is a major co- and posttranslational modification of proteins in eukaryotic cells. The biosynthesis of protein N-linked glycans starts in the endoplasmic reticulum (ER) when the oligosaccharyltransferase complex catalyzes the transfer of the Glc₃Man₉GlcNAc₂ oligosaccharide from the lipid-linked precursor to Asn residues (N-X-S/T) of nascent polypeptide chains. Subsequent N-glycan processing involves a series of highly coordinated step-by-step enzymatic conversions occurring in the ER and Golgi apparatus (Kornfeld and Kornfeld, 1985). In the first trimming reactions, α-glucosidases I (GCSI) and GCSII cleave off three glucose residues from Glc₃Man₉GlcNAc₂ to generate Man₉GlcNAc₂ (Figure 1A). The next steps of the pathway are the removal of four α1,2-linked mannose residues to provide the Man₅GlcNAc₂ substrate for the formation of complex N-glycans in the Golgi apparatus. In mammals, these mannose trimming reactions are catalyzed by class I α-mannosidases (glycosyl hydrolase family 47 of the Carbohydrate Active Enzymes database; http://www.cazy.org/). These enzymes are inverting glycosyl hydrolases that are highly specific for α1,2-mannose residues, require Ca²⁺ for catalytic activity, and are sensitive to inhibition by pyranose analogs such as 1-deoxymannojirimycin and kifunensine (Lipari et al., 1995; Gonzalez et al., 1999). Class I α-mannosidases are conserved through eukaryotic evolution and do not share sequence homology with class II α-mannosidases, such as Golgi α-mannosidase II and the catabolic lysosomal and cytoplasmic α-mannosidases (Gonzalez et al., 1999; Herscovics, 2001).Open in a separate windowFigure 1.Cartoon of Important Oligosaccharide Structures.(A) Man₉GlcNAc₂ oligosaccharide (Man9): the substrate for ER-MNSI.(B) Man₈GlcNAc₂ isomer Man8.1 according to Tomiya et al. (1991): the product of ER-MNSI and substrate for Golgi-MNSI.(C) Man₅GlcNAc₂ (Man5.1): the product of the mannose trimming reactions.The linkage of the sugar residues is indicated.[See online article for color version of this figure.]The mammalian class I α-mannosidase family consists of three protein subgroups, which have been distinguished based on their sequence similarity and proposed function: ER-α1,2-mannosidases I (ER-MNSIs), Golgi-α-mannosidases I (Golgi-MNSIs), and ER degradation-enhancing α-mannosidase (EDEM)-like proteins (Mast and Moremen, 2006). In humans, there is a single ER-MNSI, which cleaves the terminal mannose residue from the b-branch of the Man₉GlcNAc₂ oligosaccharide to create the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B). Subsequently, Golgi-MNSI (three isoforms, Golgi-MNSIA, Golgi-MNSIB, and Golgi-MNSIC, are present in humans) catalyze the removal of the remaining three α1,2-linked mannose residues to generate Man₅GlcNAc₂ (Figure 1C). The three human EDEM proteins are not directly involved in N-glycan processing but play a role in ER-associated degradation of glycoproteins (Mast et al., 2005; Hirao et al., 2006; Olivari et al., 2006).The formation of the Man₈GlcNAc₂ isomer (Man8.1), which is catalyzed by ER-MNSI, is the last N-glycan processing step that is conserved in yeast and mammals. Apart from its N-glycan processing function, ER-MNSI plays a key role in ER-mediated quality control of glycoproteins in yeasts and mammals (Mast and Moremen, 2006; Lederkremer, 2009). It has been proposed that ER-MNSI cooperates with mammalian EDEM1 to 3 or the yeast α1,2-mannosidase HTM1 to generate the signal that marks misfolded glycoproteins for degradation through the ER-associated protein degradation (ERAD) pathway. This quality control process, which finally leads to retrotranslocation to the cytoplasm and hydrolysis by the 26S proteasome, serves to prevent the secretion of aberrantly folded cargo proteins and is required to maintain protein homeostasis in the ER. Initially it was proposed that the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B) flags aberrantly folded glycoproteins for degradation; however, recent evidence suggests that further mannose trimming to Man₇GlcNAc₂ in yeast and Man_5-6GlcNAc₂ in mammals is required to trigger ERAD (Avezov et al., 2008; Clerc et al., 2009). In addition, these mannose cleavage reactions serve also to release glycoproteins from the calnexin/calreticulin quality control cycle (Caramelo and Parodi, 2008).Unlike for animals and yeast, much less is known about the biological function of plant class I α-mannosidases. Processing mannosidases have been purified and characterized from mung bean (Vigna radiata) seedlings and castor bean (Ricinus communis) cotyledons (Forsee, 1985; Szumilo et al., 1986; Kimura et al., 1991). These preparations were a mixture of different α-mannosidases, and no evidence for ER-MNSI-like activity was provided. A putative Golgi-α-mannosidase I has been cloned from soybean (Glycine max) (Nebenführ et al., 1999). A green fluorescent protein (GFP)-tagged fusion protein of the soybean enzyme has been shown to reside in the cis-stacks of the Golgi apparatus (Nebenführ et al., 1999; Saint-Jore-Dupas et al., 2006), but its role in N-glycan processing and its enzymatic properties have not been reported so far. Thus, the involvement of class I α-mannosidases in N-glycan processing as well as in glycoprotein quality control in plants is still unclear, and the existence of a plant ER-MNSI has so far been inferred only from the presence of Man₈GlcNAc₂ oligosaccharides on ER-resident glycoproteins (Pagny et al., 2000).Here, we report the molecular cloning and biochemical characterization of the enzymes accounting for ER-MNSI and Golgi-MNSI activities in Arabidopsis thaliana. We also demonstrate that disruption of these genes leads to severe cell expansion defects in roots as well as to distinct cell wall alterations. Hence, the identification of the Arabidopsis ER-type and Golgi class I α-mannosidases not only establishes the molecular basis for the missing steps in the plant N-glycan processing pathway but also provides unprecedented insights into the role of N-glycans in plant development.     

Optimum Length and Flexibility of Reovirus Attachment Protein σ1 Are Required for Efficient Viral Infection

Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.     

Acid-dependent Interleukin-1 (IL-1) Cleavage Limits Available Pro-IL-1β for Caspase-1 Cleavage

Noncommunicable diseases such as cardiovascular disease (stroke and heart attack), cancer, chronic respiratory disease, and diabetes are a leading cause of death and disability worldwide and are worsened by inflammation. IL-1 is a driver of inflammation and implicated in many noncommunicable diseases. Acidosis is also a key feature of the inflammatory microenvironment; therefore it is vital to explore IL-1 signaling under acidic conditions. A HEK-IL-1 reporter assay and brain endothelial cell line were used to explore activity of mature IL-1α and IL-1β at pH 7.4 and pH 6.2, an acidic pH that can be reached under inflammatory or ischemic conditions, alongside cathepsin D-cleaved 20-kDa IL-1β produced under acidic conditions. We report that mature IL-1 signaling at IL-1 receptor type 1 (IL-1R1) is maintained at pH 6.2, but the activity of the decoy receptor, IL-1R2, is reduced. Additionally, cathepsin D-cleaved 20-kDa IL-1β was minimally active at IL-1R1 and was not further cleaved to highly active 17-kDa IL-1β. Therefore formation of the 20-kDa form of IL-1β may prevent the generation of mature bioactive IL-1β and thus may limit inflammation.     

The Effects of Transmembrane Sequence and Dimerization on Cleavage of the p75 Neurotrophin Receptor by γ-Secretase

Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75^NTR, best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75^NTR ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75^NTR are γ-secretase substrates. However, dimerization is not a requirement for p75^NTR cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75^NTR interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75^NTR transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75^NTR. Nonetheless, heteromer interactions of p75^NTR with TrkA increase full-length p75^NTR homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75^NTR transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.     

Preconditioning of Microglia by α-Synuclein Strongly Affects the Response Induced by Toll-like Receptor (TLR) Stimulation

In recent years, it has become accepted that α-synuclein (αSyn) has a key role in the microglia-mediated neuroinflammation, which accompanies the development of Parkinson’s disease and other related disorders, such as Dementia with Lewy Bodies and Alzheimer’s disease. Nevertheless, the cellular and molecular mechanisms underlying its pathological actions, especially in the sporadic forms of the diseases, are not completely understood. Intriguingly, several epidemiological and animal model studies have revealed a link between certain microbial infections and the onset or progression of sporadic forms of these neurodegenerative disorders. In this work, we have characterized the effect of toll-like receptor (TLR) stimulation on primary murine microglial cultures and analysed the impact of priming cells with extracellular wild-type (Wt) αSyn on the subsequent TLR stimulation of cells with a set of TLR ligands. By assaying key interleukins and chemokines we report that specific stimuli, in particular Pam3Csk4 (Pam3) and single-stranded RNA40 (ssRNA), can differentially affect the TLR2/1- and TLR7-mediated responses of microglia when pre-conditioned with αSyn by augmenting IL-6, MCP-1/CCL2 or IP-10/CXCL10 secretion levels. Furthermore, we report a skewing of αSyn-primed microglia stimulated with ssRNA (TLR7) or Pam3 (TLR2/1) towards intermediate but at the same time differential, M1/M2 phenotypes. Finally, we show that the levels and intracellular location of activated caspase-3 protein change significantly in αSyn-primed microglia after stimulation with these particular TLR agonists. Overall, we report a remarkable impact of non-aggregated αSyn pre-sensitization of microglia on TLR-mediated immunity, a phenomenon that could contribute to triggering the onset of sporadic α-synuclein-related neuropathologies.     

T Cell Factor-1 Controls the Lifetime of CD4+ CD8+ Thymocytes In Vivo and Distal T Cell Receptor α-Chain Rearrangement Required for NKT Cell Development

Natural killer T (NKT) cells are a component of innate and adaptive immune systems implicated in immune, autoimmune responses and in the control of obesity and cancer. NKT cells develop from common CD4+ CD8+ double positive (DP) thymocyte precursors after the rearrangement and expression of T cell receptor (TCR) Vα14-Jα18 gene. Temporal regulation and late appearance of Vα14-Jα18 rearrangement in immature DP thymocytes has been demonstrated. However, the precise control of lifetime of DP thymocytes in vivo that enables distal rearrangements remains incompletely defined. Here we demonstrate that T cell factor (TCF)-1, encoded by the Tcf7 gene, is critical for the extended lifetime of DP thymocytes. TCF-1-deficient DP thymocytes fail to undergo TCR Vα14-Jα18 rearrangement and produce significantly fewer NKT cells. Ectopic expression of Bcl-x_L permits Vα14-Jα18 rearrangement and rescues NKT cell development. We report that TCF-1 regulates expression of RORγt, which regulates DP thymocyte survival by controlling expression of Bcl-x_L. We posit that TCF-1 along with its cofactors controls the lifetime of DP thymocytes in vivo.