Lymphotoxin, but Not TNF, Is Required for Prion Invasion of Lymph Nodes

Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1^−/− lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrP^Sc detection, we identified PrP^Sc deposits associated with HEVs in TNFR1^−/− lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs.     

Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca²⁺ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca²⁺ signals in PKCθ^−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca²⁺ release from intracellular Ca²⁺ stores. PKCθ thereby indirectly regulates activation of store-operated Ca²⁺ entry. However, PKCθ also directly and negatively regulates store-independent Ca²⁺ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ^−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry but not store-operated Ca²⁺ entry, blocked the enhanced GPVI-dependent Ca²⁺ signaling and PS exposure seen in PKCθ^−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca²⁺ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.     

Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes

The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20-like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo. Mst1^−/− lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1^−/− lymphocytes on endothelium was markedly reduced, whereas their L-selectin-dependent rolling/tethering and transition to LFA-1-mediated arrest were not affected. Mst1^−/− lymphocytes were also defective in the stabilization of adhesion through α4 integrins. Consequently, Mst1^−/− mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1^−/− lymphocytes had impaired motility over lymph node-derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration.     

Cellular prion protein and Alzheimer disease

Soluble oligomeric amyloid-β (Aβ) has been suggested to impair synaptic and neuronal function, leading to neurodegeneration that is clinically observed as the memory and cognitive dysfunction characteristic of Alzheimer disease, while the precise mechanism(s) whereby oligomeric Aβ causes neurotoxicity remains unknown. Recently, the cellular prion protein (PrP^C) was reported to be an essential co-factor in mediating the neurotoxic effect of oligomeric Aβ. Our recent study showed that Prnp^−/− mice are resistant to the neurotoxic effect of oligomeric Aβ in vivo and in vitro. Furthermore, application of an anti-PrP^C antibody or PrP^C peptide was able to block oligomeric Aβ-induced neurotoxicity. These findings demonstrate that PrP^C may be involved in neuropathologic conditions other than conventional prion diseases, i.e., Creutzfeldt-Jakob disease.     

Tunnelling nanotubes: A highway for prion spreading?

The discovery of tunnelling nanotubes (TNTs) and their proposed role in long intercellular transport of organelles, bacteria and viruses have led us to examine their potential role during prion spreading. We have recently shown that these membrane bridges can form between neuronal cells, as well as between dendritic cells and primary neurons and that both endogenous and exogenous PrP^Sc appear to traffic through these structures between infected and non-infected cells. Furthermore, prion infection can be efficiently transmitted from infected dendritic cells to primary neurons only in co-culture conditions permissive for TNT formation. Therefore, we propose a role for TNTs during prion spreading from the periphery to the central nervous system (CNS). Here, we discuss some of the key steps where TNTs might play a role during prion neuroinvasion.Key words: tunnelling nanotubes, TNTs, prion, PrP^Sc, prion spreading, dendritic cells, neuroinvasionPrion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that have been found in a number of species, including scrapie in sheep, bovine spongiform encephalopathy in cattle (BSE), Chronic wasting disease in deer and Creutzfeldt-Jacob, the Gerstmann-Straüssler-Scheinker syndrome, fatal familial insomnia and kuru in humans (reviewed in ref. ¹). Human TSEs can be sporadic, genetic or acquired by infection. A new variant of Creutzfeldt-Jakob disease (termed vCJD) was reported from the UK in 1996.² The majority of vCJD cases diagnosed to date resulted from a peripheral exposure via the consumption of BSE-contaminated food. Pathological features of TSE diseases can include gliosis, neuronal cell loss and spongiform changes, but the common feature of all members of this group of diseases is the build-up of an aberrant form of the host cellular protein PrP^C, named PrP^Sc (from scrapie). The normal cellular isoform, PrP^C, is an endogenous glycosylphosphatidyl inositol (GPI)-anchored protein present in numerous tissues in mammals, including neurons and lymphoid cells. While the exact function of PrP^C remains unclear, evidence suggest putative roles in neuroprotection, cell adhesion and signal transduction (reviewed in refs. ³ and ⁴). According to the ‘protein-only hypothesis,’ the causative agents of prion diseases are proteinaceous infectious particles (‘prions’), which are composed essentially of misfolded PrP^C, or PrP^Sc.^5,6 Prions replicate through a molecular mechanism in which abnormally folded PrP^Sc acts as a catalyst and serves as a template to convert normal PrP^C molecules into PrP^Sc.^5,6 PrP^Sc differs from PrP^C in the conformation of its polypeptide chain, which is enriched in β-sheets and is protease resistant. Although the conversion process is believed to have a predominant role in the pathogenesis of prion diseases, the cellular and molecular basis for the pathogenic conversion of PrP are still unknown.Another important question is how PrP^Sc spreads to and within the brain. After oral exposure, PrP^Sc accumulates into lymphoid tissues, such as the spleen, lymph nodes or Peyer''s patches, prior to neuroinvasion.^7–9 The exact mechanisms and specific cells involved in the spreading from the gastrointestinal track to the lymphoid system and to the peripheral nervous system (PNS), leading to neuroinvasion of the CNS remain to be elucidated. However, a range of evidence suggests that the accumulation of PrP^Sc within lymphoid tissues is necessary for efficient neuroinvasion.^9–11 In particular it has been shown that PrP^Sc accumulates first within follicular dendritic cells (FDCs)¹² and macrophages.¹³ FDCs are stromal-differentiated cells in the germinal centres of activated lymphoid follicles. A number of studies have demonstrated that FDCs play a critical role during spreading of infection since their absence greatly impaires the neuroinvasion process.^8,11,14,15 However, because FDCs are immobile cells, it is not clear how they acquire PrP^Sc and how it spreads from the FDCs to the PNS. FDCs and nerve synapses occupy different anatomical sites^16,17 and therefore the lack of physical contact between the gut and FDCs and between FDCs and the nerve periphery imply the presence of intermediate mechanisms for the transport of PrP^Sc. Dendritic cells (DCs) have been proposed to play a critical role in the transport of PrP^Sc from the gut to FDCs.¹⁸ DCs function as sentinels for incoming pathogens. Bone-marrow dendritic cells (BMDCs) are migratory cells that are able to transport proteins within Peyer''s patches and into mesenteric lymph nodes.¹⁹ Interestingly, mucosal dendritic cells which play a role in the transport of intestinal antigen for presentation to Peyer''s patches and to mesenteric lymph nodes, can also extend trans-epithelial dendrites to directly sample bacteria in the gut.^20,21 However, the transport of PrP^Sc from FDCs to the PNS remains controversial and evidence for a direct role of DCs during this process has been debated.^22,23 Several mechanisms have been proposed for the intercellular transfer of PrP^Sc, including cell-cell contact, transfer via exosomes or by GPI-painting.^24–26 For example, similar to other types of pathogens such as HIV-1, which was proposed to follow the “exosomal” pathway to be released from the cells,²⁷ it has been shown that the supernatant of prion infected cells contain large amount of PrP^Sc in membranous vesicles known as exosomes.^25,28 Thus, it was suggested that exosomes might be a way to spread prion infection in vivo.^25,28 Recently, a different type of vesicles known as plasma membrane-derived microvesicles, were also described as a potential spreading mechanism during neuroinvasion.²⁹In 2004, Rustom and colleagues discovered a new mechanism of long distance intercellular communication in mammalian cells, called tunnelling nanotubes (TNTs).³⁰ TNTs are transient, long, actin-rich projections that allow for long-distance intercellular communication (reviewed in refs. ^31–33). TNT-like structures have been described to form in vitro between numerous cell types, including neuronal and immune cells.^30,34,35 These studies demonstrated that TNT-like structures formed bridges or channels between distant cells that can be used to transfer material between cells, including Lysotracker positive or endosomal vesicles, calcium fluxes, bacteria or viruses through their cytoplasms or along the surface of the nanotubes.^31–33 Interestingly, a model GPI-anchored protein, GFP-GPI, was found to move at the surface of these tubes³⁴ and while studying the neuritic transport of prions in neuronal cells, Magalhães and colleagues noticed a strong correlation between internalized PrP-res and Lysotracker positive vesicles in neurites,³⁶ suggesting that PrP-res might also be able to transfer through TNTs during prion cell-cell spreading.The results from the studies mentioned above and random observations of TNT-like structures in neuronal model cell cultures first led us to study whether these structures could in fact provide an efficient mechanism for prion cell-cell spreading.³⁷ We initially characterized TNT-like structures in the mouse catecholaminergic neuronal cell line, Cath.a-Differentiated cells (CAD cells) a well-recognized neuronal cell model for prion infection.³⁸ Under our culturing conditions, over 40% of the CAD cells could efficiently form actin-rich TNT-like structures between differentially labelled cell populations. In CAD cells, these nanotubes were very heterogeneous, both in length and in diameters. Indeed, TNT-like structures had lengths ranging from 10 to 80 µm and while over 70% of the nanotubes had diameters smaller than 200 nm, the remaining TNT-like structures had larger diameters (200 to 800 nm). We demonstrated that vesicles of lysosomal origins, a fluorescent form of PrP (GFP-PrP), infectious Alexa-PrP^Sc, as well as both endogenous and exogenous PrP^Sc could traffic within TNTs between neuronal cells (Fig. 1). The lysosomal and GFP-PrP vesicles observed to move through TNTs had a directed movement with a speed in the range of actin-mediated motors,³⁷ consistent with previous studies suggesting the involvement of an actomyosin-dependent transport.³⁹ Interestingly, active transfer of endogenous PrP^Sc, lysosomal or GFP-PrP vesicles occurred through TNTs with larger diameters, suggesting distinct roles for the different TNT-like structures observed.³⁷ These results do not seem to be specific to CAD cells since the transfer of GFP-PrP throught TNTs was observed in different types of transfected cells, including HEK293 cells (unpublished data). Furthermore, these results were in agreement with previous observations by Onfelt and colleagues showing the presence of a fluorescent GPI model protein (GFP-GPI) in TNTs formed between EBV-transformed human B cells³⁴ suggesting that different GPI-anchored proteins can be transferred along the surface and inside vesicles within TNTs. In order to determine the relevance of this type of intercellular communication in the case of prion diseases, it was necessary to evaluate the trafficking of the pathological form of PrP (PrP^Sc) within TNTs, by analyzing the transfer of endogenous PrP^Sc between chronically infected ScCAD cells and non-infected CAD cells. By immunofluorescence after guanidium treatment, endogenous PrP^Sc was found inside TNTs and in the cytoplasm of recipient non-infected CAD cells. Similar to exogenous PrP^Sc, endogenous PrP^Sc particles were not present in non-infected CAD cells not in contact with ScCAD cells after overnight co-cultures, thus excluding exosomal transfer or protein shedding.³⁷ Similarly, no transfer was observed between cells in direct contact with one another or upon treatment with latrunculin, which inhibits TNT formation. Strikingly, the transfer of endogenous PrP^Sc was visible only when TNTs were present, demonstrating that in vitro, PrP^Sc can efficiently exploit TNTs to spread between cells of neuronal origin. These data suggested that TNTs could be a mechanism for prion spreading within the cells of the CNS.Open in a separate windowFigure 1Endogenous PrP^Sc transfer from ScCAD cells to CAD cells via TNTs. Endogenous PrP^Sc is found in punctate structures inside TNTs and in the cytoplasms of recipient cells. CAD cells were transfected with Cherry-PLAP (red) and co-cultured with ScCAD for 24 h. Cells were fixed, treated with Gnd and immunostained for PrP using SAF32 Ab (green). (A) Merge projection of Z-stacks obtained with a confocal Andor spinning-disk microscope. (B) Three-dimensional reconstruction of (A) using OsiriX software. (C) Zoom in on TNT-like structures. PrP^Sc is found in vesicular structures inside TNTs and in the cytoplasm of the recipient non-infected CAD cells (see blue arrow heads). Scale bar represents 10 µm.Interestingly, DCs were shown to form networks of TNTs both in vitro⁴⁰ and in vivo.⁴¹ In an elegant study, Watkins and Salter demonstrated that DCs could propagate calcium flux upon cell stimulation to other cells hundreds of microns away through TNTs, both between DCs and between DCs and THP-1 monocytes.⁴⁰ These data suggested the possibility that DCs could form tubular connections with neuronal cells in order to transport PrP^Sc to the PNS via TNTs. Using BMDCs in co-cultures with both cerebellar granular neurons (CGNs) and primary hippocampal neurons, we showed that BMDCs could form networks of TNTs with both types of neurons. Furthermore, these TNTs appeared to be functional, allowing for the transport of Lysotracker positive vesicles and infectious Alexa-PrP^Sc between loaded BMDCs and primary neurons, suggesting that DCs could transfer the infectious prion agent to primary neuronal cultures through TNTs. By using filters and conditions unfavorable for other mechanisms of transport, we found that moRK13 cells,²⁸ as well as CGNs (unpublished data), could be infected by co-cultures with BMDCs loaded with infectious brain homogenate.³⁷ Overall, these data indicate that TNTs could be an efficient mechanism of prion transmission between immune cells and neuronal cells, as well as between neuronal cultures. Since DCs can interact with peripheral neurons,⁴² we propose that TNTs could be involved in the process of neuroinvasion at multiple stages, from the peripheral site of entry to the PNS by neuroimmune interactions with DCs, allowing neurons to retrogradely transport prions to the CNS, and within the CNS (Fig. 2).Open in a separate windowFigure 2Transport of PrP^Sc via TNTs, an alternative spreading mechanism during neuroinvasion. Studies in our laboratory suggest that TNTs allow for the intracellular transport of PrP^Sc between dendritic cells and neurons and between neurons (see inset). The exact mechanism of transport remains to be determined. For instance, it is still not clear, whether PrP^Sc is strictly transported within endocytic vesicles, or whether it can slide along the surface or be transported as aggregosomes within the tubes. Similarly, the types of motors used, as well as the possible gated mechanisms to enter the recipient cells are not known. Because of the high propensity of DCs to form TNTs with different cell types, we propose that TNTs could play important roles in delivering PrP^Sc to the proper cell types along the neuroinvasion route. For instance, DCs could deliver PrP^Sc from the peripheral entry sites to FDCs in the secondary lymphoid tissues (2) or in a less efficient manner, they might occasionally directly transport PrP^Sc to the PNS (1). They could also bridge the immobile FDC networks and the PNS (3), since we have shown that DCs can form TNTs with nerve cells. Finally, once PrP^Sc has reached its final destination within the CNS, TNTs might play a final role in the spreading of PrP^Sc within the brain between neurons and possibly between neuronal cells and astrocytes (4).Recently, it was demonstrated that the distance between FDCs and the neighbouring PNS was critical for prion neuroinvasion.⁴³ Indeed, in the spleen of CD19^−/− mice, FDC networks were found to be 50% closer to the nerve fibers compared to wild-type mice.⁴³ The authors suggested that the increase in prion spreading efficiency in these mice was directly dependent on the reduction in the distance between the FDC networks and the PNS in these mice. These results would be consistent with a mechanism of transfer such as exosomes release. However, shortening the distance between FDCs and the PNS would also reduce the route of transport that mobile cells would have to travel and increase the chances for transfer of prions to the PNS, resulting in an increase in prion spreading efficiency. While the importance of FDCs in prion replication during the spreading to the CNS seems to be clear,^11,14,15 their specific role in the transfer of prions and their possible interactions with other mobile cells are much more debated.^22,23 In order to bridge the gap between FDCs and the PNS, a role for DCs as possible carriers of PrP^Sc has been postulated. Aucouturier and colleagues have previously shown, using RAG-1^−/− mice, which are deficient in FDCs, that CD11c⁺ DCs infected with 139A were able to carry prion infection to the CNS, without accumulation and replication in lymphoid organs,²² thus suggesting that DCs are able to transport prions from the periphery to nerve cells. Recently, however, another study using TNFR1^−/− mice, deficient for FDCs, suggested that DCs were unlikely candidates in the transport of prion to the PNS.²³ In this study, the authors showed that in TNFR1^−/− mice, ME7 or 139A infected DCs were inefficient in transferring infection to the PNS. The authors suggested that the differences between the results obtained with RAG-1^−/− mice and TNFR1^−/− mice could be due to the differences in the levels of innervation of the spleen in RAG-1^−/−mice compared to TNFR1^−/− mice. They suggested that in RAG-1^−/− mice, DCs could spread prion infection because their spleens are highly innervated, compared to TNFR1^−/− or wild-type mice, therefore increasing the propensity of DCs to encounter nerve cells and tranfer the prion agents. Because of the reduction in the levels of innervation in the spleens of wild-type mice, the authors concluded that DCs are unlikely candidate for the transport of prions directly to the PNS [see (1) in Fig. 2]. However, since these studies are using mice deficient for FDCs, it remains unclear what type of interactions might occur between FDCs and DCs, and how DCs might be able to transport prions from FDCs to the PNS in wild-type mice [see (2) in Fig. 2]. Indeed, both studies show that under the right circumstances, DCs can interact with nerve cells, similar to what was recently shown in infected mice⁴² and in agreement with our findings that DCs can form TNTs with neurons.³⁷ Within this scenario, it is clear that to determine the specific role of DCs during the spreading of prions from the gut to the PNS, the transfer mechanisms between DCs and other cell types, especially FDCs and peripheral neurons, need to be better characterized.Overall, these in vitro data strongly point toward TNTs as one possible mechanism of prion spreading. The next step will be to identify these structures in vivo and to determine whether prion spreading in vivo is the result of passive mechanisms, such as exosome release, active intercellular transport along and within TNTs or whether prions will use any means available to reach their targets. Recently, TNT-like structures were imaged in a mouse cornea,⁴¹ suggesting that while challenging, the visualization of in vivo trafficking of prions in lymphoid tissues such as in lymph nodes or in the spleen as well as in brain organotypic cultures might be possible and could be used to reveal the presence of TNTs.The discovery of the existence of nanotubular membrane bridges in vitro has opened-up a new field of research. Channels, called plasmodesmata,⁴⁴ connecting plant cells have long been known to play crucial roles in the transport of nutrients, molecules and signals during development and some of their functions were recently compared with some of the recently proposed functions of TNTs.⁴⁵ Furthermore, in vivo long, actin-rich filopodia like structures were found to be crucial during development.^46–49 For example, these structures exist in developing sea urchin embryos and were proposed to play a role in signalling and patterning during gastrulation.⁴⁷ Similar roles were proposed for thin filopodia-like structures observed during dorsal closure in drosophila.⁴⁹ In addition, TNT-like structures were observed in the mouse cornea between DCs and were shown to increase under inflammatory conditions.⁴¹ The authors postulated that these TNT-like structures could play a role in Ag-specific signalling, especially as a response to eye inflammation. Therefore, the possibility that TNTs might play numerous roles during cell development, in the immune system and as conduits for the spreading of pathogens could lead to major changes in the way we view animal cell interactions. Specifically, understanding how pathogens usurp these cellular connections to spread could allow for the screening and the identification of new therapeutic inhibitors. To this aim, characterizing the basic mechanism of TNT formation within cell model systems will be necessary to improve the knowledge of TNTs in general, to analyze the transfer of pathogens more specifically, and to identify key molecules during this process. In the case of prions, whether they hijack nanotubes to spread between cells or whether prions increase the formation of filopodia and TNT-like structures similar to some viruses^33,50 and/or the efficiency of transfer remain to be determined. Overall, in this specific field, the constant improvement of cell imaging techniques and the emergence of imaging tools to study prion spreading^{36,37,51–53} could lead to exciting new insights both in the physiology of these intercellular connections and in the pathology of these devastating diseases.     

Yeast Prions: Evolution of the Prion Concept

Prions (infectious proteins) analogous to the scrapie agent have been identified in Saccharomyces cerevisiae and Podospora anserina based on their special genetic characteristics. Each is a protein acting as a gene, much like nucleic acids have been shown to act as enzymes. The [URE3], [PSI⁺], [PIN⁺] and [Het-s] prions are self-propagating amyloids of Ure2p, Sup35p, Rnq1p and the HET-s protein, respectively. The [β] and [C] prions are enzymes whose precursor activation requires their own active form. [URE3] and [PSI⁺] are clearly diseases, while [Het-s] and [β] carry out normal cell functions. Surprisingly, the prion domains of Ure2p and Sup35p can be randomized without loss of ability to become a prion. Thus amino acid content and not sequence determine these prions. Shuffleability also suggests amyloids with a parallel in-register β-sheet structure.Key Words: Ure2, Sup35, Rnq1, HETs, PrP, prion, amyloid     

Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclic amplification with beads (PMCAb)

Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10⁻¹³ dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536^+/− mice) allowed detection of CWD agent from the 10⁻⁶ dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 10⁵. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.     

Prion pathogenesis in the absence of Toll-like receptor signalling

To reach the brain from peripheral sites, prions must colonize various cell types within the lymphoreticular compartment. However, no prion entry receptors are yet known. Toll-like receptors (TLRs) are pattern-recognition receptors that bind a multitude of pathogens and are therefore candidates as effectors of prion entry. Moreover, injection of unmethylated CpG oligodinucleotides, which stimulate TLR9, has been reported to delay peripherally initiated scrapie. We therefore studied prion infection in MyD88^−/− mice, which are defective in TLR signalling. Despite subtle defects in splenic microarchitecture, MyD88^−/− mice challenged intraperitoneally or intracerebrally were fully susceptible to disease and died of scrapie after similar incubation times to those of wild-type mice. Splenic infectivity titres rose to similar levels with the same kinetics, and brains showed similar histopathological changes. TLR signalling therefore does not have any major role in prion pathogenesis, and the protective effect of TLR stimulation is unlikely to result from direct interactions with prions.     

Role of proteolytic activation of protein kinase Cδ in the pathogenesis of prion disease

Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced proteolytic activation of PKCδ in our COSC model. Immunohistochemical analysis of scrapie-infected COSC revealed loss of PKCδ positive Purkinje cells and enhanced astrocyte proliferation. Further examination of PKCδ signaling in the RML scrapie adopted in vivo mouse model showed increased proteolytic cleavage and Tyr 311 phosphorylation of the kinase. Notably, we observed a delayed onset of scrapie-induced motor symptoms in PKCδ knockout (PKCδ^−/−) mice as compared with wild-type (PKCδ^+/+) mice, further substantiating the role of PKCδ in prion disease. Collectively, these data suggest that PKCδ signaling likely plays a role in the neurodegenerative processes associated with prion diseases.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

TNFR1 determines progression of chronic liver injury in the IKKγ/Nemo genetic model

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-κB essential modulator in hepatocytes (IKKγ/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKγ/Nemo-deleted livers. We crossed hepatocyte-specific IKKγ/Nemo knockout mice (Nemo^Δhepa) with constitutive TNFR1^−/− and TRAIL^−/− mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKγ/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury–a finding that has direct implications for treating chronic liver disease.     

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11c^creIL-4Rα^−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×10⁵ stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11c^creIL-4Rα^−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.     

Cells and prions: A license to replicate

Prion diseases are neurodegenerative, infectious disorders characterized by the aggregation of a misfolded isoform of the cellular prion protein (PrP^C). The infectious agent - termed prion - is mainly composed of misfolded PrP^Sc. In addition to the central nervous system prions can colonize secondary lymphoid organs and inflammatory foci. Follicular dendritic cells are important extraneural sites of prion replication. However, recent data point to a broader range of cell types that can replicate prions. Here, we review the state of the art in regards to peripheral prion replication, neuroinvasion and the determinants of prion replication competence.     

Early structural features in mammalian prion conformation conversion

The conversion to a disease-associated conformer (PrP^Sc) of the cellular prion protein (PrP^C) is the central event in prion diseases. Wild-type PrP^C converts to PrP^Sc in the sporadic forms of the disorders through an unknown mechanism. These forms account for up to 85% of all human (Hu) occurrences; the infectious types contribute for less than 1%, while genetic incidence of the disease is about 15%. Familial Hu prion diseases are associated with about 40 point mutations of the gene coding for the PrP denominated PRNP. Most of the variants associated with these mutations are located in the globular domain of the protein. In a recent work in collaboration with the German Research School for Simulation Science, in Jülich, Germany, we performed molecular dynamics simulations for each of these mutants to investigate their structure in aqueous solution. Structural analysis of the various point mutations present in the globular domain unveiled common folding traits that may allow a better understanding of the early conformational changes leading to the formation of monomeric PrP^Sc. Recent experimental data support these findings, thus opening novel approaches to determine initial structural determinants of prion formation.Key words: prions, prion protein, human, pathogenic mutations, structure, molecular dynamics, nuclear magnetic resonancePrion diseases have attracted much attention from researchers with different scientific backgrounds and coming from various areas of expertise. Many questions still remain unanswered in the study of these rare and yet unique neurodegenerative disorders. Central to understanding the disease is deciphering the nature of the causative agent of these disorders: the prion. In fact, the mechanism by which a prion (PrP^Sc) is formed and the structure of the latter, have posed major challenges to this field. Indeed, prion research has achieved a great deal of detailed information in understanding the pathogenesis of the disease, but until now the early events leading to the conformational change harboring prions have remained elusive.¹ In an attempt to learning how the protein may undergo this conformational rearrangement, my group and the group of Paolo Carloni at the German Research School for Simulation Science, in Jülich, Germany, reasoned that some clues might come from the study of pathogenic mutants in HuPrP. At the time of beginning our work the structures of few mutants were known.² The structure of HuPrP was used as template for our studies.³ We therefore performed molecular dynamics (MD) simulations for each of these mutants to investigate their structure in aqueous solution. In total, almost 2 µs MD data were obtained. The calculations were based on the AMBER(parm99) force field, which has been shown to reproduce very accurately the structural features of the wild-type HuPrP and a few variants for which experimental structural information was available.⁴ All the variants present structural features different from those of wild-type HuPrP and the protective dominant negative polymorphism HuPrP(E219K). These characteristics include loss of salt bridges in the α₂–α₃ regions and the loss of π-stacking interactions in the β₂-α₂ loop. In addition, in the majority of the mutants analyzed, the α₃ helix is more flexible and the residue Tyr169 is more exposed to the aqueous solvent. The biological relevance of these findings is of utmost importance. The presence of similar traits in this large spectrum of mutations hints to a role of these characteristics in their known capabilities to generate disease-causing properties. Overall, we concluded that the regions most affected by disease-linked mutations in terms of structure and/or flexibility might be those involved in the pathogenic conversion of PrP^C to the scrapie form of the protein, and ultimately, in the interaction with cellular partners.Recent reports have indicated that the alteration of PRNP sequence by pathological mutations is sufficient to generate prions in transgenic mice.⁵ Therefore, solution-state NMR studies on PrP mutants may help identifying critical regions in PrP^C structure involved in the conversion. The comparison between the structures of Q212P and V210I mutants with the wild-type HuPrP revealed that, although structures share similar globular architecture, mutations introduce novel local structural differences.⁶ The observed variations are mostly clustered in the β₂-α₂-loop region and in the α₂–α₃ inter-helical interfaces. In contrast to the wild-type protein, where the structures of Q212P and V210I mutants point to the interruption of aromatic and hydrophobic interactions between residues located at the interface of the β₂-α₂ loop and the C-terminal end of α₃ helix. A loss of contacts between the β₂-α₂-loop and the α₃ helix in the mutants results in higher exposure of hydrophobic residues to solvent. Similar findings have also been reported in the NMR structure of the E200K mutant,⁷ X-ray structures of F198S and D178N mutants⁸ and in independent MD studies.^9–11 In addition, in the two mutants here considered side chains of Phe141 and Tyr149 adopt different orientation. Our findings indicate that the structural disorder of the β₂-α₂-loop together with the increased distance between the loop and α₃ helix represent key pathological structural features and may shed light on critical epitopes on the HuPrP structure possibly involved in the conversion to PrP^Sc.Different experimental studies suggested that the conformation of the β₂-α₂-loop plays a role in the prion disease transmission and susceptibility. Several studies have indicated that mammals carrying a flexible β₂-α₂ loop could be easily infected by prions, whereas prions are poorly transmissible to animals carrying a rigid loop.¹² Importantly, horse and rabbit have so far displayed resistance to prion infections and there are no reports of these species developing spontaneous prion diseases. NMR studies showed that their PrP structures are characterized by a rigid β₂-α₂ loop and by closer contacts between the loop and α₃ helix.^13,14 Thus, it seems that prion resistance is enciphered by the amino acidic composition of the β₂-α₂-loop and its long-range interactions with the C-terminal end of the α₃ helix.Interestingly, it has been proposed a role of α₁ helix as a promoter of PrP^C aggregation.¹⁵ In support, Tyr149 in α₁ helix is part of a motif, which may be solvent exposed in PrP^Sc and involved in structural rearrangements during fibril formation.¹⁶ Pronounced stabilization of α₁ helix in the protein may represent another important factor in the prevention of spontaneous PrP^Sc formation.Comparing the structures of the wild-type protein and the mutants enabled us to detect regions on HuPrP structure that may play a key role in the pathogenic conversion. The obtained structural data indicate that the β₂-α₂ loop and, in particular, interactions of this loop with residues in the C-terminal part of α₃ helix determine the extent of exposure of hydrophobic surface to solvent, and thus could influence propensity of PrP^C for intermolecular interactions. Moreover, our results highlight the significance of the α₁ helix and its tertiary contacts in overall stabilization of HuPrP folding.Overall, the many features discussed here involve the most important regions that confer stability to wild-type HuPrP, although the mutations considered are different for position and characteristics. In particular, the β₂-α₂-loop and the α₂–α₃ regions are the most affected in terms of structural organization and flexibility of the molecule. These two subdomains are crucial for the stability of the wild-type HuPrP^C fold¹⁷ and might play a prominent role in the early unfolding events leading to PrP^Sc conversion.     

Estrogen induces two distinct cholesterol crystallization pathways by activating ERα and GPR30 in female mice

To distinguish the lithogenic effect of the classical estrogen receptor α (ERα) from that of the G protein-coupled receptor 30 (GPR30), a new estrogen receptor, on estrogen-induced gallstones, we investigated the entire spectrum of cholesterol crystallization pathways and sequences during the early stage of gallstone formation in gallbladder bile of ovariectomized female wild-type, GPR30(^−/−), ERα(^−/−), and GPR30(^−/−)/ERα(^−/−) mice treated with 17β-estradiol (E₂) at 6 µg/day and fed a lithogenic diet for 12 days. E₂ disrupted biliary cholesterol and bile salt metabolism through ERα and GPR30, leading to supersaturated bile and predisposing to the precipitation of cholesterol monohydrate crystals. In GPR30(^−/−) mice, arc-like and tubular crystals formed first, followed by classical parallelogram-shaped cholesterol monohydrate crystals. In ERα(^−/−) mice, precipitation of lamellar liquid crystals, typified by birefringent multilamellar vesicles, appeared earlier than cholesterol monohydrate crystals. Both crystallization pathways were accelerated in wild-type mice with the activation of GPR30 and ERα by E₂. However, cholesterol crystallization was drastically retarded in GPR30(^−/−)/ERα(^−/−) mice. We concluded that E₂ activates GPR30 and ERα to produce liquid crystalline versus anhydrous crystalline metastable intermediates evolving to cholesterol monohydrate crystals from supersaturated bile. GPR30 produces a synergistic lithogenic action with ERα to enhance E₂-induced gallstone formation.     

Temporary depletion of CD11c+ dendritic cells delays lymphoinvasion after intraperitonal scrapie infection

The involvement of immune cells in prion capture and transport to lymphoid tissues still remains unclear. To investigate the role of dendritic cells (DC), we used DTR^+/+ mice, a transgenic model designed to trigger short-term ablation of DC. Transient depletion of DC around the time of intraperitoneal infection delayed prion replication in the spleen, as followed by PrPsc amount, a specific hallmark of prion diseases. Consequently, neuroinvasion and incubation time of prion disease were delayed. In contrast, no differences were observed after oral infection. These results suggest that DC act as vectors for prions from the peripheral entry site to the spleen.     

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O₂, CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20–500-fold increases in the rates of O₂ dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O₂ in both the α and β chains (ΔG_{His(E7)H-bond} ≈ −8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k_O2′, k_NO′, and calculated bimolecular rates of CO entry (k_entry′) are observed. Replacing His(E7) with Trp causes further decreases in k_O2′, k_NO′, and k_entry′ to 1–2 μm⁻¹ s⁻¹ in β subunits, whereas ligand rebinding to αTrp(E7) subunits after photolysis is markedly biphasic, with fast k_O2′, k_CO′, and k_NO′ values ≈150 μm⁻¹ s⁻¹ and slow rate constants ≈0.1 to 1 μm⁻¹ s⁻¹. Rapid bimolecular rebinding to an open α subunit conformation occurs immediately after photolysis of the αTrp(E7) mutant at high ligand concentrations. However, at equilibrium the closed αTrp(E7) side chain inhibits the rate of ligand binding >200-fold. These data suggest strongly that the E7 side chain functions as a gate for ligand entry in both HbA subunits.