A Highly Toxic Cellular Prion Protein Induces a Novel,Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(ΔCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(ΔCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.Mechanisms of neuronal death have been studied intensively to gain insight into the pathological processes associated with acute and chronic neurological illnesses. Prion diseases are fatal neurodegenerative disorders of humans and animals that are accompanied by conversion of the cellular prion protein (PrP^C) into a conformationally altered isoform (PrP^Sc) that is infectious in the absence of nucleic acid.¹ Although the basic principles of prion propagation are understood, the mechanism by which abnormal forms of PrP cause neuronal death remains obscure. Membrane-anchored PrP^C is required to transduce neurotoxic signals elicited by pathogenic forms of PrP, suggesting that a normal biological activity of PrP^C may be altered during the disease process.^2,3,4,5 However, the cellular pathways and molecular components involved in this mechanism have yet to be identified.A window into the neurotoxic potential of PrP comes from transgenic mice that express PrP molecules carrying deletions within the unstructured N-terminal half of the protein. It was originally reported that mice expressing PrPΔ32-121 or Δ32-134 (collectively referred to as PrPΔN) spontaneously develop a neurodegenerative illness characterized by massive degeneration of cerebellar granule neurons (CGNs) and by white matter abnormalities.^6,7 Remarkably, this phenotype was exhibited only in the absence of endogenous PrP, and introduction of even a single Prn-p allele encoding wild-type PrP was sufficient to completely prevent the disease.⁶To further define the sequence determinants of neurotoxicity, we previously generated Tg(ΔCR) transgenic mice expressing PrP with a smaller deletion (residues 105-125) within the highly conserved central region of the protein.⁸ Tg(ΔCR) mice die within the first week of life on the Prn-p^0/0 background, and supraphysiological (5X) expression of wild-type PrP is necessary to confer survival beyond 1 year.⁸ Like Tg(PrPΔN) mice, Tg(ΔCR) animals display dramatic degeneration of CGNs and vacuolation of white matter regions.⁸ Importantly, PrP(ΔCR) is identical to PrP^C in terms of its solubility, protease sensitivity, and localization in cultured cells.^8,9 Thus, we hypothesize that deletion of critical residues in the central region of PrP^C alters a physiological activity of the protein rather than converting it to a misfolded state. Other PrP deletion mutants encompassing this region are likely to act via a similar mechanism.¹⁰To categorize the type of neuronal death induced by deleted forms of PrP, we have performed a combined biochemical, histological, and ultrastructural analysis of the brains of Tg(ΔCR) mice. We discovered that neuronal loss in these animals does not occur through either apoptosis or autophagy. By electron microscopy, we observed a novel morphology in degenerating CGNs that is reminiscent of certain forms of excitotoxic neuronal death. The same morphology was present in mice expressing PrPΔ32-134, suggesting that a common nonapoptotic mechanism may underlie the neurotoxic activity of PrP proteins lacking the critical central region. Our study has implications for understanding PrP-related cell death pathways, and it represents a starting point for designing therapeutic strategies.     

Neuroprotective Function of Cellular Prion Protein in a Mouse Model of Amyotrophic Lateral Sclerosis

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1^G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1^G93APrp−/− mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1^G93APrp+/+ mice. Additionally, during disease progression, SOD1^G93APrp−/− mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1^G93APrp−/− mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1^G93APrp−/− versus SOD1^G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-κB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1^G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling.Amyotrophic lateral sclerosis is characterized by rapid degeneration of motor neurons in the spinal cord, brain stem, and cortical Betz cells. As a result, focal muscle wasting, weakness, and spasticity develop focally. These symptoms ultimately lead to global paralysis. Patients usually die due to respiratory failure within 3 years of symptom onset.¹The causes of ALS are diverse; 10 to 15% of cases are familial with autosomal dominant inheritance, and 20% of these are related to point mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1). SOD1 is a ubiquitously expressed homodimeric protein that catalyzes the reaction of O₂⁻ to O₂ and H₂O₂, which is then further metabolized by glutathione peroxidase. Mice overexpressing human mutated SOD1 (muSOD1) linked to ALS, develop disease resembling ALS in humans by a toxic gain of function.² Several properties of muSOD1 were proposed to contribute to toxic gain of function, including enhanced peroxidase activity and formation of peroxynitrite, changes in copper and zinc binding, and aggregation of the enzyme. ALS progression is accompanied by oxidative stress processes, glutamate-induced excitotoxicity, cytoskeletal abnormalities, inflammatory processes, and toxicity via extracellular muSOD1.^2,3 The apoptotic cascade is activated in the ALS model, shown by sequential activation of caspase-1 and −3.⁴ Interestingly, Bcl-2 overexpression had a neuroprotective effect and, like intrathecal administration of caspase inhibitors, led to a slowed disease progression and increased life span in these mice.^5,6 More recently, it was proposed that not only neurons themselves contribute to the neurodegenerative process in ALS but also non-neuronal cells, particularly microglia and astrocytes.^7–10 Expression of muSOD1 in non-neuronal cells was sufficient to induce cell death in nearby motor neurons lacking muSOD1.^11,12Since an early decrease of prion protein (Prp) mRNA has been described in an ALS model,¹³ loss of Prp function might contribute to the neurodegenerative process not only in prion diseases but also in ALS. A number of physiological functions have meanwhile been attributed to Prp, including antioxidative and anti-apoptotic properties and involvement in transmembrane signaling and cell adhesion.¹⁴ Additionally, as recently reported in cell culture models, Prp regulates astrocytic signaling^15,16 and thereby might also influence neuron−glia signaling relevant for ALS pathogenesis. Clarifying the role of Prp in models of neurodegeneration is of special interest, since Prp as membrane protein might become an easily accessible drug target for treatment of neurodegenerative diseases as a spin-off of the current search for antiprion drugs.¹⁷In the present study, we analyzed the function of physiological Prp in a mouse model of ALS by cross-breeding mice transgenic for human SOD1 with G93A mutation (SOD1^G93A) with Prp knockout (Prp−/−) mice. Characterization of the SOD1^G93APrp−/− mice concerning motoric properties, disease progression, and life span, paralleled by histopathological, immunochemical, and proteomic analyses, revealed that Prp has a protective role potentially by influencing mechanisms assuring neuronal survival.     

Immunohistochemistry for the Prion Protein: Comparison of Different Monoclonal Antibodies in Human Prion Disease Subtypes

Demonstration of the abnormal form of the prion protein (PrP) in the brain confirms the diagnosis of human prion disease (PrD). Using immunohistochemistry, we have compared ten monoclonal antibodies in PrD subtypes including sporadic and variant Creutzfeldt-Jakob disease (CJD), fatal familial insomnia, Alzheimer's disease (AD), and control brains. CJD subgroups were determined using Western blot analysis for the protease-resistant PrP type in combination with sequencing to determine the genotype at the methionine/valine polymorphism at codon 129 of the prion protein gene. None of the antibodies labeled given subgroups exclusively, but the intensity of immunoreactivity varied among morphologically distinct types of deposit. Fine granular or synaptic PrP deposits stained weakly or not at all with antibodies against the N-terminus of PrP, and were visible in one case only with 12F10 and SAF54. Coarser and plaque type deposits were immunolabeled with all antibodies. The immunostaining patterns appear characteristic for the disease subgroups. Labeling of certain neurons in all cases irrespective of disease, and staining at the periphery and/or throughout the senile plaques of AD patients were also noted. Antibodies such as 6H4 and 12F10 failed to give this type of labeling and are therefore less likely to recognise non-pathological PrP material in immunohistochemistry.     

Lmb, a Protein with Similarities to the LraI Adhesin Family, Mediates Attachment of Streptococcus agalactiae to Human Laminin

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.     

Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrP^Sc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrP^Sc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrP^Sc). Although evidence suggests that sPrP^Sc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte''s ability to degrade PrP^Sc. Furthermore, the cell does not appear to distinguish between sPrP^Sc and protease-resistant PrP^Sc, suggesting that sPrP^Sc could contribute to prion infection.Prion diseases, or transmissible spongiform encephalopathies, are rare fatal neurologic disorders of mammals that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep. Prion diseases are characterized by the conversion of normal prion protein (PrP^C) into a disease-associated and aggregated isoform (PrP^Sc), which is thought to be the main component of the infectious agent or prion (reviewed in Priola and Vorberg¹). PrP^C is a glycoprotein that contains two N-linked glycosylation sites^2,3 and is bound to the plasma membrane via a glycosyl-phosphatidyl-inositol anchor.⁴ Although PrP^C is detergent soluble and fully susceptible to proteolytic degradation, PrP^Sc has an increased detergent insolubility and partial resistance to proteinase K (PK).⁵ The presence of amino-terminally truncated, PK-resistant core fragments of PrP^Sc (rPrP^Sc) after limited proteolysis is considered the most reliable diagnostic marker for prion infection,^6,7 and biochemical profiles of rPrP^Sc based on molecular mass and/or the degree of glycosylation are used to help differentiate distinct prion disease phenotypes in humans.^8–11In recent years, alternative approaches for analyzing PrP^Sc that do not rely on the enzymatic removal of PrP^C have indicated that not all forms of PrP^Sc are necessarily resistant to proteolytic treatment. The conformation-dependent immunoassay, which uses conformational differences between the N termini of PrP^C and PrP^Sc, has found that often a majority of PrP^Sc present in prion-affected brains is susceptible to proteolytic degradation.^12–15 In sporadic CJD (sCJD), this PK-sensitive species of aggregated PrP^Sc, termed sPrP^Sc, was found in some cases to account for up to 90% of the total PrP^Sc.^13,16 Careful analysis of the size distribution of PrP^Sc has also found that sPrP^Sc forms much smaller aggregates than rPrP^Sc.^17,18 Thus, sPrP^Sc appears to represent a population of PrP^Sc aggregates which tends to be both smaller and more protease sensitive than rPrP^Sc.Evidence suggests that, like rPrP^Sc, sPrP^Sc has seeding activity and can convert PrP^C to protease resistance.^18,19 It has also been associated with prion infectivity²⁰ and may influence the incubation time of prion disease.²¹ Although these data suggest that PK-sensitive disease-associated PrP aggregates may be actively involved in prion pathogenesis, no studies have been performed to determine how this population of PrP aggregates might interact with cells and influence prion infection. In this study, we have looked at the uptake and degradation of PK-sensitive and PK-resistant disease-associated PrP aggregates in an established human astrocyte cell line. Our results indicate that, despite their biochemical differences, PK-sensitive PrP aggregates are taken up and degraded similarly to PK-resistant PrP aggregates, suggesting that relative PK resistance does not significantly influence the cell''s ability to degrade PrP^Sc. Thus, the astrocyte does not appear to distinguish between sPrP^Sc and rPrP^Sc aggregates, suggesting that sPrP^Sc could be involved in prion pathogenesis.     

Mycobacterial Dormancy Regulon Protein Rv2623 as a Novel Biomarker for the Diagnosis of Latent and Active Tuberculous Meningitis

The present study was designed to investigate Rv2623 antigen, a major dormancy regulon protein of Mycobacterium tuberculosis (MTB) in CSF of suspected latent and active tuberculous meningitis (TBM) patients. A total of 100 CSF samples from TBM (n = 31), suspected latent TBM (n = 22), and suitable noninfectious control subjects (n = 47) were collected and evaluated for Rv2623 antigen level using ELISA protocol. A significantly high (P < 0.05) mean absorbance was observed in samples of suspected latent TBM and active TBM patients as compared to non-TBM control patients. However, no significant difference in Rv2623 level was observed between suspected latent TBM and TBM patients. Our preliminary findings suggest that Rv2623 may be useful as a potential biomarker for the diagnosis of the latent as well as active TBM infection. Futher evaluation of this biomarker in large number of samples is therefore needed to confirm the result.     

Ultrastructural Identification of Protein Bodies,Cellular Markers of Human Catecholamine Neurons,in a Temporal Lobe Ganglioglioma

A temporal lobe ganglioglioma, surgically removed from an 8-year-old body, and a human brainstem at the level of locus coeruleus (LC) were processed for light microscopy (LM), with formalin fixation and paraffin embedding, and for electron microscopy (EM) with glutaraldehyde fixation, potassium permanganate postfixation, phosphotungstic acid-hematoxylin block-staining, and epoxy-resin embedding. The paraffin sections were stained with toluidine blue O/rhodamine B and observed under epi-fluorescence. The thin sections for EM were viewed directly without further staining. The neuronal neoplastic cells of ganglioglioma and the neurons of LC are known to produce catecholamines. Both also contain spherical protein bodies (pb), cellular markers that identify catecholamine neurons in humans. The ultrastructural characteristics of the pb in LC were compared with those of the pb in neoplastic ganglion cells. These bodies had an identical ultrastructure, in both tissues, consisting of electron-lucent core surrounded by an electron-dense thin rim. The rhodamine B-stained sections also emphasized the identical morphology of the pb in ganglioglioma and LC. Based on the EM comparison, these brightly fluorescing spherical bodies are ideal markers for identifying in LM, the clusters of large neoplastic cells, representing neurons, which are the most important clue to the correct diagnosis of gangliogliomas.     

Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis, is a disseminated, systemic disorder that involves the lungs and other organs. The ability of the pathogen to interact with host components, including extracellular matrix (ECM) proteins, is essential to further colonization, invasion, and growth. Previously, enolase (EC 4.2.1.11) was characterized as a fibronectin binding protein in P. brasiliensis. Interaction of surface-bound enolase with plasminogen has been incriminated in tissue invasion for pathogenesis in several pathogens. In this paper, enolase was expressed in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein (recombinant P. brasiliensis enolase [rPbEno]). The P. brasiliensis native enolase (PbEno) was detected at the fungus surface and cytoplasm by immunofluorescence with an anti-rPbEno antibody. Immobilized purified rPbEno bound plasminogen in a specific, concentration-dependent fashion. Both native enolase and rPbEno activated conversion of plasminogen to plasmin through tissue plasminogen activator. The association between PbEno and plasminogen was lysine dependent. In competition experiments, purified rPbEno, in its soluble form, inhibited plasminogen binding to fixed P. brasiliensis, suggesting that this interaction required surface-localized PbEno. Plasminogen-coated P. brasiliensis yeast cells were capable of degrading purified fibronectin, providing in vitro evidence for the generation of active plasmin on the fungus surface. Exposure of epithelial cells and phagocytes to enolase was associated with an increased expression of surface sites of adhesion. In fact, the association of P. brasiliensis with epithelial cells and phagocytes was increased in the presence of rPbEno. The expression of PbEno was upregulated in yeast cells derived from mouse-infected tissues. These data indicate that surface-associated PbEno may contribute to the pathogenesis of P. brasiliensis.Microbial adhesion to host tissues is the initial event of most infectious process (39). Interaction with extracellular matrix (ECM) proteins has been correlated with the invasive abilities of different organisms (28, 40). ECM underlines epithelial and endothelial cells and surrounds connective tissues, and its major components are the collagens, laminin, fibronectin, and proteoglycans (52). After adherence, the next step must be to overcome the barriers imposed by epithelial tissues and ECM. The proteolytic activity achieved by subversion of host proteases by pathogens, such as plasmin, has been shown to be important during the process of many types of infections (47, 51).Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis that constitutes a major health problem in South America (44). Clinical manifestations of PCM are related to chronic granulomatous reactions with involvement of the lungs and the reticuloendothelial system, as well as mucocutaneous areas and other organs (22). In the soil, the fungus grows as saprobic mycelium, resulting in the formation of infectious propagules. After penetrating the host, the fungus differentiates into its yeast form, a fundamental step for the successful establishment of the disease (46).Although P. brasiliensis is not traditionally considered a typical intracellular pathogen, independent studies have demonstrated that P. brasiliensis yeast cells have the capacity to adhere and invade host cells (4, 24, 31). P. brasiliensis may actively penetrate the mucocutaneous surface and parasitize epithelial cells, thus evading the host defenses and reaching deeper tissues.Fungal ECM-binding adhesins have been characterized in different models, including P. brasiliensis. Vicentini et al. (49) showed specific binding of the protein gp43 to laminin, which is correlated to the adhesiveness of the fungus in vitro as well as to an enhancement of pathogenic potential. We have been systematically searching for new adhesion proteins in P. brasiliensis that have the potential to play roles in the fungal virulence, and proteins such as P. brasiliensis malate synthase (PbMLS) (34), PbDfg5p (defective for filamentous growth protein) (10), triosephosphate isomerase (PbTPI) (41), and glyceraldehyde-3-phosphate dehydrogenase (PbGAPDH) (4) were found to associate with ECM components. In particular, enolase from P. brasiliensis (PbEno) is a fibronectin-binding protein, as characterized by affinity ligand assays (17).The importance of plasminogen in infectious diseases is supported by the fact that many pathogens manifest the ability to bind plaminogen (47, 13). Plasminogen is a single-chain glycoprotein with a molecular mass of 92 kDa. Its protein structure comprises an N-terminal preactivation peptide, five consecutive disulfide-bonded triple-loop kringle domains, and a serine-protease domain containing the catalytic triad (48). The kringle domains of plasminogen mediate its attachment to cells surfaces by binding proteins with accessible carboxyl-terminal or internal lysine residues. The plasminogen system displays a unique role in the host defense by dissolving fibrin clots and serving as an essential component to maintain homeostasis (43). Activation of the fibrinolytic system is dependent on the conversion of plasminogen to the serine-protease plasmin by the physiological activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) (9). Plasmin is involved in fibrinolysis homeostasis and degradation of the extracellular matrix and basement membrane. The mammalian plasminogen-plasmin proteolytic system plays a crucial role in extracellular matrix degradation which is exploited by invasive pathogens, including fungi (25, 47). Microbe-derived plasminogen conversion to plasmin may promote dissemination of the pathogen within the host (1).Among several proteins, enolase has been found to play a major role in microbial recruitment of plasminogen (32). By serving as a key surface receptor for plasminogen recruitment, enolase has been shown to function as mediator of microbial virulence (6, 15). The potential of P. brasiliensis to recruit human plasminogen for invasion and virulence has not been studied until now. In this study, we demonstrated for the first time that P. brasiliensis is capable of recruiting plasminogen and activating the plasminogen fribrinolytic system in a process, at least in part, mediated by the cell wall-localized enolase. Furthermore, recombinant PbEno (rPbEno) promoted an increase in the adhesion/invasion of P. brasiliensis in in vitro models of infection, a process that seems to be associated with the enolase ability of modifying the surface of host cells. These data suggest that PbEno may play a role in mediating the P. brasiliensis recruitment of plasminogen as well as in attachment and internalization of the fungus to host tissues, potentially playing a role in the establishment of PCM.     

Analysis of the Genomic and Derived Protein Structure of a Novel Human Serum Amyloid A Gene, SAA4

We have determined the structure of the novel SAA gene, SAA4. The gene is 6.2 kb in length and comprises three introns and four exons. Introns 2 and 3 are significantly longer than those of the other human SAA genes. We have sequenced the exons and junction fragments and have shown that the sequence is the same as c-SAA[1] and does not correspond to the pseudogene carried on GSAA4[2]. The predicted SAA4 protein sequence has an eight amino acid insertion relative to the other human SAA proteins and is more closely related to rabbit and mouse SAA proteins than to the other human SAA proteins, or to those of animal species which also possess an insertion. We have analysed the predicted SAA4 protein relative to the other human SAA proteins and have identified three important structural regions. We predict that region 1 of SAA4 represents a lipid binding domain. Region 2 forms an extensive, distinctive, hydrophobic beta sheet region in place of a helical region. In region 3, SAA4 is the only SAA protein having an alpha helix which is not amphipathic. We predict that the SAA4 protein retains a modified function of the conserved region, retains the Ca2+ binding site, has an amino terminal surface site and has a potentially distinct secretion pattern. Together, these differences indicate a distinct function from those of the other SAA proteins.     

Serum Arginase,a Biomarker of Treatment Efficacy in Human African Trypanosomiasis

Arginase serum levels were increased in human African trypanosomiasis patients and returned to control values after treatment. Arginase hydrolyzes l-arginine to l-ornithine, which is essential for parasite growth. Moreover, l-arginine depletion impairs immune functions. Arginase may be considered as a biomarker for treatment efficacy.     

Human Immunodeficiency Virus (HIV) Infection of Human Macrophages Is Increased by Dopamine : A Bridge between HIV-Associated Neurologic Disorders and Drug Abuse

The prevalence of human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) that result from HIV infection of the central nervous system is increasing. Macrophages, the primary target for HIV within the central nervous system, play a central role in HIV-induced neuropathogenesis. Drug abuse exacerbates HAND, but the mechanism(s) by which this increased neuropathology results in more severe forms of HAND in HIV-infected drug abusers is unclear. The addictive and reinforcing effects of many drugs of abuse, such as cocaine and methamphetamine, are mediated by increased extracellular dopamine in the brain. We propose a novel mechanism by which drugs of abuse intensify HIV neuropathogenesis through direct effects of the neurotransmitter dopamine on HIV infection of macrophages. We found that macrophages express dopamine receptors 1 and 2, and dopamine activates macrophages by increasing ERK 1 phosphorylation. Our results demonstrate for the first time that dopamine increases HIV replication in human macrophages and that the mechanism by which dopamine mediates this change is by increasing the total number of HIV-infected macrophages. This increase in HIV replication is mediated by activation of dopamine receptor 2. These findings suggest a common mechanism by which drugs of abuse enhance HIV replication in macrophages and indicate that the drug abuse-heightened levels of central nervous system dopamine could increase viral replication, thereby accelerating the development of HAND.Human immunodeficiency virus (HIV) enters the central nervous system (CNS) soon after initial infection,¹ resulting in ongoing inflammation and neurological damage that leads to the development of HIV-associated neurocognitive disorders (HAND) in as many as 50% of infected individuals.^2,3 The prevalence of these complications is increasing despite the advent of antiretroviral therapy, due to the longer lifespan of infected individuals on antiretroviral therapies⁴ and the poor ability of most antiretroviral drugs to penetrate the blood-brain barrier.^5,6 HIV is thought to enter the brain through the transmigration of infected monocytes across the blood-brain barrier.^7,8,9,10 Within the CNS, macrophages are the primary source of HIV and the virus spreads primarily through infection of brain macrophages and microglia.^11,12 Infected macrophages produce numerous factors that are neurotoxic and contribute to the neurological damage that occurs in HIV-infected individuals.^13,14,15 Thus, HIV infection and replication within CNS macrophages plays a central role in the development of HANDs.The incidence and severity of HAND are exacerbated by drugs of abuse, such as the psychostimulants cocaine and methamphetamine,^16,17,18,19 which have been shown to increase both HIV neuropathogenesis and viral replication.^{20,21,22,23,24} However, the mechanism(s) by which drugs of abuse enhance HIV-related neuropathologies are not well understood. Dopamine (DA), a neurotransmitter involved in the control of locomotion, cognition, positive reinforcement, and neuroendocrine secretion,²⁵ is central to the action of drugs of abuse. Psychostimulants such as cocaine and methamphetamine exert addictive and reinforcing effects through elevation of extracellular DA levels in the CNS.^{26,27,28,29,30} The use of both cocaine and methamphetamine generates extracellular CNS dopamine levels far higher than those found in the brains of non-drug-abusers.^{26,31,32,33,34,35,36}Dopamine acts through dopamine receptors, which are members of the G-protein coupled seven transmembrane domain family of receptors. Dopamine receptors (DRs) are divided into two subtypes designated D1-like DRs, comprised of dopamine receptor 1 (D1R) and D5R, and D2-like DRs, comprised of D2R, D3R, and D4R.²⁵ Classically, DRs have been studied on neurons, but DR expression has also been reported in several types of peripheral blood leukocytes, including T lymphocytes and monocytes.^37,38,39 Dopamine receptors have been shown to modulate the immune function of T lymphocytes.^25,40,41 A recent study showed D1R on human macrophages,²⁴ but the expression of other DRs and the functions of DRs in this cell type have not been well characterized.In studies with simian immunodeficiency virus-infected macaques, injection with or oral administration of L-DOPA, a DA precursor that crosses the blood-brain barrier, or selegiline, a blocker of DA breakdown by monoamine oxidase, resulted in increased levels of simian immunodeficiency virus in the CNS.^42,43 In addition, infected macaques exhibited an increased incidence of simian immunodeficiency virus encephalitis and induction of a spongiform polioencephalopathy in dopaminergic regions of the CNS.^42,43 These studies suggest that the enhanced extracellular DA elicited by use of drugs like cocaine and methamphetamine could be sufficient to increase HIV replication in the CNS and exacerbate HIV neuropathogenesis. Macrophages play a central role in the development of HIV-induced neuropathology. Thus, examination of DA modulation of HIV infection of macrophages, as well as the characterization of intracellular signaling pathways that are involved in the DA-mediated increase in HIV infectivity, are important to the identification of mechanisms by which drugs of abuse enhance the development of HAND.This report demonstrates that primary human monocyte-derived macrophages (MDMs) inoculated with HIV in the presence of DA exhibit increased levels of viral replication when compared with MDMs inoculated with HIV in the absence of DA. The DA-induced increase in viral replication correlated with an increase in the percentage of MDMs infected with HIV. HIV infection in the presence of the D2R agonist, quinpirole, increased viral replication similarly to DA, while infection in the presence of the D1R agonist, SKF 82958, did not alter HIV replication, suggesting that D2R is involved in the DA-mediated increase in HIV replication. The data also confirm that uninfected MDMs expressed both D1R and D2R on the cell surface and show that endogenous macrophage D2R was active by demonstrating that DA induced extracellular signal regulated kinase 1 (ERK 1) phosphorylation in macrophages through D2R. These results suggest that dopamine-induced increases in HIV replication in macrophages may be an important mechanism by which specific drugs of abuse, characterized by their ability to increase extracellular DA levels in the CNS, exacerbate the neuropathogenesis of HIV infection.     

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.Extracellular matrix (ECM) calcification can be either a physiological or a pathological process depending on site and time of occurrence. Physiological ECM calcification is restricted to bone and to the hypertrophic zones of growth plate cartilage, whereas pathological or ectopic ECM calcification, defined as inappropriate biomineralization occurring in soft tissues and consisting of calcium phosphate salts that include hydroxyapatite, is an abnormal process that can occur virtually in any tissue of the body.¹ However, skin, kidney, tendons, and the cardiovascular system appear particularly prone to develop this pathology.²First considered to be a passive process occurring as a nonspecific response to tissue injury or necrosis, recent evidence now indicates that ECM calcification is a naturally occurring process that must be actively inhibited and starts to appear as soon as inhibitors are removed from the matrix.^1,3,4 In a healthy organism, cells appear to synthesize natural inhibitors of mineralization that prevent ectopic calcification, which initiates when disequilibrium occurs between expression of calcification inhibitors and enhancers, emphasizing the need for a tight regulation to prevent ectopic calcifications.Key genes known to be involved in the regulation of this complex process are those acting as calcification inhibitors such as matrix Gla protein (MGP), osteocalcin (BGP), bone sialoprotein (BSP), osteoprotegerin (Opg), and fetuin.^1,3 Among those, MGP, a vitamin K-dependent protein (VKD), is widely accepted as playing a pivotal role in preventing soft tissue calcification, local mineralization of the vascular wall,⁵ and more recently, skin elastic fiber mineralization in pseudoxanthoma elasticum (PXE)^6,7,8 and in scleroderma with and without calcinosis.⁹ It is also known that several factors, such as insufficient intake of vitamin K, mutations in the γ-carboxylase enzyme, and warfarin treatment, which can all induce arterial^10,11,12 and skin calcifications,^7,13,14,15 may act by reducing or abolishing γ-carboxylation of VKD proteins. Those pathologies have also been associated with a loss of MGP function, until now considered to be the central Gla protein for prevention of connective tissue mineralization, both in the vascular system and skin. Although many efforts have been made to understand the mechanisms controlling these abnormal calcifications, the existence of other potential, still unknown, calcification inhibitors has been suggested to explain some reported phenotypes and occurrences that are not completely justified by the presence or absence of MGP.^1,16,17We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), with an unprecedented high content of Gla residues and uncommonly high capacity to bind calcium, with orthologs in all taxonomic groups of vertebrates and highly conserved throughout evolution (78% identity between sturgeon and human GRP).¹⁸ GRP mRNA was found to be highly expressed in sturgeon cartilaginous tissues, and in rat skeletal tissues, both cartilage and bone, which invalidated the concept that this protein could be solely a specific marker for distal chondrocytes, as previously proposed by others.¹⁸ In this study we show, for the first time, that GRP is a circulating protein also expressed and accumulated in soft tissues like skin and vascular system of rats and humans and that it is clearly associated with calcification pathologies in these tissues, being highly accumulated at sites of ectopic mineral deposits. Furthermore, the extensive number of Gla residues (16 Gla residues in sturgeon and, by comparison, 15 in all mammals) and the absence of other identifiable functional domains, together with our in vivo and in vitro evidence for a high mineral binding affinity, strongly suggest that GRP might be a potent physiological modulator of soft tissue calcification, acting by directly influence mineral formation and or recruitment, and an important new player in the complexity of phenotypes involving connective tissue mineralization, whose mechanisms and regulatory pathways remain to be fully understood.     

Novel Laminin-Binding Protein of Streptococcus pyogenes, Lbp, Is Involved in Adhesion to Epithelial Cells

The lbp gene, which encodes a laminin-binding protein (Lbp) of Streptococcus pyogenes, was found in all S. pyogenes M types. An Lbp-deficient mutant showed a significantly lower efficiency of adhesion to HEp-2 cells than did the wild-type strain. These results indicate that Lbp is one of the important S. pyogenes adhesins.