The oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 influences intracellular signaling.

Signal transduction by the Toll-like receptors (TLRs) is a key component of innate immunity against many pathogens and also underlies a large burden of human diseases. Therefore, the mechanisms and regulation of signaling from the TLRs are of considerable interest. Here we seek to determine the molecular mechanism by which TLR2 and TLR4, members of the Toll-like receptor family, are activated by bacterial LPS, hyperoxia, and zymosan respectively. Our central hypothesis is that the oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 are critical for pathogen-initiated intracellular signaling as well in hyperoxia. Cysteine thiols of TLR4 and its co-receptor MD2 have been shown to aid binding between the two molecules and also bacterial LPS binding to the receptor complex. We extend these findings by demonstrating the oxidation of free thiols on the ectodomain of hTLR4, after exposure to LPS or hyperoxia suggesting that the cysteines on the ectodomain of TLR4 could form intra- or intermolecular disulfide bonds. We also demonstrated blockade of intracellular signaling from TLR4 and TLR2 by thiol-modifying compounds which suggest a novel therapeutic intervention for sepsis, hyperoxia-induced cell injury and yeast infection. In these experiments CHO-3E10, HEK293 cells expressing hTLR2 or hTLR4 and mouse peritoneal macrophages cells were pretreated with cell impermeable maleimides to alkylate thiols on the extracellular domain of TLRs, cells were then exposed to LPS, hyperoxia or zymosan. In all of these models, we detected decreased intracellular signaling from TLR2 or TLR4. Furthermore, incubation with phenyl arsine oxide - which forms stable complexes with vicinal cysteine residues - prevented LPS induced HEK293/hTLR4 intracellular signaling which was reversed by DMPS. Sequence analysis of different TLRs revealed Leucine-Rich Repeat C-terminal (LRRCT) domain that contains 4 conserved cysteines. Further work is required to pinpoint the role of each cysteine in receptor dimerization, pathogen binding, hyperoxia modulation, and intracellular signaling.

CFTR regulates B cell activation and lymphoid follicle development.

BACKGROUND: Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear. METHODS: The number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr -/-) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr -/- and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS). RESULTS: There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr -/- mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr -/- mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr -/- mice produced increased IL-6 when stimulated with LPS compared to wild type controls. CONCLUSIONS: These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.

Apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs.

Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln-/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln-/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln-/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr-/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

Complement Receptor C5aR1 Plays an Evolutionarily Conserved Role in Successful Cardiac Regeneration.

BACKGROUND: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration. METHODS: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control. RESULTS: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1. CONCLUSIONS: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration.

The Epithelial Sodium Channel Is a Modifier of the Long-Term Nonprogressive Phenotype Associated with F508del CFTR Mutations.

Cystic fibrosis (CF) remains the most lethal genetic disease in the Caucasian population. However, there is great variability in clinical phenotypes and survival times, even among patients harboring the same genotype. We identified five patients with CF and a homozygous F508del mutation in the CFTR gene who were in their fifth or sixth decade of life and had shown minimal changes in lung function over a longitudinal period of more than 20 years. Because of the rarity of this long-term nonprogressive phenotype, we hypothesized these individuals may carry rare genetic variants in modifier genes that ameliorate disease severity. Individuals at the extremes of survival time and lung-function trajectory underwent whole-exome sequencing, and the sequencing data were filtered to include rare missense, stopgain, indel, and splicing variants present with a mean allele frequency of <0.2% in general population databases. Epithelial sodium channel (ENaC) mutants were generated via site-directed mutagenesis and expressed for Xenopus oocyte assays. Four of the five individuals carried extremely rare or never reported variants in the SCNN1D and SCNN1B genes of the ENaC. Separately, an independently enriched rare variant in SCNN1D was identified in the Exome Variant Server database associated with a milder pulmonary disease phenotype. Functional analysis using Xenopus oocytes revealed that two of the three variants in δ-ENaC encoded by SCNN1D exhibited hypomorphic channel activity. Our data suggest a potential role for δ-ENaC in controlling sodium reabsorption in the airways, and advance the plausibility of ENaC as a therapeutic target in CF.

Neurokinin-1 receptor signalling impacts bone marrow repopulation efficiency.

Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1(-/-)) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 (-/-), Tac4 (-/-) and Tac1 (-/-)/Tac4 (-/-) mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.

Neprilysin null mice develop exaggerated pulmonary vascular remodeling in response to chronic hypoxia.

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

Pharmacological inhibition of CB1 cannabinoid receptor protects against doxorubicin-induced cardiotoxicity.

OBJECTIVES: We aimed to explore the effects of pharmacologic inhibition of cannabinoid-1 (CB1) receptor in in vivo and in vitro models of doxorubicin (DOX)-induced cardiotoxicity. BACKGROUND: Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Endocannabinoids mediate cardiodepressive effects through CB1 receptors in various pathophysiological conditions, and these effects can be reversed by CB1 antagonists. METHODS: Left ventricular function was measured by Millar pressure-volume system. Apoptosis markers, CB1/CB2 receptor expression, and endocannabinoid levels were determined by immunohistochemistry, Western blot, reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, flow cytometry, fluorescent microscopy, and liquid chromatography/in-line mass spectrometry techniques. RESULTS: Five days after the administration of a single dose of DOX (20 mg/kg intraperitoneally) to mice, left ventricular systolic pressure, maximum first derivative of ventricular pressure with respect to time (+dP/dt), stroke work, ejection fraction, cardiac output, and load-independent indexes of contractility (end-systolic pressure-volume relation, preload-recruitable stroke work, dP/dt-end-diastolic volume relation) were significantly depressed, and the myocardial level of the endocannabinoid anandamide (but not CB1/CB2 receptor expression) was elevated compared with vehicle-treated control mice. Treatment with the CB1 antagonists rimonabant or AM281 markedly improved cardiac dysfunction and reduced DOX-induced apoptosis in the myocardium. Doxorubicin also decreased cell viability and induced apoptosis in the H9c2 myocardial cell line measured by flow cytometry and fluorescent microscopy, which were prevented by the preincubation of the cells with either CB1 antagonist, but not with CB1 and CB2 agonists and CB2 antagonists. CONCLUSIONS: These data suggest that CB1 antagonists may represent a new cardioprotective strategy against DOX-induced cardiotoxicity.

Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

Measles virus (MV), a morbillivirus that remains a significant human pathogen, can infect the central nervous system, resulting in rare but often fatal diseases, such as subacute sclerosing panencephalitis. Previous work demonstrated that MV was transmitted trans-synaptically and that, while a cellular receptor for the hemagglutinin (H) protein was required for MV entry, it was dispensable for subsequent cell-to-cell spread. Here, we explored what role the other envelope protein, fusion (F), played in trans-synaptic transport. We made the following observations: (1) MV-F expression in infected neurons was similar to that seen in infected fibroblasts; (2) fusion inhibitory peptide (FIP), an inhibitor of MV fusion, prevented both infection and spread in primary neurons; (3) Substance P, a neurotransmitter with the same active site as FIP, also blocked neuronal MV spread; and (4) both genetic deletion and pharmacological inhibition of the Substance P receptor, neurokinin-1 (NK-1), reduced infection of susceptible mice. Together, these data implicate a role for NK-1 in MV CNS infection and spread, perhaps serving as an MV-F receptor or co-receptor on neurons.

Neurokinin-1 receptor blockade and murine lung tumorigenesis.

RATIONALE: Analogous to the adenoma-carcinoma sequence in the colon, it has been proposed that adenocarcinoma (AC) in the lung arises from adenomatous hyperplasia that progresses through atypical adenomatous hyperplasia to AC. However, the data supporting this sequence are largely circumstantial and the almost impossible task of identifying these lesions before resection rules out any longitudinal study in humans. OBJECTIVES, METHODS, AND RESULTS: We show in mice that the loss of function of the neurokinin-1 receptor (NK-1R)-due to either a pharmacologic or genetic manipulation-results in a sequence of morphologic changes in response to bleomycin treatment that precede the development of AC. We also demonstrate that a series of alterations in gene expression of proliferation markers (i.e., PCNA and Ki-67) and cell cycle regulators (i.e., FHIT, p53, and p21) characterizes the sequence of the precursor lesions. The loss of function of the NK-1R results in changes of the apoptotic rate and in a delay of DNA break recovery of alveolar epithelial cells following bleomycin treatment. The NK-1R blockade interferes with a caspase-independent pathway of apoptosis by affecting both the translocation of Nur77 into the cytoplasm and the expression of some important Bcl2 family members such as Bcl2 and Bak. CONCLUSIONS: To our knowledge, this is the first model to demonstrate a role for NK-1R in lung epithelial cell death and tumorigenesis. This animal model may provide new information on the biology of AC and will facilitate designing and testing of new therapeutic interventions.

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2.

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5a(desArg) is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.

Lack of neurokinin-1 receptor expression affects tissue mast cell numbers but not their spatial relationship with nerves.

A spatial association between mast cells and nerves has been described in both the gastrointestinal and genitourinary tracts. However, the factors that influence the anatomic relationship between mast cells and nerves have not been completely defined. It has been suggested that the high-affinity receptor for substance P [neurokinin-1 (NK1)] might modulate this interaction. We therefore assessed mast cell-nerve relationships in tissues isolated from wild-type and NK1 receptor knockout (NK1-/-) mice. We now report that, in the complete absence of NK1 receptor expression, there is a significant increase in the number of mast cells without a change in the anatomic relationship between mast cell and nerves in stomach and bladder tissues at the light microscopic level. We next determined whether transplanted mast cells would maintain their spatial distribution, number, and contact with nerve elements. For this purpose, mast cell-deficient Kit(W)/Kit(W-v) mice were reconstituted with wild-type or NK1-/- bone marrow. No differences in mast cell-nerve contact were observed. These results suggest that NK1 receptor expression is important in the regulation of the number of mast cells but is not important in the interaction between mast cells and nerves. Furthermore, the interaction between mast cells and nerves is not mediated through NK1 receptor expression on the mast cell. Further studies are needed to determine the molecular pathway involved in mast cell migration and interaction with nerve elements, but the model of reconstitution of Kit(W)/Kit(W-v) mice with mast cells derived from different genetically engineered mice is a useful approach to further explore these mechanisms.

Presynaptic localization of neprilysin contributes to efficient clearance of amyloid-beta peptide in mouse brain.

A local increase in amyloid-beta peptide (Abeta) is closely associated with synaptic dysfunction in the brain in Alzheimer's disease. Here, we report on the catabolic mechanism of Abeta at the presynaptic sites. Neprilysin, an Abeta-degrading enzyme, expressed by recombinant adeno-associated viral vector-mediated gene transfer, was axonally transported to presynaptic sites through afferent projections of neuronal circuits. This gene transfer abolished the increase in Abeta levels in the hippocampal formations of neprilysin-deficient mice and also reduced the increase in young mutant amyloid precursor protein transgenic mice. In the latter case, Abeta levels in the hippocampal formation contralateral to the vector-injected side were also significantly reduced as a result of transport of neprilysin from the ipsilateral side, and in both sides soluble Abeta was degraded more efficiently than insoluble Abeta. Furthermore, amyloid deposition in aged mutant amyloid precursor protein transgenic mice was remarkably decelerated. Thus, presynaptic neprilysin has been demonstrated to degrade Abeta efficiently and to retard development of amyloid pathology.

Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines.

Among the first cells to invade a site of infection, polymorphonuclear neutrophils (PMN) play an important role in the control of numerous infections. While PMN are considered critical for control of acute infections, their role in chronic infections remains less well understood. Here we report that PMN are essential for accurate early granuloma formation during chronic M. tuberculosis infection without influencing mycobacterial growth restriction. The PMN-mediated regulation of granuloma formation depended on chemokines signaling through CXCR3, in particular MIG, as indicated by immune histochemical analysis of lung sections from C57BL/6 wild-type and CXCR3(-/-) mutant mice and supported by microarray transcriptome analysis. Hence, PMN play a central role in regulating the focal granulomatous response in the lung, and this early granuloma formation can be segregated from long-term protection against pulmonary M. tuberculosis infection.

C5L2, a nonsignaling C5A binding protein.

C5a anaphylatoxin, a potent inflammatory mediator, is known to act through a specific G protein coupled receptor. However, some of the complex effects of C5a in vivo may not be explained solely by the deletion of the known receptor. Here, we show that an orphan receptor, identified as C5L2, is a high affinity C5a binding protein. Unlike the previously described C5aR, C5L2 is obligately uncoupled from heterotrimeric G proteins, in part by virtue of an amino acid alteration in the so-called DRY sequence at the end of the third transmembrane segment. Both human and murine C5L2 bear a leucine for arginine replacement at this site. C5L2, when transfected into several cell types, is weakly phosphorylated in transfected cells following binding of C5a but does not induce significant activation of MAP kinases, mediate calcium flux, or stimulate chemotaxis. Bone marrow cells from wild type respond robustly to C5a with induction and suppression of a number of inflammation related genes. In contrast, C5a receptor deficient mice, which bear C5L2 alone, do not respond to C5a with changes in gene transcription by microarray analyses. Biophysical properties of the C5L2, including slow ligand on and off rates, absence of internalization, and relatively high affinity for the C5a des Arg metabolite, suggest that this receptor may serve to modulate C5a biological functions in vivo. Finally, in contrast to previous reports, we find absolutely no interaction of C5L2 with other anaphylatoxins C3a and C4a.

Neurokinin 1 receptors and neprilysin modulation of mouse bladder gene regulation.

Neurokinin 1 (NK(1)) receptors play a fundamental role in neurogenic inflammation. We sought to determine the mechanisms downstream from NK(1) receptor (NK(1)R) activation using cDNA arrays and a novel statistical method to analyze gene expression. We used female NK(1)R(-/-) and wild-type (WT) mice that were sensitized actively by intraperitoneal injections of dinitrophenol 4 (DNP(4))-human serum albumin. Cystitis was induced by intravesical instillation of antigen of DNP(4)-ovalbumin, and control mice were challenged with saline. At 1, 4, and 24 h after instillation, bladders were removed for 1) RNA extraction (n = 3), 2) replicate of RNA extraction (n = 3), and 3) morphological analysis (n = 6). For cDNA array experiments, three bladders from each group were homogenized, and total RNA was obtained. DNase-treated RNA was reverse-transcribed to cDNA, labeled with [alpha-(32)P]dATP and hybridized to Atlas Mouse 1.2 Arrays (Clontech). After calculating the mean and SD for background spots, each experimental value was assigned a normalized score S using the formula S' = (S - Av)/SD, where S' is the original pixel value, and Av and SD are the mean and standard deviation of background spots, respectively. Only genes that expressed 3 SD values above background were used. Hypervariable genes were sorted by cluster analysis. Matrices of correlation coefficients were calculated and represented in a connectivity mosaic. As results, we found that in WT mice the most prominent gene cluster had neprilysin in a central position and positively correlated to a group of activator protein-1 (AP-1)-responsive genes, including laminin-alpha3, tissue plasminogen activator 11, fos-B, and TNF-beta. In WT mice, antigen-induced bladder inflammation led to a downregulation in neprilysin expression. In contrast, NK(1)R(-/-) mice failed to mount an inflammatory reaction and presented neprilysin negatively correlated with the same genes described in WT. In conclusion, this work indicates an overriding participation of NK(1)R and neprilysin in bladder inflammation, provides a working model for the involvement of AP-1 transcription factor, and evokes testable hypotheses regarding the role of NK(1)R and neprilysin in inflammation.

Mast cells mediate substance P-induced bladder inflammation through an NK(1) receptor-independent mechanism.

The role of neurokinin-1 receptors (NK1R) in the interaction between mast cells and substance P (SP) in bladder inflammation was determined. Mast cell-deficient Kit(W)/Kit(W-v), congenic normal (+/+), and Kit(W)/Kit(W-v) mice that were reconstituted with bone marrow cells isolated from NK1R(-/-) mice were challenged by instillation of SP, antigen, or saline into the urinary bladder. Twenty-four hours after challenge, the bladders were prepared for morphological assessment and gene expression. SP-induced bladder inflammation was mast cell dependent and did not require NK1R expression on the mast cell. Cluster analysis identified functionally significant genes that were dependent on the presence of mast cells for their upregulation regardless of stimulus. Those include serine protein inhibitor 2.2, maspin, mitogen- and stress-activated protein kinase 2, and macrophage colony-stimulating factor 1. Our findings demonstrate that while mast cells are essential for both antigen- and SP-induced bladder inflammation, there are common genes and unique genes expressed in each type of inflammatory reaction. When combined with unique animal models, gene array analysis provides a useful approach for identifying and characterizing pathways involved in bladder inflammation.

Tyrosine-sulfated peptides functionally reconstitute a CCR5 variant lacking a critical amino-terminal region.

Entry of most primary human immunodeficiency virus, type 1 (HIV-1) isolates into their target cells requires the cellular receptor CD4 and the G protein-coupled chemokine coreceptor CCR5. An acidic, tyrosine-rich, and tyrosine-sulfated domain of the CCR5 amino terminus plays a critical role in the ability of CCR5 to serve as an HIV-1 coreceptor, and tyrosine-sulfated peptides based on this region physically associate with the HIV-1 envelope glycoprotein gp120 and slow HIV-1 entry into CCR5-expressing cells. Here we show that the same tyrosine-sulfated peptides, but not their unsulfated analogs, can restore the HIV-1 coreceptor activity of a CCR5 variant lacking residues 2-17 of its amino terminus. Additionally, these sulfated peptides restored the ability of this CCR5 variant to mobilize calcium in response to the chemokines macrophage inflammatory factors 1alpha and 1beta. These observations show that a tyrosine-sulfated region of the CCR5 amino terminus can function independently to mediate association of chemokines and the HIV-1 envelope glycoprotein with the remaining domains of CCR5.

Abeta-degrading endopeptidase, neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology.

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.