Metabolic Profiling of Mice with Deletion of the Orphan G Protein-Coupled Receptor, GPR37L1.

Understanding the neurogenic causes of obesity may reveal novel drug targets to counter the obesity crisis and associated sequelae. Here, we investigate whether the deletion of GPR37L1, an astrocyte-specific orphan G protein-coupled receptor, affects whole-body energy homeostasis in mice. We subjected male Gpr37l1-/- mice and littermate wildtype (Gpr37l1+/+, C57BL/6J background) controls to either 12 weeks of high-fat diet (HFD) or chow feeding, or to 1 year of chow diet, with body composition quantified by EchoMRI, glucose handling by glucose tolerance test and metabolic rate by indirect calorimetry. Following an HFD, Gpr37l1-/- mice had similar glucose handling, body weight and fat mass compared with wildtype controls. Interestingly, we observed a significantly elevated respiratory exchange ratio in HFD- and chow-fed Gpr37l1-/- mice during daylight hours. After 1 year of chow feeding, we again saw no differences in glucose and insulin tolerance or body weight between genotypes, nor in energy expenditure or respiratory exchange ratio. However, there was significantly lower fat mass accumulation, and higher ambulatory activity in the Gpr37l1-/- mice during night hours. Overall, these results indicate that while GPR37L1 may play a minor role in whole-body metabolism, it is not a viable clinical target for the treatment of obesity.

Involvement of GPR37L1 in murine blood pressure regulation and human cardiac disease pathophysiology.

Multiple mouse lines lacking the orphan G protein-coupled receptor, GPR37L1, have elicited disparate cardiovascular phenotypes. The first Gpr37l1 knockout mice study to be published reported a marked elevation in systolic blood pressure (SBP; ∼60 mmHg), revealing a potential therapeutic opportunity. The phenotype differed from our own independently generated knockout line, where male mice exhibited equivalent baseline blood pressure to wild type. Here, we attempted to reproduce the first study by characterizing the cardiovascular phenotype of both the original knockout and transgenic lines alongside a C57BL/6J control line, using the same method of blood pressure measurement. The present study supports the findings from our independently developed Gpr37l1 knockout line, finding that SBP and diastolic blood pressure (DBP) are not different in the original Gpr37l1 knockout male mice (SBP: 130.9 ± 5.3 mmHg; DBP: 90.7 ± 3.0 mmHg) compared with C57BL/6J mice (SBP: 123.1 ± 4.1 mmHg; DBP: 87.0 ± 2.7 mmHg). Instead, we attribute the apparent hypertension of the knockout line originally described to comparison with a seemingly hypotensive transgenic line (SBP 103.7 ± 5.0 mmHg; DBP 71.9 ± 3.7 mmHg). Additionally, we quantified myocardial GPR37L1 transcript in humans, which was suggested to be downregulated in cardiovascular disease. We found that GPR37L1 has very low native transcript levels in human myocardium and that expression is not different in tissue samples from patients with heart failure compared with sex-matched healthy control tissue. These findings indicate that cardiac GPR37L1 expression is unlikely to contribute to the pathophysiology of human heart failure.NEW & NOTEWORTHY This study characterizes systolic blood pressure (SBP) in a Gpr37l1 knockout mouse line, which was previously reported to have ∼60 mmHg higher SBP compared with a transgenic line. We observed only a ∼27 mmHg SBP difference between the lines. However, when compared with C57BL/6J mice, knockout mice showed no difference in SBP. We also investigated GPR37L1 mRNA abundance in human hearts and observed no difference between healthy and failing heart samples.

Endogenous and Borrowed Proteolytic Activity in the Borrelia.

The Borrelia spp. are tick-borne pathogenic spirochetes that include the agents of Lyme disease and relapsing fever. As part of their life cycle, the spirochetes traffic between the tick vector and the vertebrate host, which requires significant physiological changes and remodeling of their outer membranes and proteome. This crucial proteome resculpting is carried out by a diverse set of proteases, adaptor proteins, and related chaperones. Despite its small genome, Borrelia burgdorferi has dedicated a large percentage of its genome to proteolysis, including a full complement of ATP-dependent proteases. Energy-driven proteolysis appears to be an important physiological feature of this dual-life-cycle bacterium. The proteolytic arsenal of Borrelia is strategically deployed for disposal of proteins no longer required as they move from one stage to another or are transferred from one host to another. Likewise, the Borrelia spp. are systemic organisms that need to break down and move through host tissues and barriers, and so their unique proteolytic resources, both endogenous and borrowed, make movement more feasible. Both the Lyme disease and relapsing fever Borrelia spp. bind plasminogen as well as numerous components of the mammalian plasminogen-activating system. This recruitment capacity endows the spirochetes with a borrowed proteolytic competency that can lead to increased invasiveness.

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases.

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal 'tethered' counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.

Deletion of Orphan G Protein-Coupled Receptor GPR37L1 in Mice Alters Cardiovascular Homeostasis in a Sex-Specific Manner.

GPR37L1 is a family A orphan G protein-coupled receptor (GPCR) with a putative role in blood pressure regulation and cardioprotection. In mice, genetic ablation of Gpr37l1 causes sex-dependent effects; female mice lacking Gpr37l1 (GPR37L1-/-) have a modest but significant elevation in blood pressure, while male GPR37L1-/- mice are more susceptible to cardiovascular dysfunction following angiotensin II-induced hypertension. Given that this receptor is highly expressed in the brain, we hypothesize that the cardiovascular phenotype of GPR37L1-/- mice is due to changes in autonomic regulation of blood pressure and heart rate. To investigate this, radiotelemetry was employed to characterize baseline cardiovascular variables in GPR37L1-/- mice of both sexes compared to wildtype controls, followed by power spectral analysis to quantify short-term fluctuations in blood pressure and heart rate attributable to alterations in autonomic homeostatic mechanisms. Additionally, pharmacological ganglionic blockade was performed to determine vasomotor tone, and environmental stress tests were used to assess whether cardiovascular reactivity was altered in GPR37L1-/- mice. We observed that mean arterial pressure was significantly lower in female GPR37L1-/- mice compared to wildtype counterparts, but was unchanged in male GPR37L1-/- mice. GPR37L1-/- genotype had a statistically significant positive chronotropic effect on heart rate across both sexes when analyzed by two-way ANOVA. Power spectral analysis of these data revealed a reduction in power in the heart rate spectrum between 0.5 and 3 Hz in female GPR37L1-/- mice during the diurnal active period, which indicates that GPR37L1-/- mice may have impaired cardiac vagal drive. GPR37L1-/- mice of both sexes also exhibited attenuated depressor responses to ganglionic blockade with pentolinium, indicating that GPR37L1 is involved in maintaining sympathetic vasomotor tone. Interestingly, when these mice were subjected to aversive and appetitive behavioral stressors, the female GPR37L1-/- mice exhibited an attenuation of cardiovascular reactivity to aversive, but not appetitive, environmental stimuli. Together, these results suggest that loss of GPR37L1 affects autonomic maintenance of blood pressure, giving rise to sex-specific cardiovascular changes in GPR37L1-/- mice.

Polymicrobial Nature of Tick-Borne Diseases.

Tick-borne diseases have doubled in the last 12 years, and their geographic distribution has spread as well. The clinical spectrum of tick-borne diseases can range from asymptomatic to fatal infections, with a disproportionate incidence in children and the elderly. In the last few years, new agents have been discovered, and genetic changes have helped in the spread of pathogens and ticks. Polymicrobial infections, mostly in Ixodes scapularis, can complicate diagnostics and augment disease severity. Amblyomma americanum ticks have expanded their range, resulting in a dynamic and complex situation, possibly fueled by climate change. To document these changes, using molecular biology strategies for pathogen detection, an assessment of 12 microbes (9 pathogens and 3 symbionts) in three species of ticks was done in Suffolk County, New York. At least one agent was detected in 63% of I. scapularis ticksBorrelia burgdorferi was the most prevalent pathogen (57% in adults; 27% in nymphs), followed by Babesia microti (14% in adults; 15% in nymphs), Anaplasma phagocytophilum (14% in adults; 2% in nymphs), Borrelia miyamotoi (3% in adults), and Powassan virus (2% in adults). Polymicrobial infections were detected in 22% of I. scapularis ticks, with coinfections of B. burgdorferi and B. microti (9%) and of B. burgdorferi and A. phagocytophilum (7%). Three Ehrlichia species were detected in 4% of A. americanum ticks. The rickettsiae constituted the largest prokaryotic biomass of all the ticks tested and included Rickettsia amblyommatis, Rickettsia buchneri, and Rickettsia montanensis The high rates of polymicrobial infection in ticks present an opportunity to study the biological interrelationships of pathogens and their vectors.IMPORTANCE Tick-borne diseases have increased in prevalence in the United States and abroad. The reasons for these increases are multifactorial, but climate change is likely to be a major factor. One of the main features of the increase is the geographic expansion of tick vectors, notably Amblyomma americanum, which has brought new pathogens to new areas. The clinical spectrum of tick-borne diseases can range from asymptomatic to fatal infections, with a disproportionate incidence in children and the elderly. In addition, new pathogens that are cotransmitted by Ixodes scapularis have been discovered and have led to difficult diagnoses and to disease severity. Of these, Borrelia burgdorferi, the agent of Lyme disease, continues to be the most frequently transmitted pathogen. However, Babesia microti, Borrelia miyamotoi (another spirochete), Anaplasma phagocytophilum, and Powassan virus are frequent cotransmitted agents. Polymicrobial infection has important consequences for the diagnosis and management of tick-borne diseases.

HtrA of Borrelia burgdorferi Leads to Decreased Swarm Motility and Decreased Production of Pyruvate.

Borrelia burgdorferi HtrA (HtrABb) is a serine protease that targets damaged or improperly folded proteins. In our previous studies, HtrABb specifically degraded basic membrane protein BmpD, chemotaxis phosphatase CheX, and outer membrane protein P66. In addition, HtrABb degrades virulence factor BB0323 and components of the extracellular matrix fibronectin and aggrecan. A proteomics-based analysis (two-dimensional difference gel electrophoresis [2-D DIGE], liquid chromatography-mass spectrometry [LC-MS]) of an HtrABb-overexpressing strain of B. burgdorferi (A3HtrAOE) revealed that protein levels of P66 were reduced in comparison to wild-type B. burgdorferi, confirming its status as an HtrABb substrate. Hbb, a P66-DNA-binding transcription factor, was specifically degraded by HtrABb, providing supportive evidence for a role for both in the regulation of P66. A3HtrAOE exhibited reduced motility in swarm assays, a possible link between overabundance of HtrABb and its enzymatic specificity for P66. However, the ΔP66 strain did not have reduced motility in the swarm assays, negating a role for this protein. The proteomics analyses also identified three enzymes of the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycerol-3-phosphate dehydrogenase (GPDH), and glycerol kinase (GK), and one enzyme involved in carbohydrate metabolism, diphosphate-fructose-6-phosphate 1-phosphotransferase, which were reduced in A3HtrAOE. Consistent with its reduced protein levels of these glycolytic enzymes, A3HtrAOE was also deficient in production of pyruvate. We propose a model for a role for HtrABb in contributing to a decrease in metabolic activity of B. burgdorferiIMPORTANCE Being a vector-borne bacterium, B. burgdorferi must remodel its protein content as it transfers from tick to mammal. Proteolysis is a mechanism whereby remodeling can be accomplished. HtrABb degrades a number of proteins whose disappearance may help in preparing this organism for a stage of low metabolic activity.

Orphan receptor GPR37L1 contributes to the sexual dimorphism of central cardiovascular control.

BACKGROUND: Over 100 mammalian G protein-coupled receptors are yet to be matched with endogenous ligands; these so-called orphans are prospective drug targets for the treatment of disease. GPR37L1 is one such orphan, abundant in the brain and detectable as mRNA in the heart and kidney. GPR37L1 ablation was reported to cause hypertension and left ventricular hypertrophy, and thus, we sought to further define the role of GPR37L1 in blood pressure homeostasis. METHODS: We investigated the cardiovascular effects of GPR37L1 using wild-type (GPR37L1wt/wt) and null (GPR37L1KO/KO) mice established on a C57BL/6J background, both under baseline conditions and during AngII infusion. We profiled GPR37L1 tissue expression, examining the endogenous receptor by immunoblotting and a ß-galactosidase reporter mouse by immunohistochemistry. RESULTS: GPR37L1 protein was abundant in the brain but not detectable in the heart and kidney. We measured blood pressure in GPR37L1wt/wt and GPR37L1KO/KO mice and found that deletion of GPR37L1 causes a female-specific increase in systolic, diastolic, and mean arterial pressures. When challenged with short-term AngII infusion, only male GPR37L1KO/KO mice developed exacerbated left ventricular hypertrophy and evidence of heart failure, while the female GPR37L1KO/KO mice were protected from cardiac fibrosis. CONCLUSIONS: Despite its absence in the heart and kidney, GPR37L1 regulates baseline blood pressure in female mice and is crucial for cardiovascular compensatory responses in males. The expression of GPR37L1 in the brain, yet absence from peripheral cardiovascular tissues, suggests this orphan receptor is a hitherto unknown contributor to central cardiovascular control.

GPCRs in context: sexual dimorphism in the cardiovascular system.

Cardiovascular disease (CVD) remains the largest cause of mortality worldwide, and there is a clear gender gap in disease occurrence, with men being predisposed to earlier onset of CVD, including atherosclerosis and hypertension, relative to women. Oestrogen may be a driving factor for female-specific cardioprotection, though androgens and sex chromosomes are also likely to contribute to sexual dimorphism in the cardiovascular system (CVS). Many GPCR-mediated processes are involved in cardiovascular homeostasis, and some exhibit clear sex divergence. Here, we focus on the G protein-coupled oestrogen receptor, endothelin receptors ETA and ETB and the eicosanoid G protein-coupled receptors (GPCRs), discussing the evidence and potential mechanisms leading to gender dimorphic responses in the vasculature. The use of animal models and pharmacological tools has been essential to understanding the role of these receptors in the CVS and will be key to further delineating their sex-specific effects. Ultimately, this may illuminate wider sex differences in cardiovascular pathology and physiology. LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins.

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.

The G protein-coupled receptor N-terminus and receptor signalling: N-tering a new era.

G protein-coupled receptors (GPCRs) are a vast family of membrane-traversing proteins, essential to the ability of eukaryotic life to detect, and mount an intracellular response to, a diverse range of extracellular stimuli. GPCRs have evolved with archetypal features including an extracellular N-terminus and intracellular C-terminus that flank a transmembrane structure of seven sequential helices joined by intracellular and extracellular loops. These structural domains contribute to the ability of a GPCR to be correctly synthesised and inserted into the cell membrane, to interact with its cognate ligand(s) and to couple with signal-transducing heterotrimeric G proteins, allowing the activated receptor to selectively modulate a number of signalling cascades. Whilst well known for its importance in receptor translation and trafficking, the GPCR N-terminus is underexplored as a participant in receptor signalling. This review aims to discuss and integrate recent advances in knowledge of the vital roles of the GPCR N-terminus in receptor signalling.

Orphan receptor ligand discovery by pickpocketing pharmacological neighbors.

Understanding the pharmacological similarity of G protein-coupled receptors (GPCRs) is paramount for predicting ligand off-target effects, drug repurposing, and ligand discovery for orphan receptors. Phylogenetic relationships do not always correctly capture pharmacological similarity. Previous family-wide attempts to define pharmacological relationships were based on three-dimensional structures and/or known receptor-ligand pairings, both unavailable for orphan GPCRs. Here, we present GPCR-CoINPocket, a novel contact-informed neighboring pocket metric of GPCR binding-site similarity that is informed by patterns of ligand-residue interactions observed in crystallographically characterized GPCRs. GPCR-CoINPocket is applicable to receptors with unknown structure or ligands and accurately captures known pharmacological relationships between GPCRs, even those undetected by phylogeny. When applied to orphan receptor GPR37L1, GPCR-CoINPocket identified its pharmacological neighbors, and transfer of their pharmacology aided in discovery of the first surrogate ligands for this orphan with a 30% success rate. Although primarily designed for GPCRs, the method is easily transferable to other protein families.

Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.

Niclosamide blocks glucagon phosphorylation of Ser552 on ß-catenin in primary rat hepatocytes via PKA signalling.

Recently, it has been found that glucagon is able to activate the ß-catenin signalling pathway leading to increased cyclin D1 and c-Myc expression in liver. Therefore the main aim of the present study is to determine whether the effect of glucagon activating ß-catenin signalling leading to increased target gene expression is mediated through cAMP activation of PKA (protein kinase A). Primary rat hepatocytes were incubated with insulin, glucagon or adrenaline (epinephrine) and a range of inhibitors of PI3K (phosphoinositide 3-kinase), Wnt, mitochondrial uncoupler (niclosamide) or PKA inhibitors to dissect out the pathway leading to increased Ser(552) phosphorylation on ß-catenin following glucagon exposure. In primary rat hepatocytes, we found that short exposure to glucagon or adrenaline caused a rapid increase in Ser(552) phosphorylation on ß-catenin that leads to increased cyclin D1 and c-Myc expression. A range of PI3K and Wnt inhibitors were unable to block the effect of glucagon phosphorylating ß-catenin. Interestingly, both niclosamide and the PKA inhibitor H89 blocked the glucagon effect on ß-catenin signalling, leading to a reduction in target gene expression. Likewise, niclosamide inhibited cAMP levels and the direct addition of db-cAMP (dibutyryl-cAMP sodium salt) also resulted in Ser(552) phosphorylation of ß-catenin. We have identified a new pathway via glucagon signalling that leads to increased ß-catenin activity that can be reversed with the antihelminthic drug niclosamide, which has recently shown promise as a potential treatment of T2D (Type 2 diabetes). This novel finding could be useful in liver cancer treatment, particularly in the context of T2D with increased ß-catenin activity.

Borrelia burgdorferi†HtrA: evidence for twofold proteolysis of outer membrane protein p66.

In prokaryotes, members of the High Temperature Requirement A (HtrA) family of serine proteases function in the periplasm to degrade damaged or improperly folded membrane proteins. Borrelia burgdorferi, the agent of Lyme disease, codes for a single HtrA homolog. Two-dimensional electrophoresis analysis of B. burgdorferi B31A3 and a strain that overexpresses HtrA (A3HtrAOE) identified a downregulated protein in A3HtrAOE with a mass, pI and MALDI-TOF spectrum consistent with outer membrane protein p66. P66 and HtrA from cellular lysates partitioned into detergent-resistant membranes, which contain cholesterol-glycolipid-rich membrane regions known as lipid rafts, suggesting that HtrA and p66 may reside together in lipid rafts also. This agrees with previous work from our laboratory, which showed that HtrA and p66 are constituents of B. burgdorferi outer membrane vesicles. HtrA degraded p66 in vitro and A3HtrAOE expressed reduced levels of p66 in vivo. Fluorescence confocal microscopy revealed that HtrA and p66 colocalize in the membrane. The association of HtrA and p66 establishes that they could interact efficiently and their protease/substrate relationship provides functional relevance to this interaction. A3HtrAOE also showed reduced levels of p66 transcript in comparison with wild-type B31A3, indicating that HtrA-mediated regulation of p66 may occur at multiple levels.

The lipid raft proteome of Borrelia burgdorferi.

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft-associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC-MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 (http://proteomecentral.proteomexchange.org/dataset/PXD002365).

Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.

As little as a decade ago, generation of a single knockout mouse line was an expensive and time-consuming undertaking available to relatively few researchers. The International Knockout Mouse Consortium, established in 2007, has revolutionized the use of such models by creating an open-access repository of embryonic stem (ES) cells that, through sequential breeding with first FLP1 recombinase and then Cre recombinase transgenic mice, facilitates germline global or conditional deletion of almost every gene in the mouse genome. In this Case Study, we describe our experience using the repository to create mouse lines for a variety of experimental purposes. Specifically, we discuss the process of obtaining germline transmission of two European Conditional Mouse Mutagenesis Program (EUCOMM) "knockout-first" gene targeted constructs and the advantages and pitfalls of using this system. We then outline our breeding strategy and the outcomes of our efforts to generate global and conditional knockouts and reporter mice for the genes of interest. Line maintenance, removal of recombinase transgenes, and cryopreservation are also considered. Our approach led to the generation of heterozygous knockout mice within 6 months of commencing breeding to the founder mice. By describing our experiences with the EUCOMM ES cells and subsequent breeding steps, we hope to assist other researchers with the application of this valuable approach to generating versatile knockout mouse lines.

Hypercholesterolemia and ApoE deficiency result in severe infection with Lyme disease and relapsing-fever Borrelia.

The Lyme disease (Borrelia burgdorferi) and relapsing-fever (Borrelia hispanica) agents have distinct infection courses, but both require cholesterol for growth. They acquire cholesterol from the environment and process it to form cholesterol glycolipids that are incorporated onto their membranes. To determine whether higher levels of serum cholesterol could enhance the organ burdens of B. burgdorferi and the spirochetemia of B. hispanica in laboratory mice, apolipoprotein E (apoE)-deficient and low-density lipoprotein receptor (LDLR)-deficient mice that produce large amounts of serum cholesterol were infected with both spirochetes. Both apoE- and LDLR-deficient mice infected with B. burgdorferi had an increased number of spirochetes in the joints and inflamed ankles compared with the infected wild-type (WT) mice, suggesting that mutations in cholesterol transport that result in high serum cholesterol levels can affect the pathogenicity of B. burgdorferi. In contrast, elevated serum cholesterol did not lead to an increase in the spirochetemia of B. hispanica. In the LDLR-deficient mice, the course of infection was indistinguishable from the WT mice. However, infection of apoE-deficient mice with B. hispanica resulted in a longer spirochetemia and increased mortality. Together, these results argue for the apoE deficiency, and not hypercholesterolemia, as the cause for the increased severity with B. hispanica. Serum hyperlipidemias are common human diseases that could be a risk factor for increased severity in Lyme disease.