A bioluminescent-based probe for in vivo non-invasive monitoring of nicotinamide riboside uptake reveals a link between metastasis and NAD+ metabolism.

Nicotinamide riboside (NR) is a form of vitamin B3 and is one of the most studied compounds for the restoration of cellular NAD+ levels demonstrating clinical potential in many metabolic and age-related disorders. Despite its wide commercial availability as a powerful nutraceutical, our understanding of NR uptake by different cells and tissues is greatly limited by the lack of noninvasive in vivo imaging tools limiting its clinical translation. Here, we report the development and validation of a bioluminescent NR uptake probe (BiNR) for non-invasive longitudinal imaging of NR uptake both in vitro and in vivo. In addition, we optimized an assay that allows monitoring of NR flux without the need to transfect cells with the luciferase gene, enabling the use of the BiNR probe in clinical samples, as demonstrated with human T cells. Lastly, we used BiNR to investigate the role of NR uptake in cancer prevalence and metastases formation in triple negative breast cancer (TNBC) animal model. Our results demonstrate that NR supplementation results in a significant increase in cancer prevalence and metastases of TNBC to the brain. These results outline the important role of powerful nutraceuticals like NR in cancer metabolism and the need to personalize their use in certain patient populations.

Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals.

Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.

Noninvasive imaging and quantification of bile salt hydrolase activity: From bacteria to humans.

The microbiome-produced enzyme bile salt hydrolase (BSH) plays a central role in human health, but its function remains unclear due to the lack of suitable methods for measuring its activity. Here, we have developed a novel optical tool based on ultrasensitive bioluminescent imaging and demonstrated that this assay can be used for quick and cost-effective quantification of BSH activity across a broad range of biological settings including pure enzymes and bacteria, intact fecal slurries, and noninvasive imaging in live animals, as well as for the assessment of BSH activity in the entire gastrointestinal tract of mice and humans. Using this assay, we showed that certain types of prebiotics are capable of increasing BSH activity of the gut microbiota in vivo and successfully demonstrated potential application of this assay as a noninvasive diagnostic test to predict the clinical status of inflammatory bowel disease (IBD) patients.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo.

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

Bioluminescent-based imaging and quantification of glucose uptake in vivo.

Glucose is a major source of energy for most living organisms, and its aberrant uptake is linked to many pathological conditions. However, our understanding of disease-associated glucose flux is limited owing to the lack of robust tools. To date, positron-emission tomography imaging remains the gold standard for measuring glucose uptake, and no optical tools exist for non-invasive longitudinal imaging of this important metabolite in in vivo settings. Here, we report the development of a bioluminescent glucose-uptake probe for real-time, non-invasive longitudinal imaging of glucose absorption both in vitro and in vivo. In addition, we demonstrate that the sensitivity of our method is comparable with that of commonly used 18F-FDG-positron-emission-tomography tracers and validate the bioluminescent glucose-uptake probe as a tool for the identification of new glucose transport inhibitors. The new imaging reagent enables a wide range of applications in the fields of metabolism and drug development.

DNMT3B overexpression contributes to aberrant DNA methylation and MYC-driven tumor maintenance in T-ALL and Burkitt's lymphoma.

Aberrant DNA methylation is a hallmark of cancer. However, our understanding of how tumor cell-specific DNA methylation patterns are established and maintained is limited. Here, we report that in T-cell acute lymphoblastic leukemia (T-ALL) and Burkitt's lymphoma the MYC oncogene causes overexpression of DNA methyltransferase (DNMT) 1 and 3B, which contributes to tumor maintenance. By utilizing a tetracycline-regulated MYC transgene in a mouse T-ALL (EµSRα-tTA;tet-o-MYC) and human Burkitt's lymphoma (P493-6) model, we demonstrated that DNMT1 and DNMT3B expression depend on high MYC levels, and that their transcription decreased upon MYC-inactivation. Chromatin immunoprecipitation indicated that MYC binds to the DNMT1 and DNMT3B promoters, implicating a direct transcriptional regulation. Hence, shRNA-mediated knock-down of endogenous MYC in human T-ALL and Burkitt's lymphoma cell lines downregulated DNMT3B expression. Knock-down and pharmacologic inhibition of DNMT3B in T-ALL reduced cell proliferation associated with genome-wide changes in DNA methylation, indicating a tumor promoter function during tumor maintenance. We provide novel evidence that MYC directly deregulates the expression of both de novo and maintenance DNMTs, showing that MYC controls DNA methylation in a genome-wide fashion. Our finding that a coordinated interplay between the components of the DNA methylating machinery contributes to MYC-driven tumor maintenance highlights the potential of specific DNMTs for targeted therapies.

Behavioral and physiological characterization of PKC-dependent phosphorylation in the Grin2a∆PKC mouse.

Activity-dependent plasticity in NMDA receptor-containing synapses can be regulated by phosphorylation of serines and tyrosines in the C-terminal domain of the receptor subunits by various kinases. We have previously identified S1291/S1312 as important sites for PKC phosphorylation; while Y1292/Y1312 are the sites indirectly phosphorylated by PKC via Src kinase. In the oocyte expression system, mutation of those Serine sites to Alanine (that cannot be phosphorylated) in the GluN2A subunit, resulted in a decreased PKC stimulated current enhancement through the receptors compared to wild-type NMDA receptors. To investigate the behavioral and physiological significance of those PKC-mediated phosphorylation sites in vivo, the Grin2a∆PKC mouse expressing GluN2A with four mutated amino acids: S1291A, S1312A, Y1292F and Y1387F was generated using homologous recombination. The Grin2a∆PKC mice exhibit reduced anxiety in the open field test, light dark emergence test, and elevated plus maze. The mutant mice show reduced alternation in a Y maze spontaneous alternation task and a in a non-reinforced T maze alternation task. Interestingly, when the mutant mice were exposed to novel environments, there was no increase in context-induced Fos levels in hippocampal CA1 and CA3 compared to home-cage Fos levels, while the Fos increased in the WT mice in CA1, CA3 and DG. When the SC-CA1 synapses in slices from mutant mice were stimulated using a theta-burst protocol, there was no impairment in LTP. Overall, these results suggest that at least one of those PKC-mediated phosphorylation sites regulates NMDAR-mediated signaling that modulates anxiety.

Multi-Modal Imaging with a Toolbox of Influenza A Reporter Viruses.

Reporter viruses are useful probes for studying multiple stages of the viral life cycle. Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography-computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate in vitro and in vivo influenza virus studies.

Conditional deletions refine the embryonic requirement for Dlk1.

Numerous studies have implicated Delta-like 1 (DLK1), a transmembrane protein that shares homology with Notch ligands, in embryonic growth and differentiation. Dlk1 expression is widespread, though not ubiquitous, during early development, but is confined to a few specific cell types in adults. Adult Dlk1-expressing tissues include the Insulin-producing ß-cells of the pancreas and the Growth hormone-producing somatotrophs of the pituitary gland. Previously generated Dlk1 null mice (Dlk1(Sul-pat)), display a partially penetrant neonatal lethality and a complex pattern of developmental and adult phenotypes. Here we describe the generation of a conditional Dlk1 mouse line (Dlk1(flox)) to facilitate cell type-specific deletion of the Dlk1 gene, providing a powerful system to explore each aspect of the Dlk1 null phenotype. Four tissue-specific Cre mouse lines were used to produce individual Dlk1 deletions in pancreatic ß-cells, pituitary somatotrophs and the endothelial cells of the embryo and placenta, key candidates for the Dlk1 phenotype. Contrary to expectations, all of these conditional mice were fully viable, and none recapitulated any aspect of the Dlk1(Sul-pat) null mice. Dlk1 expression is therefore not essential for the normal development of ß-cells, somatotrophs and endothelial cells, and the tissues responsible for the Dlk1 null phenotype remain to be identified. Dlk1(flox) mice will continue to provide an important tool for further research into the function of Dlk1.

MYC phosphorylation, activation, and tumorigenic potential in hepatocellular carcinoma are regulated by HMG-CoA reductase.

MYC is a potential target for many cancers but is not amenable to existing pharmacologic approaches. Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) by statins has shown potential efficacy against a number of cancers. Here, we show that inhibition of HMG-CoA reductase by atorvastatin (AT) blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced hepatocellular carcinoma (HCC) as well as in human HCC-derived cell lines. To confirm specificity, we show that the antitumor effects of AT are blocked by cotreatment with the HMG-CoA reductase product mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we show that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors.

Dlk1 is necessary for proper skeletal muscle development and regeneration.

Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-κB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy.

Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain.

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.

Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region.

BACKGROUND: The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting. RESULTS: A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Delta5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Delta5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Delta5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects. CONCLUSION: These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Delta5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.

Dlk1 expression marks developing endothelium and sites of branching morphogenesis in the mouse embryo and placenta.

The protein product of the Delta-like 1 (Dlk1) gene belongs to the Delta-Notch family of signaling molecules, proteins involved in cell fate determination in many tissues during development. The DLK1 protein is believed to function as a growth factor, maintaining the proliferative state of undifferentiated cells, and is usually down-regulated as immature cells differentiate. The expression pattern of the DLK1 protein has been described in certain human tissues; however, Dlk1 expression is not well understood in the mouse, the most tractable mammalian genetic model system. To better understand the role of Dlk1 in embryonic development, the tissue-specific expression pattern of Dlk1 mRNA during mouse embryogenesis was analyzed by in situ hybridization. In embryonic day 12.5 (e12.5) embryos, high levels of Dlk1 were found in the developing pituitary, pancreas, lung, adrenal, and many mesodermally derived tissues. Strikingly, Dlk1 expression also marks the growing branches of organs that develop through the process of branching morphogenesis. At e16.5, Dlk1 expression is down-regulated in most tissues but remains in the pituitary, the adrenal gland, and in skeletal muscle. In the placenta, expression of Dlk1 is detected in endothelial cells lining the fetal blood vessels of the labyrinth. This pattern is distinct from that seen in the human placenta and suggests a role for Dlk1 in regulating maternal-fetal interactions.

A 178-kb BAC transgene imprints the mouse Gtl2 gene and localizes tissue-specific regulatory elements.

The regulation of genomic imprinting, the allele-specific expression of an autosomal gene, is complex and poorly understood. Imprinted genes are organized in clusters, where cis-acting regulatory elements are believed to interact to control multiple genes. We have used BAC transgenesis in the mouse to begin to delineate the region of DNA required for proper expression and imprinting of the mouse Delta-like1 (Dlk1) and Gene-trap locus2 (Gtl2) imprinted genes. We demonstrate that the Gtl2 gene is expressed from a BAC transgene in mouse embryo and placenta only upon maternal inheritance, as is the endogenous Gtl2 gene. Gtl2 is therefore properly imprinted on the BAC in an ectopic chromosomal location and must carry with it all necessary imprinting regulatory elements. Furthermore, we show that the BAC Gtl2 gene is expressed at levels approaching those of the endogenous gene only in the brain of adult animals, not in other sites of endogenous expression such as the pituitary, adrenal, and skeletal muscle. These data localize the enhancer(s) for brain Gtl2 expression, but not those for other tissues, to the DNA contained within the BAC clone. As the Dlk1 gene is not expressed from the BAC in any tissues, it must require additional elements that are different from those necessary for Gtl2 expression. Our data refine the interval for future investigation of Gtl2 imprinting and provide evidence for distinct regulation of the linked Dlk1 and Gtl2 genes.

Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line.

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1-Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1-Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.