Poster boards and push pins will be provided. The boards measure 4' by 4'. All boards will be well-marked with the poster numbers.
Five poster awards will be awarded: sponsored by AUM LifeTech, Charles River Laboratories, New England BioLabs, and the Biomedical Posdoctoral Council.
Biochemistry / Biophysics
1-05 Yann Bikard
1-06 Meredith Jackrel
1-07 Mariana Torrente
1-18 Rachana Garg
1-19 Laura Barrio-Real
1-20 Sofia Lisanti
1-21 Hem Chandra Jha
Cell / Developmental Biology
1-29 Veronique Giroux
1-30 Elizabeth Hennessy
1-31 Jianping Li
1-32 Wenshan Wang
1-33 Lauren Manderfield
1-34 Chiamin Liao
Clinical / Translational
1-37 Pavitra Ramachandran
1-38 Shruthi Ravimohan
Genetics / Genomics
2-07 Kathryn Driesbaugh
2-08 Pazit Beckerman
2-09 Jacque Young
2-10 Amanda Zacharias
2-11 Elisabeth Mlynarski
Microbiology / Immunology
Neuroscience / Psychology
2-34 Zuo-Fei Yuan
2-35 Joshua Slee
Using reverse micelle encapsulation to improve fragment based drug discovery
Brian Fuglestad1, Trevor M. Penning2, and A. Joshua Wand1
1Johnson Research Foundation, Department of Biochemistry and Biophysics and 2Center of Excellence in Environmental Toxicology and Department of Pharmacology, University of Pennsylvania Perelman School of Medicine
Human respiratory syncytial virus (RSV) leads to illness in infants, children, and high-risk adults. Effective vaccines and therapeutic methods are currently unavailable. In order to develop strategies to better serve patients, a better understanding of the interactions between virus replication and the host immune response is needed. Our laboratory recently showed that defective viral genomes (DVGs) generated at the time of high virus replication are the primary danger signals that trigger the immune response during Sendai virus infection in mice. Therefore, we hypothesized that DVGs generated during RSV infection stimulate the antiviral response and influence virus virulence in humans. To investigate this function of DVGs, we generated virus stocks with a high content of DVGs (HD) and DVG-free virus (LD). HD not only strongly stimulated the expression of antiviral genes, but also potently inhibited virus replication in vitro and ex vivo. Purified defective viral particles (pDPs) from HD were supplemented to LD infection, showing that pDPs phenotypically mimicked RSV-HD. In vivo, mice infected with HD developed a strong and rapid antiviral response as well and showed reduced pathology. To determine whether DVGs are present in human patients, we analyzed pediatric respiratory secretions from RSV-infected children admitted to The Children’s Hospital of Philadelphia. We observed DVGs in more than 45% (20/41) of specimens. Excitingly, DVG-positive samples showed a robust antiviral response, similar to what we observed in mice and in human lung tissue ex vivo. Taken together, our data demonstrate that RSV DVGs are potent triggers of the host antiviral response to RSV in humans and that DVGs may be a good target for preventive and therapeutic treatments.
EGFR in a sticky situation: discovery of a constitutively dimeric receptor that remains regulated by ligand
Daniel M. Freed1, Diego Alvarado1,2, and Mark A. Lemmon1
1Department of Biochemistry & Biophysics, Perelman School of Medicine, University of Pennsylvania; 2Present address: Kolltan Pharmaceuticals, New Haven, CT
The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases controls multiple cellular processes including growth, proliferation, and motility, and the importance of these receptors as oncogenic drivers in several cancers makes understanding their regulatory mechanism highly significant. It has been widely believed for many years that EGFR signaling is regulated exclusively by ligand-induced receptor dimerization. However, several recent reports have provided clues that EGFRs on the cell surface can exist as inactive dimers even in the absence of ligand – arguing that the mechanism cannot be so simple. Using a variety of biophysical methods, we show that the highly homologous EGFR from C. elegans (LET-23) is constitutively dimeric, yet inactive until ligand binds – providing the ‘smoking gun’ that EGFRs are not regulated simply by reversible dimerization. Analytical ultracentrifugation analyses coupled with mutagenesis were used to map areas of the extracellular region responsible for LET-23 dimerization. Intriguingly, the deletion of domain IV in the extracellular module prevents dimerization only in the absence of ligand – essentially reconfiguring LET-23 for ligand-induced dimerization. Our results suggest that EGFRs are not regulated by dimerization per se, but instead by allosteric changes within receptor dimers – much like the insulin receptor. We are currently focusing on the structural details of these allosteric changes, with a view towards identifying new, more effective strategies of EGFR inhibition in cancer.
Defining Kapβ2 as a protein disaggregase for ALS disease protein FUS
Lin Guo, Hejia Wang, Nikita Singh, and James Shorter
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania
Amyotrophic lateral sclerosis (ALS, also called Lou Gehrig’s disease) is a debilitating, fatal neurodegenerative disease that is characterized by a severe and selective devastation of upper and lower motor neurons, progressive muscle weakness, paralysis, and death. Recently, ALS-linked mutations have been uncovered in the gene encoding the RNA-binding protein FUS (Fused in Sarcoma). Ordinarily, FUS is localized to the nucleus. However, in ALS, FUS mutations in the PY-NLS (proline/tyrosine-nuclear localization signal) lead to impaired nuclear import of FUS, with mislocalization of the protein to the cytoplasm and formation of cytoplasmic inclusions in degenerating motor neurons. In neurons, FUS can be transported into the nucleus by interaction with the nuclear import receptor Karyopherinβ2 (Kapβ2). Interestingly, FUS mutants that have mutations in the PY-NLS display decreased affinity for Kapβ2 binding. In the current study, we investigated the interaction between FUS and Kapβ2. Our results indicate that Kapβ2 effectively inhibits and reverses WT FUS aggregation in vitro. The activity of Kapβ2 against FUS aggregates is slightly impaired in FUS disease mutants that have mutations in PY-NLS and is severely impaired in FUS disease mutants that lack the PY-NLS. We also demonstrated in this study that Kapb2 is effective against FUS aggregates that have different morphologies (i.e. amorphous, fibrils, and protein hydrogel). Moreover, Kapβ2 is shown to inhibit the aggregation of another ALS disease protein with PY-NLS: hnRNPA1. Although Kapβ2 is less effective in disaggregating hnRNPA1 fibrils, our results show that it has the potential to be developed as a general protein disaggregase for proteins bearing a PY-NLS.
Application of the entropy meter to new protein systems
Jackwee Lim, Matthew A. Stetz, Kyle W. Harpole, Vignesh Kasinath and A. Joshua Wand
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia
The physical basis for protein-ligand interactions is complex and involves an interplay between different thermodynamic parameters. Among these are changes in conformational entropy, which may be coupled to different protein events such as ligand binding and thermal stability. It has recently been shown in two model systems (calmodulin and catabolite activator protein) that protein conformational entropy contributes significantly to the free energy of protein-ligand interactions. The generality of protein conformational entropy, however, has yet to be verified. Using the recently established “entropy meter” based on the quantitative relationship between conformational entropy and Nuclear Magnetic Resonance (NMR)-derived generalized order parameters, we intend to investigate its contribution to thermodynamics processes in a broader variety of proteins. The protein-based systems of interest include β-lactamase, neutrophil gelatinase-associated lipocalin (NGAL), histamine-binding protein (HBP) and RNaseH.
Regulation of epithelial ion channels CFTR and ENAC by the ER luminal chaperone ERp29
Yann Bikard1, Yael S. Grumbach1, Laurence Suaud1, Jonathan Litvak1 and Ronald C. Rubenstein1,2
1Division of Pulmonary Medicine and Cystic Fibrosis Center, The Children’s Hospital of Philadelphia; 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania
ERp29 is an endoplasmic reticulum (ER) 29 kD thioredoxin-homologous protein. Interestingly, ERp29 has only a single Cysteine instead of the usual C-X-X-C thioredoxins motif. ERp29 displays chaperone-like properties at both the biophysical and cellular levels and plays a role in regulating connexin 43 hemichannel biogenesis and assembly. Our group has also demonstrated that ERp29 promotes CFTR biogenesis. As the biogenesis of CFTR and ENaC share similar features, the present studies tested the hypothesis that ERp29 would also regulate ENaC biogenesis and functional expression, as well as further probed the mechanism by which ERp29 promotes ENaC and CFTR biogenesis. In MDCK epithelial cells expressing αβγ-ENaC, overexpression of wild type ERp29 increased ENaC functional expression in Ussing chamber experiments. In contrast, overexpression of a mutant ERp29 lacking its single Cysteine (C157S) decreased ENaC functional expression. These observations were not associated with altered expression of ENaC at the apical surface, suggesting that ERp29 may modulate ENaC open probability. ERp29 overexpression also promoted the interaction of both ENaC and CFTR with the coat complex II ER exit machinery, whereas C157S ERp29 overexpression decreased this interaction. Finally, as we have previously shown that ERp29 is found at the surface of epithelial cells, we have begun to test the hypothesis that ERp29’s escape from ER retention is critical for its regulation of CFTR biogenesis by mutating ERp29’s KEEL ER retention motif to KDEL, which should enhance ER retention, or KDEV or deletion of KEEL, which should decreased ER retention. Our findings suggest a key role for ERp29 in the biogenesis of ENaC and CFTR and our ongoing studies will interrogate ERp29’s mechanism of action.
Potentiated Hsp104 variants antagonize diverse proteotoxic misfolding events
Meredith E. Jackrel1, Morgan E. DeSantis1,2, Bryan A. Martinez4, Laura M. Castellano1,3, Rachel M. Stewart1, Kim A. Caldwell4, Guy A. Caldwell4, and James Shorter1,2,3
1Department of Biochemistry and Biophysics, 2Biochemistry and Molecular Biophysics Graduate Group, and 3Pharmacology Graduate Group, Perelman School of Medicine, University of Pennsylvania; 4Department of Biological Sciences, University of Alabama, Tuscaloosa, AL.
There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD). Treatments for these disorders remain palliative, and no therapeutics exists that address their underlying cause. However, not all protein misfolding events are associated with toxicity. Yeast harness amyloid for beneficial purposes, and thus have mechanisms to tightly regulate the construction and disassembly of amyloid. Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no homolog in animals and only limited activity against human neurodegenerative disease proteins. We have re-engineered Hsp104 to rescue TDP-43, FUS, and alpha-synuclein proteotoxicity by mutating single residues in either middle or small domain of nucleotide-binding domain 1. These potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations thus reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Furthermore, these variants suppress the toxicity of disease-associated versions of TDP-43, FUS, and alpha-synuclein. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregates can restore proteostasis and mitigate neurodegeneration.
Suramin inhibits Hsp104 ATPase and disaggregase activity
Mariana P. Torrente1, Laura M. Castellano1,2, and James Shorter1,2
1Department of Biochemistry and Biophysics; 2Pharmacology Graduate Group; Perelman School of Medicine; University of Pennsylvania
Hsp104 is a hexameric AAA+ protein that utilizes energy from ATP hydrolysis to dissolve disordered protein aggregates as well as amyloid fibers. Interestingly, Hsp104 orthologs are found in all kingdoms of life except animals. Thus, Hsp104 could represent an interesting drug target. Specific inhibition of Hsp104 activity might antagonize non-metazoan parasites that depend on a potent heat shock response, while producing little or no side effects to the host. However, no small molecule inhibitors of Hsp104 are known except guanidinium chloride. Here, we screen over 16,000 small molecules and identify 16 novel inhibitors of Hsp104 ATPase activity. Excluding compounds that inhibited Hsp104 activity by non-specific colloidal effects, we defined Suramin as an inhibitor of Hsp104 ATPase activity. Suramin is a polysulphonated naphthylurea and is used as an antiprotozoal drug for African Trypanosomiasis. Suramin also interfered with Hsp104 disaggregase, unfoldase, and translocase activities, and the inhibitory effect of Suramin was not rescued by Hsp70 and Hsp40. Suramin does not disrupt Hsp104 hexamers and does not effectively inhibit ClpB, the E. coli homolog of Hsp104, establishing yet another key difference between Hsp104 and ClpB behavior. Intriguingly, a potentiated Hsp104 variant, Hsp104A503V, is more sensitive to Suramin than wild-type Hsp104. By contrast, Hsp104 variants bearing inactivating sensor-1 mutations in nucleotide-binding domain (NBD) 1 or 2 are more resistant to Suramin. Thus, Suramin depends upon ATPase events at both NBDs to exert its maximal effect. Suramin could develop into an important mechanistic probe to study Hsp104 structure and function.
Noninvasive quantification of regional tissue pH
Rajat K. Ghosh*, Stephen J. Kadelcek, Mehrdad Pourfathi, Rahim R. Rizi
Department of Radiology, Perelman School of Medicine, University of Pennsylvania
Internal pH is tightly regulated in healthy tissue. However, in a number of fundamental pathologic conditions such as ischemia, inflammation, infection, and cancer the acid-base balance is altered despite the presence of endogenous intra- and extracellular buffering agents. Non-invasive regional measurement of pH would provide a valuable tool for assessing local therapeutic response in patients exhibiting these disease states. Extracellular pH can be used as a quantitative metric to assess tissue malignancy, and determine whether intervention is required. This can be especially useful for tumor locations and types, such as intracranial neoplasm where biopsies would be especially invasive.
While techniques such as magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), and positron emission tomography (PET) have been utilized in a preclinical environment to image pH in vivo, none have thus far been successfully translated to the clinic. Each of these techniques displays drawbacks that have limited administration in a patient setting.
Hyperpolarized (HP) molecules have shown promise in determining in vivo pH. Dynamic nuclear polarization (DNP) increases the nuclear spin polarization of substrate by a factor of 104 ex vivo. The agent is subsequently injected and interrogated with MRI. The acid-base balance is determined through assessing the pH dependent ratio of bicarbonate to CO2.
We have successfully used HP MRI to generate pH images of lung tissue. This approach can be used for convenient simultaneous imaging of bicarbonate, pyruvate, and acetate in order to extract data on both pH and metabolism. Our approach has generated images with in plane resolution of 0.9 mm x 0.9 mm, and can be increased further by 4 fold.
3D measurement of lower limb parameters in children with limb deformity
Saba Pasha, Richard Davidson
Division of Orthopedic Surgery, The Children’s Hospital of Philadelphia
Several parameters are used in lower extremity reconstructive surgery. However these parameters are measured on 2D X-ray images The 2D measurement of a 3D deformity may result in overestimation or underestimation of the deformity which in turn adversely impacts the surgical outcome. The objective of this project is to determine 3D parameters that can characterize the form of the limb deformities. Six models of tibia and femur with deformity were scanned in a slop scanning device that permits simultaneous bi-planar X-ray acquisition in upright position. Sawbone models were placed in a Plexiglas scaffold that permits axial rotation of the model with respect to the X-ray detectors in sagittal and frontal planes. Three scans were taken from each model in 0°, 10°, 20° rotation angles in EOS. Same scaffold was placed in computer tomography (CT) scanner and sawbone models were scanned at three tilt angles, 0°, 10°, 20°. These angles present different soft tissue thicknesses at the hip level which impacts the femur alignment when the patient lies down on her/his back. A manual scanner was used to generate the 3D reconstruction of the bone and 3D parameters of the model were calculated in MATLAB. 3D reconstructions of the CT scans and X-rays were generated in dedicated softwares and statistically compared. Uni-planar parameters showed more reproducibility when different imaging modalities were used. Torsion, rotation, translation, length, and magnitude of the deformity changed significantly as the sawbone models were rotated in SSD. These changes were less prominent in CT. This suggests the importance of patient positioning with respect to the SSD. This provides guidlines toward incorporating 3D parameters in lower extremity reconstructive surgery.
MRI-derived porosity index provides quantitative insight into cortical pore architecture
Mahdieh Bashoor-Zadeh, Chamith S. Rajapakse, Cheng Li, Wenli Sun, Alexander C. Wright, Felix W. Wehrli
Laboratory for Structural NMR Imaging, Department of Radiology, University of Pennsylvania
Small changes in the cortical bone (CB) pore network exert significant influence on bone mechanical properties than do similar changes in trabecular bone. Microstructure of CB pores cannot be resolved with current bone imaging modalities in vivo. Ultra-short echo time (UTE) MRI allows the assessment of CB through the detection of proton signal arising from pore water (residing in pores) and bound water (bound to collagen matrix). This study aims to develop a clinically feasible method to assess the CB pore network by measuring the signal decay at only two echo times (TE) as part of a single 3D UTE MRI scan. CB specimens of 36mm thickness were harvested from the maximum CB thickness of the tibial diaphysis of 16 cadavers (27-97 YO). CB specimens underwent 3D UTE MRI generating images at TE=50 and 2000µs (0.5mm voxel size). A porosity index (PI), defined as the ratio between long and short-TE image intensities, was computed pixel by pixel. To investigate the association between PI and pore size, segmented μCT images (8.6µm voxel size) at the center of each specimen were analyzed. Pores were divided into 4 groups: normal (diameter: D<82μm), large (82μm≤D<172μm), extra-large (172μm≤D<385μm) and giant (D≥385μm). The contribution of each group to the total pore space was calculated as area fraction (AF) of each group. The AF of normal pores decreased with age, while that of giant pores increased. PI correlated positively with AF occupied by giant pores (R2=0.68) and negatively with AF occupied by normal pores (R2=0.78). The data show that the age-related increase in porosity is the result of expansion of existing pores rather than creation of new pores. In addition the MRI PI provides a new biomarker that provides insight into pore size distribution of CB.
Dextran coated bismuth-iron oxide nanohybrid contrast agents for computed tomography and magnetic resonance imaging
Pratap C. Naha1, Ajlan Al Zaki2, Elizabeth Hecht1, Michael Chorny3, Peter Chhour1,2, Walter Witschey1, Harold I. Litt1,4, Andrew Tsourkas2, David P. Cormode1,2,4
1Department of Radiology, Perelman School of Medicine, University of Pennsylvania; 2Department of Bioengineering, Perelman School of Medicine, University of Pennsylvania; 3The Children’s Hospital of Philadelphia; 4Department of Cardiology, Perelman School of Medicine, University of Pennsylvania
Nanoparticle based imaging probes for computed tomography (CT) and magnetic resonance imaging (MRI) are of great current interest. Bismuth nanoparticles have been shown to be a potent contrast agent for CT. However, synthetic routes to biocompatible bismuth nanoparticles are few. Dextran coated iron oxides are clinically approved for MRI. Combining iron and bismuth in the same formulation would result in a novel nanohybrid contrast agent for both CT and MRI. We herein present the synthesis, characterization, cell viability, biodistribution, biodegradation and in vivo imaging results of dextran coated bismuth-iron oxide nanoparticles (BION) as a contrast agent for both CT and MRI. We synthesized a range of BION formulations and characterized them using various analytical methods. CT phantom imaging confirmed that the X-ray attenuation of the different BION formulations was directly proportional to the amount of bismuth present in the nanoparticle. The MRI contrast produced decreased with increasing bismuth content. No cytotoxicity response was observed in Hep G2 and BJ5ta cells after 24 hours incubation with BION. Strong X-ray attenuation was observed in the heart and blood vessels over a sustained period, indicating that Bi-30 has a prolonged circulation half-life. Considerable signal loss in T2-weighted MR images was observed in the liver compared to pre-injection scan, indicating efficacy in this imaging modality. In vitro and in vivo data indicated that the BION are degradable and excretable. In conclusion, dextran coated BION are biocompatible, biodegradable, excretable, possess strong X-ray attenuation and also perform well as T2-weighted MR contrast agents. As a result, BION can be used as a dual imaging probe for both CT and MRI.
Detection of inflammation in bleomycin-induced lung injury using hyperpolarized 13C MRS
Hoora Shaghaghi, Stephen Kadlecek, Sarmad Siddiqui, Mehrdad Pourfathi, Hooman Hamedani, Harrilla Profkaa, and Rahim Rizia
Department of Radiology, Perelman School of Medicine, University of Pennsylvania
It is generally accepted that inflammatory cell activity and particularly that of neutrophils can exacerbate acute lung injury (ALI). It would be useful to visualize and quantify the progression of lung inflammation to directly inform a wide variety of clinical decisions. Unfortunately, direct imaging of inflammatory cell infiltration or activity is difficult. In this work, we have demonstrated the use of non-ionizing, hyperpolarized (HP) 13C spectroscopy to detect pulmonary inflammation. We have illustrated that the uptake and metabolism of HP 1-13C pyruvate by inflammatory cells is rapid and displays sufficient additional signal to be easily distinguished from metabolism by the healthy lung tissue. We have used intratracheal instillation of bleomycin since this is a well-established model of acute pulmonary inflammation. The overall apparent activity of lactate dehydrogenase is shown to increase significantly (3.3-fold) at the 7 day acute stage and to revert substantially to baseline at 21 days, while other markers indicating monocarboxylate uptake and transamination rate are unchanged. Elevated lung lactate signal levels correlate well with phosphodiester levels as determined with 31P spectroscopy and with the presence of neutrophils as determined by histology, consistent with a relationship between intracellular lactate pool labeling and the density and type of inflammatory cells present. Comparison of spectroscopic and histologic results suggested that the most probable source of the observed signal increase is direct uptake and metabolism of pyruvate by inflammatory cells and primarily neutrophils. This signal is seen in high contrast to the low baseline activity of the lung. The overall signal levels are raised such that imaging applications become feasible.
T cell dependent rejection of pancreatic cancer in tumor-bearing mice treated with agonistic CD40 and chemotherapy
Katelyn T. Byrne and Robert H. Vonderheide
Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania
Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive cancer that is resistant to most treatments. Although the FDA recently approved nanoparticle albumin-bound paclitaxel, Abraxane (Abrx) in combination with gemcitabine (Gem), the objective response rate was only 23%. Given that CD40 activation can reverse tumor-induced immune suppression, we administered agonistic CD40 antibody (FGK45) with Gem/Abrx chemotherapy to mice bearing subcutaneous PDA tumors established using cell lines derived from the KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre mouse model of PDA. We found only rare tumor regressions in mice treated with chemotherapy or FGK45 alone, whereas more than 50% of tumors regressed in mice treated with Gem/Abrx/FGK45 (p<0.0001). This effect was T cell dependent, with some mice remaining tumor-free for over 100 days. The proportion of T regulatory cells (Tregs) was reduced 6.9-fold after treatment with Gem/Abrx/FGK45 (p<0.05, versus single therapies), with a concurrent 1.75-fold increase in activated T helper (Th) cells (p<0.01), resulting in increased intratumoral ratios of CD4 Th or CD8 T effector cells to Tregs. Furthermore, after Gem/Abrx/FGK45 treatment, antigen-presenting cells in the tumor and draining lymph node expressed increased CD86 and MHCII (3.7-fold versus control, p<0.0001), and produced more TNF-alpha (1.6-fold versus control, p<0.0001). Interestingly, responses to Gem/Abrx/FGK45 were independent of the TLR4 and MyD88 pathways, which previously reported to mediate chemotherapy-induced anti-tumor immune responses. These studies highlight the clinical potential of adding CD40 activation to standard-of-care chemotherapy as a novel strategy for PDA, and suggest T cells are effective against highly immunosuppressive tumors if triggered by robust vaccination.
Converting pancreatic cancer from non-immunogenic to immunogenic through activation of the IFN signaling pathway
Dawson Knoblock1 and Gregory L. Beatty2
1Division of Hematology-Oncology, 2Department of Medicine, Perelman School of Medicine, University of Pennsylvania
Pancreatic cancer is a leading cause of cancer-related deaths with few effective treatment options. Tumor immunotherapy, which harnesses the ability of cytotoxic T lymphocytes (CTL) to recognize and eliminate tumor cells, has shown great promise for the treatment of many advanced cancers. However, in pancreatic ductal adenocarcinoma (PDAC), immunotherapy has been largely ineffective. We hypothesized that pancreatic tumors may evade immune elimination due to an intrinsic lack of immunogenicity. To test this hypothesis, we developed several cancer cell lines from a clinically relevant genetic model of PDAC. We found by flow cytometry that PDAC cells display undetectable levels of MHC class I molecules and low levels of the T cell inhibitory ligand PDL1. In vitro treatment of PDAC cells with IFN-γ produced a marked upregulation of MHC molecules as well as PDL1 expression without impacting tumor cell viability or growth. However, PDAC cells pre-treated with IFN-γ in vitro (versus untreated PDAC cells) showed delayed tumor outgrowth in vivo in a subcutaneous implantation model. This effect was further enhanced by combining IFN-γ pre-treatment of PDAC tumor cells with in vivo blockade of the PDL1 receptor, PD1, which is upregulated on activated T cells. Ongoing studies are now examining the role of T cells in delaying the outgrowth of tumors pre-treated with IFN-γ. Overall, our findings suggest that PDAC is a poorly immunogenic tumor that can be converted to an immunogenic tumor through activation of the IFN signaling pathway.
CCL2 is necessary for macrophage-dependent fibrosis degradation in pancreatic ductal adenocarcinoma
Kristen B. Long, Whitney L. Gladney, and Gregory L. Beatty
Abramson Cancer Center, Department of Medicine, Division of Hematology-Oncology, Perelman School of Medicine, University of Pennsylvania
Pancreatic ductal adenocarcinoma (PDA) is an incurable, rapidly progressing disease. It is resistant to chemotherapy, as the fibrotic tumor stroma of PDA is believed to act as a barrier preventing effective drug delivery and therefore limiting the benefit to patients. We have previously shown in a mouse model of PDA that immunotherapy with an agonist CD40 monoclonal antibody (mAb) can induce tumor stroma degradation in a manner that is dependent on extratumoral macrophages. Here, we investigated the mechanism by which macrophages facilitate anti-tumor effects using the KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) genetically engineered mouse model of PDA. In this model, we observed high levels of the extracellular matrix proteins fibronectin, hyaluronic acid and type I collagen present within the surrounding PDA stroma. Treatment with the CD40 mAb resulted in an extratumoral macrophage-dependent degradation of both fibronectin and type I collagen, but had no effect on hyaluronic acid. To understand the activation status of extratumoral macrophages after CD40 mAb treatment, we examined serum cytokine levels. We found a significant increase in CCL2, which was almost completely absent when extratumoral macrophages were depleted prior to CD40 mAb treatment. We hypothesized, then, that CCL2 produced by extratumoral macrophages is an activating component necessary for matrix degradation. To examine this relationship, we blocked CCL2 prior to CD40 mAb treatment and found that CCL2 ablation abrogated the anti-stromal effects of CD40 mAb. Our findings suggest that extratumoral macrophages mediate such anti-stromal effects through the production of CCL2. This finding has important implications for the design of novel strategies that exploit the anti-fibrotic potential of macrophages in an adjuvant setting.
G0-like cells support an oral cancer stem cell pool by transitioning to a JARID1B-hi state
Nicole Facompre1, Zachary Belden1, Anil Rustgi2, Meenhard Herlyn3, Hiroshi Nakagawa2, Devraj Basu1,3,4
1Department of Otorhinolaryngology, and 2Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania;
3Wistar Institute; 4VA Medical Center
Oral squamous cell carcinomas (OSCCs) are proposed to contain a subset of cells whose stem cell-like molecular and functional properties are required to sustain tumor growth. Growing evidence supports the existence of a heterogeneous CSC pool with multiple tumorigenic subsets persisting in dynamic equilibrium. To explore such plasticity within an oral CSC pool, we evaluated tumor cells in a “G0-like” state previously found to have high growth potential and innate drug resistance. G0-like cells isolated from OSCCs exhibited the hallmarks of low reactive oxygen species (ROS), high Hes1, low Akt, and resistance to phosphoinositide 3-kinase (PI3K) inhibition. Despite lacking molecular stem cell markers, these cells displayed functional properties of efficient in vitro sphere formation and in vivo tumorigenicity. To evaluate the contribution of G0-like cells to a CSC population, a GFP reporter system was used to isolate CSCs based on high expression of JARID1B, an H3K4me3 demethylase associated with multiple normal and malignant stem cell phenotypes. JARID1B-hi cells highly expressed CD44 and possessed both molecular and functional CSC traits, as well as sensitivity to PI3K inhibition. Silencing JARID1B expanded the G0-like subset but abrogated its functional CSC properties, suggesting these cells mediate such functions by returning to a JARID1B-hi CSC state. These findings reveal a JARID1B-mediated mechanism underlying a contribution of G0-like cells to a CSC pool and provide insight into how targeting the PI3K pathway, which has central importance in OSCC, can have disparate effects on subsets of tumor cells. Together, these observations may provide a basis for more effective OSCC treatment targeting both CSCs and non-CSCs that can efficiently return to a CSC state.
Elucidating the role of DNA methylation in Renal cell cancer (RCC)
Kriti Gaur, Ae Seo Deok Park, and Katalin Susztak
Renal Electrolyte and Hypertension Division, Perelman School of Medicine, University of Pennsylvania
Kidney cancer is among the 10th most common cancers in both men and women, with the American Cancer Society estimating 60000 new cases and 13000 deaths in the U.S in 2014. Though organ confined or locally advanced RCC is cured by surgical resection, patients with metastatic disease have a 5 year survival rate under 10%. Present knowledge of the molecular basis of cancer indicates that epigenetic alterations, e.g aberrant promoter methylation, make an important contribution to the biological behavior of tumor. In fact, methylome analysis of clear cell RCC samples demonstrated a distinct epigenetic profile which is dissimilar from normal samples (Hu et al., 2014), and was not influenced by patient demographic, age, gender, VHL mutational status or history of hypertension and diabetes mellitus. We generated mice with deletions in methyl cytosine hydroxylase enzyme, Ten-Eleven translocation-2 (TET2) as well as DNA methyltransferases Dnmt1, Dnmt 3a, Dnmt3b and examined their effects in modulating signaling pathways, like VHL and Notch, that are known to play key role in RCC progression and also monitored the methylation status of individual loci and other epigenetically regulated targets. Our mouse model will not only provide mechanistic insight into the epigenetic basis of RCC development but also allow for testing of potential therapies for renal neoplasms.
CXCL13 as a mediator of oncogenic PKCe in prostate cancer
Rachana Garg1, Martin Abba2 and Marcelo G. Kazanietz1
1Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.; 2Centro de Investigaciones
Inmunológicas Básicas y Aplicadas (CINIBA), Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Calle 60 y 120, La Plata, CP:1900, Argentina.
Protein kinase C epsilon (PKCe) has emerged as an oncogenic kinase and is known to be up-regulated in prostate cancer. We recently showed that transgenic PKCe overexpression in the mouse prostate leads to hyperplasia and prostatic intraepithelial neoplastic lesions but no cancer. In this study, we intercrossed PKCe prostate transgenic mice with mice haploinsufficient for Pten (another alteration in prostate cancer). Interestingly, the resulting mutant mice develop fully invasive adenocarcinoma. To explore mechanistic aspects of observed cooperativity between PKCe overexpression and Pten deficiency, we overexpress PKCe in mouse prostate epithelial lines that are either heterozygous (P8) or homozygous (CaP8) for Pten deletion. PKCe overexpression leads to significant enhancement in cell proliferation and levels of p-Akt, p-Erk, and p-mTOR; these effects are more pronounced in CaP8 cells. Migration, invasion, clonogenic, and soft agar colony formation assays also show synergism between PKCe overexpression and Pten loss. Furthermore, microarray analysis reveals changes in genes related to EMT, adhesion, metabolism, and invasiveness following PKCe overexpression and Pten loss. Particularly, the chemokine,CXCL13 is majorly up-regulated in CaP8-PKCe cells. CXCL13 measurement in the conditioned medium (CM) shows increased levels in PKCe overexpressing or Pten depleted cells. Remarkably, while CM from CaP8-PKCe cells causes significant induction in growth and motility of P8 cells; CXCL13 RNAi depletion in CaP8-PKCe cells reduce their proliferative and migratory capacity, thus establishing the relevance of CXCL13 in the phenotypic effects of PKCe overexpression and Pten loss. Our results therefore argue for the role of CXCL13 in mediating effects of oncogenic PKCe in prostate cancer.
Subtype-specific overexpression of the Rac-GEF P-Rex1 in breast cancer is associated with promoter hypomethylation
Laura Barrio-Real1, Lorena G. Benedetti1, Nora Engel2, Soonweng Cho3, Saraswati Sukumar3, and Marcelo G. Kazanietz1
1Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania; 2Fels Institute for Cancer Research and Molecular Biology, Temple University; 3Department of Oncology, Johns Hopkins University, Baltimore, MD
P-Rex1, a PI3K- and Gbg-dependent Rac-GEF, is an essential mediator of ErbB signaling in breast cancer and plays important roles in breast tumorigenesis and metastasis. In a previous study, we determined that P-Rex1 is essentially undetectable in normal human mammary tissue, however it is markedly up-regulated in human breast tumors particularly in the luminal subtype (Sosa et al., Mol. Cell 40:877-892, 2010). The mechanisms behind the P-Rex1 up-regulation in breast cancer are unknown. We observed that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and its methylation inversely correlates with P-Rex1 expression in mammary cell lines. Expression levels of P-Rex1 in normal MCF-10A and basal-like MDA-MB-231 cells could be restored to those observed in luminal breast cancer cell lines upon treatment with the demethylating agent 5-aza-2’-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A. A comprehensive analysis of human breast cancer cell lines and tumors revealed a significant demethylation of the PREX1 promoter in ER-positive, luminal subtypes whereas high methylation is retained in basal-like breast cancer and normal mammary tissue. Moreover, we observed a strong impact of methylation status of PREX1 on breast cancer patient survival as low methylation of the PREX1 promoter was associated with poor prognosis. Our results establish differential methylation of the PREX1 gene as a key mechanism for the control of P-Rex1 expression in human breast cancer subtypes and suggest that its demethylation contributes to the aberrant overexpression of this Rac-GEF.
Deletion of the mitochondrial chaperone trap-1 uncovers global reprogramming of metabolic networks
Sofia Lisanti1,2, Michele Tavecchio1,2, Young Chan Chae1,2, Qin Liu2, Angela K. Brice3, Madhukar L. Thakur4, Lucia R. Languino1,5
and Dario C. Altieri1,2
1Prostate Cancer Discovery and Development Program and 2Molecular and Cellular Oncogenesis Program, The Wistar Institute; 3Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania; 4Department of Radiology and 5Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University.
Reprogramming of metabolic pathways contributes to human disease, especially cancer, but the regulators of this process are unknown. Here, we generated mice knockout for the mitochondrial chaperone TRAP-1, a regulator of bioenergetics in tumors. TRAP-1-/- mice are viable and showed reduced incidence of age-associated pathologies, including obesity, inflammatory tissue degeneration, dysplasia and spontaneous tumor formation. This was accompanied by global upregulation of oxidative phosphorylation and glycolysis transcriptomes, causing deregulated mitochondrial respiration, oxidative stress, impaired cell proliferation and a switch to glycolytic metabolism, in vivo. These data identify TRAP-1 as a central regulator of mitochondrial bioenergetics, and this pathway could contribute to metabolic rewiring in tumors.
KSHV-mediated regulation of Par3 and SNAIL contributes to B-cell transformation
Hem Chandra Jha, Santosh Kumar Upadhyay, Sanket Shukla, Zhiguo Sun, and Erle S. Robertson
Department of Microbiology and Tumor Virology Program, Abramson Cancer Center, Perelman School of Medicine at the University
KSHV is involved mainly in two types of cancers, B-cells and endothelial cells. Infection with KSHV can induce cell migration, invasion and disease progression. Protease-activated receptors (PARs) are proteins that have seven transmembrane domains similar to G protein-coupled receptors, which are activated through proteolytic cleavage by serine proteases. PARs represent a component of the body defense system, and aggressively participate in the inflammatory responses. In many cancer cell lines, SNAIL is upregulated and also plays a critical role in the development of malignancies. However, the mechanism by which latency associated nuclear antigen (LANA) encoded by KSHV regulates PAR-3 and SNAIL during viral-mediated oncogenesis is yet to be explored. We now demonstrate that PAR-3 is significantly up-regulated in KSHV-infected primary cells and KS tissues. Further, PAR-3 can interact with LANA in KSHV positive and LANA expressing cells which ultimately led to translocation of PAR-3 from cell periphery to a predominantly nuclear signal. PAR-3 is stabilized by LANA and PAR-3 knockdown led to reduced cell proliferation in colony formation assays and increased apoptotic induction. We also demonstrated that SNAIL transcript and protein was elevated in the presence of LANA or PAR-3 in KSHV infected primary cells. Further Ku70, Ku80 and DNAPKcs were important for translocation of PAR-3 to the nucleus in KSHV-infected cells. Importantly, SNAIL inhibition suppressed its downstream targets on KSHV infection suggesting a role in establishment of KSHV latency. Knockdown of PAR-3 and SNAIL in KSHV positive cells led to apoptosis of KSHV associated cancers. Overall, our study revealed for the first time that Par-3 and SNAIL are crucial for establishment of KSHV-associated B-cell lymphomas.
Notch1, but not Notch2, regulates podocyte survival and dedifferentiation during diabetic glomerulosclerosis
Mariya T. Sweetwyne, Katalin Susztak
Department of Renal-Electrolyte and Hypertension, Perelman School of Medicine, University of Pennsylvania
Over 110 L of blood/day filter through the glomeruli of the kidneys. A unique cell, the podocyte, wraps around glomerular capillaries to form the filtration slit. Injury to podocytes causes loss of the filtration slit and leads to renal failure. Diabetes is the #1 cause of renal failure. Therefore, understanding the podocyte response to diabetes should lead to the prevention of renal failure. Multiple Notch protein ligands and receptors are regulated in diabetic glomeruli. Proving that podocyte Notch signaling is critical during glomerulosclerosis, podocyte-specific deletion of a global Notch transcriptional co-activator, Rbpj, protected glomeruli from diabetic injury. Here we determined the responsible Notch receptor isoform by probing the specific role of Notch1 and 2 in podocytes with diabetic and pro-fibrotic stimuli. Mice expressing floxed Notch1, or 2, and Podocin-Cre generated animals with podocyte-specific deletion of Notch1 or 2. In vivo, streptozocin injection induced diabetes. Mice were examined for glomerulosclerosis. In vitro, primary podocytes were cultured and treated with TGF-b1, a pro-fibrotic cytokine highly upregulated in diabetic glomeruli, to probe Notch signaling. Podocyte-specific deletion of Notch1, but not 2, protected diabetic mice from podocyte loss, albuminuria and glomerular injury. In vitro, Notch1, but not Notch2, was activated by TGF-b1 in wildtype (WT) podocytes. TGF-b1 also increased dedifferentiation (stress fiber formation and Snail1 transcriptional regulator expression) and apoptosis (Caspase 3/7) of WT and Notch2-null podocytes. Notch1-null podocytes were protected from these changes. Thus, we find specific upregulation of podocyte Notch1 induces the formation of diabetic glomerulorsclerosis via podocyte dedifferentiation and loss.
Diurnal changes in autophagy: a role for the clock?
S. C. McLoughlin, G. Yang, G. Grant, G.A. FitzGerald
Institute for Translational Medicine and Therapeutics, University of Pennsylvania
Autophagy is a cellular process whereby the cell digests its own components as a means of removing defective organelles, providing a source of amino acids or lipids in times of need, controlling inflammation and defending against invading pathogens. Reduced autophagy is implicated in a variety of conditions ranging from neurodegenerative diseases to cancer. In vivo, autophagy is increased during the sleep phase and reduced during the active phase. The control of such diurnal variability is not fully understood and the relevance of a day/night pattern is unknown. Disrupted circadian rhythms affect a variety of cellular processes including metabolism and inflammation. Here, we investigate the effects of disrupted circadian rhythms on autophagy gene expression, basal autophagy function and induced autophagic response in liver and macrophages. We show arrhythmic and reduced expression of a subset of autophagy genes in the liver of global, postnatal BMAL1 knockout mice. This indicates that disrupted autophagy in the absence of BMAL1 is not secondary to the premature aging phenotype of the conventional BMAL1 knockout mice. Peritoneal and bone-marrow derived macrophages from BMAL1 knockout mice show altered levels of autophagy gene expression and an abnormal autophagic response, which may contribute to the inflammatory phenotype of these mice. Intriguingly, primary macrophages cultured from both wildtype and BMAL1 knockout mice do not exhibit rhythmic expression of autophagy genes that are reported to cycle in macrophages in vivo. Further studies will elucidate the mechanism by which circadian disruption leads to abnormal autophagy and the role of the clock, if any, in diurnal autophagy rhythms in vivo.
Rapamycin blocks induction of the thermogenic program in white adipose tissue
Cassie Tran1,2, Sarmistha Mukherjee1,2, James G. Davis1,2, and Joseph A. Baur1,2
1Department of Physiology, Perelman School of Medicine, University of Pennsylvania;2Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania
White adipose depots contain a subset of cells that display features of brown adipocytes, including expression of the classical marker uncoupling protein 1 (UCP1). These cells, termed beige or brite adipocytes, accumulate in response to multiple stimuli, including cold and beta adrenergic signaling. Because of their ability to metabolize glucose and fatty acids and dissipate the resulting energy as heat, induction and stimulation of beige adipocytes is considered a promising strategy to combat obesity and diabetes. Here we report that rapamycin, a drug that extends life, but paradoxically, causes glucose intolerance and lipid dysregulation, blocks the induction of beige adipocytes by cold exposure or a beta3-adrenergic agonist (CL316,243). Rapamycin acutely inhibits mechanistic target of rapamycin complex I (mTORC1), but can also inhibit mTORC2 under chronic conditions. We find that acute exposure is sufficient to partially block induction of thermogenic markers, suggesting the involvement of mTORC1. Inhibition occurs downstream of the canonical beta adrenergic signaling cascade, since acute responses to CL316,243, including lipolysis, insulin secretion, and glucose depression, remain intact. These findings reveal a critical role for mTOR signaling in the determination of adipocyte fate, and suggest that inhibition of the thermogenic program may contribute to the detrimental effects of rapamycin on metabolism.
Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2
Manuela Lavorato1, Tai-Qin Huang2, Venkat Ramesh Iyer3, Stefano Perni1, Gerhard Meissner2, and Clara Franzini-Armstrong1
1Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania; 2Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC; 3Division of Cardiology, Children’s Hospital of Philadelphia
In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca2+ homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2ADA/ADA) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca2+ sequestration, and early cardiac hypertrophy and death. We have examined the ultrastructure and RyR2 immunolocalization in wild type (WT) and Ryr2ADA/ADA hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage. Some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15% in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. These data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2ADA/ADA mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.
Altered force sensing and cell-cell adhesion by mutant ALK2 FOP cells – implications for heterotopic ossification
Julia Haupt1,3, Brian Cosgrove1,4, Claire McLeod1,4, Andria Culbert1,3, Robert L. Mauck1,4,5,6, and Eileen M. Shore1,2,3
1Departments of Orthopedics, 2Departments of Genetics, 3Center for Research in FOP and Related Disorders, Perelman School of Medicine, University of Pennsylvania; 4Departments of Bioengineering 5Departments of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania; 6Translational Musculoskeletal Research Center, Philadelphia VA Medical Center
The rare genetic disease fibrodysplasia ossificans progressiva (FOP) misregulates progenitor cells to form heterotopic bone in soft tissues. Microenvironment rigidity modulates lineage specification to direct cell fates. Soft substrates induce cells towards neuro-/adipogenic fates while stiff substrates promote chondro-/osteogenesis. Pathologic tissue stiffening occurs in fibrotic diseases when damaged tissue aberrantly acquires increased rigidity during wound healing. Injury-induced FOP lesions similarly exhibit excessive fibroproliferation. Gain-of-function R206H mutations in the BMP receptor ALK2/ACVR1 cause FOP. Since BMP signaling regulates and is regulated by cell tensional force, we hypothesized that altered ALK2 signaling from the FOP mutation alters mechanical force sensing in progenitor cells and lowering the threshold for bone formation. We used immortalized mouse embryonic fibroblasts (iMEFs) from ACVR1R206H knock-in and WT embryos on varied matrix elasticity (soft/5kPa; moderate/15kPa, stiff/55kPa) and analyzed cell size, aspect ratio (AR), circularity, and solidity at low cell density. WT cells responded to increasing stiffness as expected with increased cell size and AR, and decreased circularity and solidity. However, FOP cells on soft substrates were similar to WT on stiff substrates, and FOP cells were less responsive to substrate rigidity. FOP iMEFs also showed loss of contact inhibition, reduced cell-cell contacts, and β-catenin loss at cell membranes. The data support that the combination of increased BMP signaling and misinterpretation of biomechanical signals in FOP cells lowers their threshold for commitment to chondro-/osteogenic lineages, resulting in an aberrant tissue repair response that leads to ectopic bone formation.
VAMP7 trafficking in melanosome biogenesis: tubular transport into and out of melanosomes
Megan K. Dennis1, Cédric Delevoye2,3, Amanda Acosta-Ruiz1, Geoffrey G. Hesketh4, J. Paul Luzio5, David J. Owen5, Graça Raposo2,3, and Michael S. Marks1
1Pathology & Laboratory Medicine, University of Pennsylvania and Children’s Hospital of Philadelphia; 2Institut Curie, Centre de Recherche, and 3Structure & Membrane Compartments, Centre National de la Recherche Scientifique, Paris, France; 4Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Toronto, Ontario; 5Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK
Melanosomes are lysosome related organelles that coexist in pigmented cells alongside classical endolysosomes. Melanosomal membrane proteins are directed towards maturing melanosomes via membranous transport carriers that are derived from early endosomes, but neither the fusion machinery for transport carriers with melanosomes nor a pathway for recycling of components to the site of origin for additional transport are known. We focused on VAMP7 as a candidate vSNARE for melanosome transport. VAMP7 localizes to melanosomal membranes, and depletion of VAMP7 results in mistrafficking of melanosomal cargoes and hypopigmentation. To determine the pathway through which VAMP7 is targeted to melanosomes, we assessed the localization of VAMP7 versus the endosomal SNARE syntaxin13 (STX13) in melanocytes from models of Hermansky-Pudlak syndrome (HPS), wherein melanosomal maturation is impaired. VAMP7 is retained in endosomes in HPS melanocytes and is found in STX13-positive tubules upon rescue of trafficking towards melanosomes in cells re-expressing the absent HPS protein. This suggests that VAMP7 is targeted to melanosomes in STX13-containing endosomal tubules and is likely the vSNARE on this cargo transport pathway. To assess VAMP7 recycling from melanosomes, we exploited live-cell imaging of VAMP7 in wild-type melanocytes. VAMP7 tubules were seen exiting melanosomes. These tubules are not labeled with STX13 and instead are marked by the presence of VARP, a scaffolding protein that maintains VAMP7 in an inactive conformation and is largely localized at steady state to puncta associated with melanosomes. As expected for recycling structures, these tubules lacked melanosomal cargoes. These data support a role for VARP in recycling VAMP7 from melanosomes following cargo delivery.
Interactions between Hedgehog and inflammatory signaling reproduce aspects of biliary atresia
Zenobia C. Cofer, Shuang Cui, Valerie Sapp, and Randolph Matthews
The Children’s Hospital of Philadelphia
Biliary Atresia (BA) is a progressive, fibro-inflammatory liver disorder that exclusively affects infants. Without timely surgery to restore bile flow, ongoing damage to the biliary tract will lead to fibrosis and eventual cirrhosis. Even in surgically corrected patients future liver failure may occur. Thus, BA is the leading indicator of liver transplantation in children. What causes BA remains unknown. However, patient livers show infiltration of immune cells typical of an inflammatory response. Previous reports suggest autoimmunity as a potential cause. Other reports show Hedgehog (Hh) pathway overexpression and epigenetic changes as potential factors in BA development. Using the zebrafish hepatobiliary models ahcy and dnmt1, we show epigenetics and inflammation as possible mechanisms important in BA development. Targeted knockdown of DNA methyltransferase 1 (dnmt1) induces upregulation of components of the inflammatory pathway. Livers from ahcy larvae also show increased expression of tumor necrosis factor alpha (TNFα). Injection of TNFα into fish larvae causes liver abnormalities. TNFα injected larvae also show increased expression of the Hh receptor, Smo. Inhibition of Hh signaling by cyclopamine treatment rescues liver morphology in ahcy and dnmt1 larvae. Furthermore, treatment of larvae with the Hh pathway agonist purmorphamine produces biliary defects and increases expression of the inflammatory pathway. Our results suggest a potential crosstalk between Hh signaling and inflammatory pathways may result in defects observed in BA patients.
Keratin15 annotates a unique population in esophageal squamous epithelium
Veronique Giroux1,3, Jiri Kalabis1,3, George Cotsarelis2, Anil K Rustgi1,3,4
1Division of Gastroenterology, 2Department of Dermatology, 3Abramson Cancer Center and 4Department of Genetics, Perelman School of Medicine, University of Pennsylvania
Background: Stem cells and progenitor cells reside within the proliferating basal cell compartment of the esophageal squamous epithelium. We have identified a potential esophageal stem cell population localized in the basal compartment that is CD34+ with self-renewal capacity and gives rise to all differentiated lineages in the suprabasal and superficial compartments of the epithelium.
Methods: Herein, we investigated the role of hair follicle stem cell marker, K15, in the murine esophageal epithelium. We determined the expression and localization of K15. Next, we performed lineage tracing using RU486-inducible Cre recombinase under control of K15 promoter mice (K15-crePR1) crossed with Rosa Reporter mice (RosamTomato/mGFP). GFP staining was then performed to visualize K15 labeled cells.
Results: We demonstrated that K15 is located predominantly in the basal compartment of the esophagus epithelium. K15 expression is correlated inversely with K13 expression, which is associated with differentiated cells, suggesting that K15 expression is restricted to undifferentiated cells. Furthermore, Ki67+ cells express K15, but K15 expression is not restricted to Ki67+ cells, suggesting that K15 expression is not dependent exclusively upon cellular proliferation. We also found that K15 expression is decreased by 11.7 fold in the stem cell side-population and by 9.6 fold in CD34+ cells. We next performed lineage tracing experiments. Interestingly, at 1, 3, 5, 7 and 10 days following Cre recombination, we observed an increasing number of GFP+ cells in the esophagi of K15-crePR1;Rosa mice.
Conclusions: K15 marks a novel subpopulation of cells in the mouse esophageal epithelium.
Circadian regulation of lipid metabolism by long non-coding RNAs
Elizabeth J. Hennessy and Garret A. FitzGerald
The Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania
Studies in humans and mice have shown that a dysfunctional molecular clock, brought about by shift work, jet lag or mutations in clock signaling genes, results in an increased prevalence of metabolic diseases signifying that environmental variables can have drastic effects on health. The importance of the molecular clockwork is suggested not only by its high degree of conservation and functional redundancy but also by the multiple levels of regulation of the system. The rapid temporal turnover of genes involved in the feed forward and feedback loops in the circadian signaling pathway suggests the requirement of fine-tuning mechanisms. The contribution of non-coding RNAs to the regulation of gene expression has been demonstrated with miRNAs Long non-coding RNAs (lncRNAs) are being established as potent regulators of gene expression at many different levels. A previous study in the FitzGerald lab revealed that specific lipid metabolism gene cassettes oscillate in mouse aorta but do not oscillate in other tissues such as liver indicating a distinct role for the aorta in the maintenance of lipid homeostasis and the clock. To further understand the tissue specific link between circadian rhythm and lipid metabolism we have measured transcript levels of lncRNAs in aorta and the liver, another important tissue in lipid metabolism. Because lncRNAs are notoriously poorly conserved between species, we hypothesize that in a system as highly conserved as the circadian clock, we have identified lncRNAs essential for the maintenance of the system. The discovery of non-coding RNAs and the realization that this class of molecule is not just dark matter of the genome has opened new doors for therapeutic opportunity.
Activation of epidermal cells inhibits intradermal adipogenesis via paracrine Wnt signaling
Jianping Li, Daisuke Suzuki, N. Adrian Leu and Makoto Senoo
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine
It has been well recognized that the hair growth cycle, epidermal cell activation and intradermal adipogenesis are synchronized. Although adipocyte-derived factors contribute to the hair cycle, and conversely, that epidermal cells partially promote adipogenesis through the secretion of proadipogenic factors, the inhibitory mechanism of adipogenesis remains unclear. We find that epidermal overexpression of p63 in mice inhibits adipogenesis, leading to hair cycle arrest and delayed hair regrowth after clipping. In addition, we demonstrate that activation of epidermal cells by tape-stripping wounding in wild type mice enhances p63 expression in the epidermis, followed by the inhibition of adipogenesis in the dermis. Finally, we show that p63-dependent secreted factors from epidermal cells inhibit adipogenesis, and we identify several Wnt ligands as anti-adipogenic factors. Our results indicate that epidermal Wnt ligands are critical paracrine mediators that inhibit intradermal adipogenesis, and they highlight the role of activated epidermal cells as an active participant in synchronizing adipogenesis with the hair cycle.
Ebf2 is a selective marker of brown and beige adipogenic precursor cells
Wenshan Wang1,2, Megan Kissig1,2, Sona Rajakumari1,2, Li Huang1,2, Hee-Woong Lim1,3, Kyoung-Jae Won1,3, Patrick Seale1,2
1Institute for Diabetes, Obesity & Metabolism; 2Department of Cell and Developmental Biology; and 3Genetics Department, Perelman School of Medicine at the University of Pennsylvania
Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α+, myogenic factor 5Cre- lineage–marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2GFP embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.
Pax3 and Hippo signaling coordinate gene expression in neural crest
Lauren J. Manderfield1, Kurt A. Engleka1, Haig Aghajanian1, Steven Yang1, Li Li1, Julie E. Baggs2, John B. Hogenesch2, Eric N. Olson3 and Jonathan A. Epstein1,4
1Department of Cell and Developmental Biology, University of Pennsylvania; 2Department of Pharmacology, University of Pennsylvania; 3Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX; 4Penn Cardiovascular Institute, University of Pennsylvania
Loss of Pax3, a developmentally regulated transcription factor expressed in pre-migratory neural crest cells, results in severe developmental defects and embryonic lethality.Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation capacity exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional co-activators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the related Hippo effector proteins Taz and Yap65 as Pax3 co-activators. Synergistic co-activation of target genes such as Mitf by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead-independent, and is regulated by the Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 co-localize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 recapitulates Pax3/7-related neural crest defects and expression of the Pax3 target gene, Mitf, is decreased. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.
Differentiation of human endodermal stem cell cells into lung lineage cells
Chiamin Liao1, Edward Morrisey2, Paul Gadue1,3
1Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, and 2Department of Medicine and Cell and
Developmental Biology, Perelman School of Medicine, University of Pennsylvania; 3Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia
Pluripotent stem cells (PSCs), while offering a powerful system for studying disease, face several challenges. First, undifferentiated PSCs have the potential to form teratomas, complicating future therapeutic applications. Second, it can be difficult to differentiate PSCs into mature functional cell types, especially in the case of anterior endoderm derivatives such as thyroid, thymus, and lung. To overcome these obstacles, our lab has generated a new stem cell population termed endoderm progenitor (EP) cells, derived from human PSC lines. EP cells can be expanded in culture, are restricted in developmental potential to endodermal derivatives, and do not generate teratomas. We have previously shown that EP cells can differentiate into hepatocytes, pancreatic beta cells, and intestinal cells. Importantly, EP-derived pancreatic beta cells are functionally responsive to glucose in vitro, unlike those generated directly from ES cells. These results suggest that EP cells provide a superior and safer starting point for generating endodermal cell types. Our goal is to examine the ability of EP cells to generate lung lineage cells and to determine if, like beta cells, EP cells generate higher quality differentiated lung progeny. Our data show that undifferentiated EP cells express the lung marker FOXP2, and that we can anteriorize EP cells as indicated by up-regulation of SOX2, FOXA2 and loss of immature endoderm markers such as SOX17 and EOMES. Our transplantation studies have demonstrated that anteriorized EP cells can spontaneously differentiate into lung lineage cells as demonstrated by up-regulation of NKX2.1 and markers of differentiated lung lineage cells (SOX9, P63 and SPC) and EP cells can generate close to 100% FOXA2+SOX2+ anteriorized endodermal cells.
Depressed cerebral blood flow response to hypercapnia in children with obstructive sleep apnea syndrome
David R. Busch1,2, Jennifer M. Lynch2, Madeline E. Winters2, Ann L. McCarthey4, Mary Anne Cornaglia3, Arjun G. Yodh1,
Carole L. Marcus3, Daniel J. Licht2, Rui Xiao5, Ignacio E. Tapia3
1Dept. Physics and Astronomy; 2Division of Neurology; 3Division of Pulmonology Children’s Hospital of Philadelphia and Hospital of the University of Pennsylvania; 4Temple University School of Medicine; 5Dept. of Biostatistics and Epidemiology, University of Pennsylvania
Obstructive sleep apnea syndrome (OSAS) is characterized by episodes of repetitive upper airway collapse during sleep, resulting in intermittent hypercapnia, hypoxemia, and frequent arousals from sleep. Children with OSAS are chronically exposed to hypercapnia during sleep, therefore it is possible that they have abnormal cerebral blood flow (CBF) regulation during wakefulness, compared to snorers and controls. We further hypothesize that this disregulation is manifested by blunted CBF response to induced hypercapnia.
This abnormal CBF regulation may contribute to poor neurobehavioral outcomes. Diffuse optical spectroscopy may provide a non-invasive tool to identify those children most likely to develop deleterious sequela while these conditions are still treatable.
Translating RNAi Therapy for Spinocerebellar Ataxia Type 1
Megan S. Keiser1, Ryan L. Boudreau2, Jeffrey H. Kordower3, and Beverly L. Davidson1
1Department of Hematology, Children’s Hospital of Philadelphia; 2Department of Pulmonary, University of Iowa, Iowa City, IA;
3Department of Neurological Sciences, Rush University Medical Center, Chicago, IL
Spinocerebellar ataxia 1 (SCA1) is one of nine polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective treatment strategies for this disease. RNA interference (RNAi) is a naturally occurring process that mediates gene silencing and is currently being investigated as therapy for dominant diseases such as SCA1. Adeno-associated viral (AAV) delivery of a microRNA (miRNA) targeting human and rhesus ataxin-1 (miS1) to SCA1 transgenic mouse cerebellum and brainstem improved neuropathological and motor phenotypes. QPCR analysis revealed 70% knock-down of human Atxn1 in miS1 treated mice compared to control treated mutant mice. Suppression of Atxn1 by miS1 led to rescue in Purkinje cell dendritic integrity. Similar rescue was observed in SCA1 mice overexpressing Atxn1l. Finally, both treatments rescued multiple behavioral phenotypes including balance and gait coordination. As the next step in transitioning to clinical trials we have initiated studies in non-human primates (NHPs) to evaluate the biodistribution, safety and efficacy of AAV.miS1 delivery to NHPs. We are currently mapping the extent of transduction by immunohistochemistry and evaluating the therapeutic safety and efficacy of AAV.miS1. These studies will provide important data to move RNAi therapy for SCA1 to the clinic.
Assessing the tropism of adeno-associated virus serotypes in the primate retina for gene therapy
Pavitra Ramachandran, Alexandra Zezulin, Jeannette Bennicelli, Therese Cronin, Zhangyong Wei, Shangzhen Zhou, Albert M. Maguire
and Jean Bennett
F.M. Kirby Center for Molecular Ophthalmology and Center for Advanced Retinal and Ophthalmic Therapeutics, Perelman School of Medicine, University of Pennsylvania
The success of gene therapy largely depends on delivery of the therapeutic molecule to the target tissue or cell type. Efficient gene delivery can be achieved using viral vectors such as adeno-associated viruses (AAV). AAV has become a popular gene delivery vehicle as these are non-pathogenic viruses and have not been observed to cause an adverse immune response in the human body. There are many AAV (1-9) serotypes with distinct tropisms to different cell and tissue types. In addition, mutations made to the AAV capsid can enhance or alter cellular tropism. We are interested in identifying the tropism of different AAV serotypes in the retina that will be applicable to retinal gene therapy. AAV serotype analyses have been carried out extensively in the mouse retina. However, AAV can display altered cellular tropism across different species. Hence, to assess the tropism of the various AAV serotypes in primates, we delivered AAVs expressing a reporter gene from a cytomegalovirus (CMV) promoter and chicken beta actin enhancer to the rhesus macaque retina. We have used different delivery routes and different doses to assess the efficacy and tropism of the different AAVs in the retina. These studies will be directly applicable to determining AAV gene delivery approaches for the many retinal diseases with no treatment options.
Pre-ART immunologic profiles characterize risk of early mortality and TB-IRIS in HIV/TB co-infected adults
Shruthi Ravimohan1,4, Neo Tamuhla4, Andrew P. Steenhoff1,3,4, Rona Letlhogile4, Kebatshabile Nfanyana4,
Tumelo Rantleru4, Robert Gross1,2,4, Drew Weissman1,4, and Gregory P. Bisson1,2,4
1Perelman School of Medicine, and 2Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania; 3The Children’s Hospital of Philadelphia; 4Botswana-UPenn Partnership, Gaborone, Botswana
Advanced HIV/TB patients are at high risk of early mortality and TB-immune reconstitution inflammatory syndrome (TB-IRIS) after antiretroviral therapy (ART) initiation. As the immunopathogenesis of the two outcomes appear to differ, we hypothesized that patients who experience these outcomes have distinct pre-ART immunologic profiles. We conducted a prospective cohort study in Botswana among advanced (CD4 counts <125 cells/μl) HIV-infected, ART-naïve adults with active TB. Exposures included clinical variables and 29 soluble biomarker levels in plasma pre-ART quantified by Luminex. Patients were separated based on outcomes observed within 6 months of ART initiation: early mortality, paradoxical TB-IRIS using standard definitions, and non-TB-IRIS survivors (controls). Clinical factors associated with early mortality and TB-IRIS relative to controls were compared by rank-sum or chi square tests. Luminex log10 transformed variables were analyzed in a logistic regression. Among 170 patients initiating ART, 120 (70%) survived without TB-IRIS; 33 (19%) developed TB-IRIS, and 18 (11%) died. Female sex, initiating nevirapine, non-TB opportunistic infections, and pre-ART CD4 counts <50 cells/μl were significantly associated with early mortality (p<0.01). Most pre-ART cytokine levels were higher among deaths vs. controls, and elevated pre-ART IL-10 and IL-6 levels were independently associated with death. In contrast, most pre-ART cytokine values were lower in TB-IRIS patients vs. controls, and lower levels of GM-CSF, IL-10, IL-6, and IL-12p70 were independently associated with TB-IRIS. Pre-ART risk profiling of each outcome is possible and should be investigated in order to direct appropriate interventions to each patient population.
Using protein mRNA tethering to identify novel splicing factors
Benjamin Cieply, Tatiana Karakasheva, Russ Carstens
Department of Genetics, Perelman School of Medicine, University of Pennsylvania
Over 400 RNA binding proteins (RBPs) with canonical RNA binding domains or motifs exist in the human genome but the functions of many of these proteins, including splicing regulatory activity, have not been defined. We hypothesize that novel splicing factors, including tissue-specific examples, reside within this set of genes and our broad goal is to identify them. Greatly confounding the achievement of this goal is the task of determining which RBPs to test on which transcripts. We are therefore using protein/mRNA tethering via the lambda-N/BoxB system to recruit RBPs downstream of a test exon in an EGFP reporter and screen for their ability to activate splicing in co-transfection assays. We have tested about 250 different RBP-lambda-N fusion proteins in this manner and identified many examples of known splicing factors such as ELAV1, TIA-1, NOVA1 and RB-FOX2 as well as candidate novel splicing factors. A mutant BoxB, which abolishes tethering, is also being employed as a secondary screen to eliminate factors with off target effects. Preliminary validations of the screen via shRNA mediated knockdown in cell lines of two candidate splicing factors, ZCCHC24 and PEG10, have revealed splicing changes in the FLNB and ITGA6 transcripts respectively. mRNA-seq is in progress to identify genome wide alternative splicing regulated by these factors. We plan to complete the screen for splicing activation and silencing in order to establish a greatly expanded list of RBPs that are splicing factors. Several of the factors that activated splicing in our screen, thus far, exhibit tissue/cell type specific expression patterns in, for example, testes, placenta, and T-cells. This work will open new avenues for the investigation of the role of alternative splicing in development and disease.
Detection of cell-free tumor DNA circulating in blood of mouse model of medulloblastoma
Mateusz Koptyra, Tom Curran
Children’s Hospital of Philadelphia Research Institute
Tumors acquire mutations during clonal evolution and as a response to therapeutic challenge. Particularly for brain tumors, it is not possible to obtain repeat biopsies to study this process. In addition, tumor heterogeneity increases genomic complexity. Recent studies have shown that genomic alterations in solid tumors can be characterized by analyzing circulating cell-free tumor DNA. We used mouse medulloblastoma model to investigate cell-free tumor DNA circulating in blood. Using real-time PCR and whole genome sequencing, we detected tumor specific DNA sequences in the plasma of tumor-bearing animals. We could distinguish the signal obtained from tumor DNA from the normal DNA. Moreover, we could monitor the therapy response and tumor re-growth in animals. These findings may help in the development of a diagnostic test for medulloblastoma brain tumor.
Copy number variants encompassing disease genes in an Amish family segregating bipolar disorder
Rachel L. Kember1, Benjamin Georgi1, Joan E. Bailey-Wilson7, Dwight Stambolian2, Steven M. Paul4,5,6 and Maja Bucan1,3
1Departments of Genetics, 2Ophthalmology, 3Psychiatry, Perelman School of Medicine, University of Pennsylvania;4Departments of
Neuroscience, 5Pharmacology and 6Psychiatry, Weill Cornell Medical College, New York, NY; 7Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Baltimore, MD
Bipolar affective disorder (BP) is a common, highly heritable psychiatric disorder characterized by periods of depression and mania. Genetic factors have been shown to be of importance in the development of BP, but identifying specific genetic variants remains a challenge. Copy number polymorphisms (CNVs) are a common class of genetic variation that can change gene expression and have been previously associated with human disease. Using dense SNP genotype data, we characterized CNVs in 388 members of an Old Order Amish Pedigree with bipolar disorder. We identified CNV regions arising from common ancestral mutations by utilizing the pedigree information. By combining this analysis with whole genome sequence data in the same individuals, we also explored the role of compound heterozygosity. We describe 541 inherited CNV regions, of which 268 are rare in a control population of European origin but present in a large number of Amish individuals. In addition, we highlight a set of CNVs found at higher frequencies in BP individuals, and within genes known to play a role in human development and disease. As in prior reports, we find no evidence for an increased burden of CNVs in BP individuals, but we report a trend towards a higher burden of CNVs in known Mendelian disease loci in bipolar individuals (BPI and BPII, p=0.06). We conclude that these CNVs may be contributing factors in the phenotypic presentation of disorder in this family. These results reinforce the hypothesis of a complex genetic architecture underlying BP disorder, and suggest that the role of CNVs should continue to be investigated in BP data sets.
Exploring novel treatment strategies for Rett syndrome
Janine Lamonica, Deborah Kwon, Darren Goffin, Yingtao Zhao, Brian Johnson, Yue Cui, and Zhaolan (Joe) Zhou
Department of Genetics, Perelman School of Medicine, University of Pennsylvania
Rett syndrome (RTT) is a progressive neurological disease that affects multiple aspects of cognitive development, learning, and motor function, and is one of the leading causes of intellectual disability in females. RTT is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2), with ~10% of cases the result of a single point mutation, T158M. We recently developed a knockin (KI) mouse model mimicking the RTT-associated MeCP2 T158M mutation and found that these mice recapitulate many clinical features of RTT. Surprisingly, we found that the T158M mutation impairs the binding of MeCP2 to methylated DNA in vivo and concomitantly destabilizes MeCP2 protein. This result raised the possibility that either stabilizing MeCP2 T158M protein or elevating MeCP2 T158M protein levels may rescue MeCP2 function and ameliorate symptoms. To test this, we developed transgenic mice expressing different amounts of MeCP2 T158M protein (T158MTg). Upon crossing T158MTg mice to T158M KI mice, animals with elevated levels of T158M protein were generated and subjected to a variety of behavioral assessments. We found that male mice with elevated levels of MeCP2 T158M protein displayed a partial rescue in many areas, including body weight, motor function, and longevity. Thus, increasing MeCP2 T158M protein level improves the health of T158M KI mice and partially alleviates RTT-like phenotypes. This suggests that although it carries the T158M mutation, stabilizing MeCP2 protein, could be a potential treatment strategy. Future experiments will examine the viability of this therapeutic approach in female mice, investigate the pathways involved in MeCP2 degradation, and test novel pharmacologic interventions for RTT based on these pathways.
Assembly of the Golden Orb Weaver spider (Nephila clavipes) genome enables full characterization of silk gene repertoire
Paul L. Babb1, and Benjamin F. Voight1,2
1Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania; 2Department of Genetics, Perelman School of Medicine, University of Pennsylvania
Spider silk is one of the toughest biomaterials that exists (>150 MJ/m3), possesses extreme extensibility, and has wide utility for medical and industrial applications. Orb-weaving spiders spin different “spidroin” proteins together for discrete functional needs (dragline, capture, cement, etc.). Yet, many questions surround the biology of spider silk. What is the genetic basis of strong silk? How does spidroin diversity – number of loci, sequence composition, repeat structure, and tissue-of-origin – relate to the function of silk? To address these questions, we require (1) a complete orb-weaver genome, (2) a full catalog of silk genes, (3) their complete sequences, and (4) knowledge of silk-gland tissue expression. Thus, we sequenced the entire genome (3.5 Gb) of a spider with exceptionally strong silk: the Golden Orb-Weaver (Nephila clavipes). We then used RNA-Seq to build 16 tissue-specific transcriptomes to annotate the genome. We assembled 2.04 Gb of non-repetitive genome on 117,053 scaffolds (N50: 81 kb) at 49x coverage. After computational annotation of >25,000 genes, we identified 14 silk genes from five common spidroin classes (major and minor ampullate, aciniform, piriform, tubliform) and two orb-weaver-specific classes (flagelliform, aggregate), and we detected gland-specificity of spidroin transcripts in 10 silk-gland transcriptomes. We obtained entire sequences for 9 of the silk genes and observed a fourfold increase in the number of repeats in N. clavipes spidroins compared to other spider species. These repeats increase the number of structural beta-sheets in spidroins previously shown to correspond with tough silk and ostensibly providing an adaptive phenotype for N. clavipes, which relies on the successful capture of large prey for survival.
SNPs in the coding sequence and 3’-UTR disrupt microRNA-mediated regulation of target mRNAs
Shyam Ramachandran1,2, Tin-Yun Tang1,2, Ryan M Spengler3, and Beverly L Davidson1,2
1Department of Pathology and Laboratory Medicine, and 2Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia; 3Internal Medicine, University of Iowa, Iowa City, IA
MicroRNAs (miRNAs) are 21 nt long, non-coding RNAs that induce post-transcriptional gene silencing by binding to the target mRNA at the miRNA recognition element (MRE). MiRNA binding generally requires perfect base pairing of nucleotides 2-8 in the miRNA (seed sequence). Our lab recently generated a transcriptome-wide map of MREs in the human brain. In addition to the expected 3’UTR binding sites, we also identified a number of MREs in the coding exons of genes. Some miRNA/MRE interactions were in genes with known disease-associated SNPs, and furthermore, these SNPs overlapped the miRNA binding site. We hypothesize that when SNPs are present in MREs, they either reduce miRNA binding (protein expression increases) or enhance it (protein expression decreases). Through a series of carefully selected filters, we identified 32 mRNA:miRNA interactions that are either disrupted or enhanced by a SNP in the MRE seed. This list comprises 32 unique genes and 21 unique miRNAs. Twenty genes have the MRE in the coding sequence while the remaining MREs reside in the 3’-UTR. We are using a novel reporter assay to determine 1) if the miRNAs repress gene expression at the site of predicted interaction, and 2) how the SNP impacts miRNA binding. Promising candidates will be taken forward for further studies with respect to the biological and disease relevance of the SNP and its target. Our studies will complement earlier work identifying associations between miRNA dysregulation and neurodegenerative disorders. In addition, the potential of disease-associated SNPs overlapping MREs may shed light on whether these SNPs are of therapeutic interest or pathological importance.
Myxomatous Mitral Valve Disease surgical patients have a high distribution of the serotonin transporter-LL polymorphism
Kathryn H. Driesbaugh PhD1, Juan Grau MD1,2, Joseph E. Bavaria MD1, Farrah Alkhaleel1, Eric Lai1, Richard Shaw PhD2, Robert C. Gorman MD1, Joseph H. Gorman III1, Robert J. Levy, MD3, and Giovanni Ferrari PhD1
1Division of Cardiovascular Surgery, Department of Surgery, University of Pennsylvania School of Medicine; 2Valley Heart Center, Columbia University; 3Division of Cardiology, The Children’s Hospital of Philadelphia
The prolapse of the Mitral Valve (MV) leaflet is a hallmark of several cardiac disorders such as Myxomatous MV Disease (MMVD), or ischemic MV regurgitation (MR), and is only treated surgically. Elevated levels of serotonin (5HT) have been associated with valvulopathies. Our current studies suggest that 5HT signaling plays an important role in the progression of MMVD. In addition, a 44 base polymorphism of a repetitive element in the promoter region of the serotonin transporter (SERT) gene, designated as a short (S) or long (L) allele, has been reported. It has been shown that the LL form results in an increased 5HT cellular reuptake compared to the SS form. We hypothesized that patients with MMVD have an enhanced 5HT signaling due to a high frequency of the LL polymorphism.
The echocardiogram and surgical reports of 254 MV surgical patients were reviewed and grouped based on MV diagnosis and Carpentier Functional Classification [Type I: Ischemic MVP (N=59), Type II: MMVD (N=152), Type III: rheumatic MVP (N=41)]. DNA was extracted and genomic fragment analysis was performed to determine allelic frequencies. A chi-square statistical test was performed.
While Type I and Type III patients show the expected polymorphism distribution, patients with MMVD have a higher than expected frequency of LL-SERT polymorphism (34% vs. 25%). MMVD patients also have an increased level of 5HT-receptors and SERT in surgically resected MV tissues. Notably, the frequency of SERT-LL is significantly enhanced (53.4% vs. 25%) (p = 0.009) in MMVD patients under 60 years old.
The SERT-genotype may constitute a novel means of characterizing patients with MMVD into a subgroup with increased risk for rapid progression that may benefit from pharmacotherapy that can alter 5HT-related mechanisms.
Understanding cell identity in the kidney using novel cell type specific epigenome mapping
Pazit Beckerman, Yi-An Ko, Katalin Susztak
Department of Renal-Electrolyte and Hypertension, Perelman School of Medicine, University of Pennsylvania
Background: A single genome gives rise to over 200 cell types in the human body. The epigenome determines the unique gene expression program in each cell type through its effect on chromosomal architecture and transcription factor (TF) accessibility to DNA. Histone modifications serve both activating and silencing roles in transcription. Determining genome-wide distribution of histone marks in different cells can decipher their role in cell type specific transcriptional regulation. Such maps are lacking in the kidney research. The aim of this study is to construct the first epigenome maps of the human kidney and of different kidney cell types.
Methods: Human kidney cortical tissue samples were processed to single cells. Human renal proximal tubule (HKC8) and podocyte cell lines were cultured. Chromatin was crosslinked, lysed and sonicated to fragments of around 200bp. Chromatin mmunoprecipitation (ChIP) was preformed using antibodies for several histone modifications. Libraries of ChIP-enriched DNA and controls were prepared and sequenced.
Results: Sequences obtained from human kidney tissue, proximal tubule and podocyte ChIP-seq were aligned to the human genome and histone modification maps were constructed. Using computational approaches, we integrated the chromatin datasets to build the first annotated epigenome maps of those cells, defining cell specific regulatory regions.
Conclusions: We have constructed novel kidney-specific epigenomic regulatory maps. Their crucial significance lies in our ability to define kidney cell-specific critical genes and regulatory networks. Comparing epigenomic maps of different kidney cells will help us understand kidney cell identity. We will be able to identify key TFs that take critical part in kidney cell regulation and in the pathogenesis of kidney disease.
Viral metagenomics reveal blooms of anelloviruses in the respiratory tract of lung transplant recipients
Jacque C. Young1, Christel Chehoud1, Kyle Bittinger1, Aubrey Bailey1, Joshua M. Diamond2, Edward Cantu3, Andrew R. Haas2, Arwa Abbas1, Laura Frye2, Jason D. Christie2,4,5, Frederic D. Bushman1* and Ronald G. Collman1,2
1Department of Microbiology, 2Pulmonary, Allergy and Critical Care Division, 3Department of Surgery, 4Center for Clinical Epidemiology and Biostatistics and, 5Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania
Few studies have examined the lung virome in health and disease. Outcomes of lung transplantation are known to be influenced by several recognized respiratory viruses, but global understanding of the virome of the transplanted lung is incomplete. To define the DNA virome within the respiratory tract following lung transplantation we carried out metagenomic analysis of allograft bronchoalveolar lavage (BAL), and compared to healthy and HIV+ subjects. Viral concentrates were purified from BAL and analyzed by shotgun DNA sequencing. All of the BAL samples contained reads mapping to anelloviruses, with high proportions in lung transplant samples. Anellovirus populations in transplant recipients were complex, with multiple concurrent variants. Q-PCR quantification revealed that anellovirus sequences were 56-fold more abundant in BAL from lung transplant recipients compared with healthy controls or HIV+ subjects (p<0.0001). Anellovirus sequences were also more abundant in upper respiratory tract specimens from lung transplant recipients than controls (p=0.006). Comparison to metagenomic data on bacterial populations showed that high anellovirus loads correlated with dysbiotic bacterial communities in allograft BAL (p=0.00816). Thus the respiratory tracts of lung transplant recipients contain high levels and complex populations of anelloviruses, warranting studies of anellovirus lung infection and transplant outcome.
Transmitotic persistence of Wnt pathway activity diversifies gene expression in C. elegans embryos
Amanda L. Zacharias1, Travis Walton1, Elicia Preston1, and John Isaac Murray1,2
1Department of Genetics, Perelman School of Medicine, University of Pennsylvania; 2Penn Genome Frontiers Institute
Quantitative differences in signaling pathway activity are a powerful mechanism for diversifying cell fates during development. However, few examples of this mechanism have been identified in vivo, due to the challenges of performing quantitative assays. The Wnt signaling pathway plays a conserved role during animal development, transcriptionally regulating distinct targets in different stages and cell types (i.e. contexts). This dependence of targets on context could reflect not only interactions with differentially expressed transcription factors, but also context-specific differences in the activity of the Wnt pathway itself. We investigated the role of Wnt pathway activity in target expression by using time-lapse microscopy and automated lineage tracing of Caenorhabditis elegans embryos to quantify expression of Wnt ligands, target genes, and nuclear localization of transcriptional effectors in vivo at single cell resolution throughout development. We measured the Wnt pathway-dependence of candidate targets and identified over a dozen important developmental regulators as new Wnt targets. We found that most targets require the Wnt-effector transcription factor POP-1/TCF for either activation or repression but not both. Contrary to existing models, we observed that Wnt-mediated transcriptional activation is strongest in cells that received a Wnt signal in two or more consecutive divisions. We found that these repeatedly signaled cells have higher nuclear β-catenin concentrations and are more likely to express targets that require POP-1 for transcriptional activation. Taken together, these results suggest that the persistence of Wnt signaling across mitosis can integrate lineage history and allow Wnt to activate distinct targets in different developmental contexts.
Copy number variants and congenital heart defects in the 22q11.2 deletion syndrome
Elisabeth E. Mlynarski1, Molly B. Sheridan1, Michael Xie2, Silvia E. Racedo3, Donna M. McDonald McGinn1,
Marcella Devoto1,4,5,6, Elaine Zackai1,4, Bernice E. Morrow3, and Beverly S. Emanuel1,4
1Division of Human Genetics, Children’s Hospital of Philadelphia, 2Center for Biomedical Informatics, Children’s Hospital of Philadelphia, 3Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA, 4Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, 5Department of Biostatistics and Epidemiology, The Perelman School of Medicine, University of Pennsylvania, 6Department of Molecular Medicine, University of Rome La Sapienza, Rome 00185, Italy
22q11DS is the most common microdeletion syndrome, affecting approximately 1 in 2,000-4,000 individuals. The 22q11DS phenotype attributed to the 3 Mb deletion is highly variable, and in addition to congenital heart defects (CHDs) includes dysmorphic facial features, palatal anomalies, hypocalcaemia, immunodeficiency, cognitive impairment and various neuropsychiatric disorders. Approximately 75% of 22q11DS patients have been reported with a CHD and/or an aortic arch defect. The etiology of this phenotypic variability is not currently known. The variable expressivity and reduced penetrance of CHDs is likely influenced by genetic factors as 22q11DS patients with a CHD are more likely to have an unaffected relative with an isolated CHD than those 22q11DS patients with normal cardiac anatomy. These findings are not explained by the inherited chromosome 22, suggesting that variants that influence the development of CHD in these families lie outside of the 22q11.2 region. We hypothesize that individuals with 22q11DS possessing cardiac malformations have structural genetic variants outside of the 22q11.2 deleted region that affect their risk of being born with a CHD. Copy number variant (CNV) analysis was performed using Affymetrix SNP 6.0 array data from two cohorts of 22q11DS patients: the discovery cohort consisted of 569 subjects and a second cohort of 387 subjects. A common duplication of SLC2A3 was the most frequent CNV identified in the discovery cohort, found in significantly more subjects with CHDs than without (p=0.0057, Fisher’s exact test). The SLC2A3 duplication was subsequently detected and found to be significantly enriched in replication cohort subjects with CHDs (p=0.0011, Fisher’s exact test), further suggesting the SLC2A3 duplication may be involved in 22q11DS CHDs, possibly as a genetic modifier.
Atorvastatin partially reverses HIV-induced downregulation of heme oxygenase-1 in human monocyte-derived macrophages
Melanie R. Duncan1, Alexander J. Gill2, Anjana Yadav1, Dennis L. Kolson2, and Ronald G. Collman1
1Department of Medicine, and 2Department of Neurology, Perelman School of Medicine, University of Pennsylvania
HIV-associated neurocognitive disorder (HAND) is a collection of neurological deficits characterized by cognitive, motor, and behavior abnormalities, and remains a significant problem in ART-treated infected individuals. Neurological injury in HAND is an indirect consequence of persistent immune activation, chronic inflammation, and the release of neurotoxins such as glutamate by HIV-infected and/or activated macrophages and microglia into the brain. Previous studies have implicated several macrophage enzymes in the process, including heme oxygenase -1 (HO-1), glutaminase, and indoleamine 2,3-dioxygenase (IDO) that are disturbed by HIV infection. Statins (HMG-CoA reductase inhibitors), used to treat atherosclerosis have pleiotropic effects independent from its cholesterol-lowering action, such as downregulating monocyte/macrophage activation and inflammation. Therefore, we hypothesized that statin drugs will modulate the levels of proteins associated with inflammation and HIV-1 infection in human monocyte-derived macrophages (MDM). We used an in vitro model of HIV-infected MDM. Cells were infected with the macrophage-tropic HIV-1 strain YU-2. After 4-8 days, following establishment of infection, cells were treated with atorvastatin (10 uM). After 6-48 hours, cells were lysed and immunoblotted for HO-1, glutaminases, IDO, and a housekeeping protein. We found that treatment with atorvastatin increases the level of HO-1, partially reversing the effect of HIV infection. In contrast, atorvastatin had no significant effect on two isoforms of glutaminase, KGA and GAC, or on IDO. Atorvastatin partially reverses the deleterious effect of HIV-1 on HO-1 in macrophages. Thus, statin drugs could be useful as an adjunctive therapy for HAND in HIV-infected ART-treated individuals.
Characterizing gut microbiota variation across diverse rural African populations
Matthew Hansen1, Meagan Rubel1, Aubrey Bailey2, Kyle Bittinger2, Alice Laughlin2, Alessia Ranciaro1, Simon R. Thompson1,
F.D. Bushman2, and Sarah A. Tishkoff1
1Department of Genetics, and 2Department of Microbiology, Perelman School of Medicine, University of Pennsylvania
Africans harbor the greatest levels of human genetic variation, and differences in diet are likely to have produced distinct selection pressures resulting in genetic adaptations. An additional important source of variation that can influence both health and disease is the gut microbiome. To better understand how genetic adaptations, culture, and diet interact, we analyzed the gut microbiomes from ethnically diverse rural Africans and urban European and African Americans in Philadelphia using ribosomal marker classification (16s RNA V1/V2) from fecal samples. The African cohort is composed of populations with different diets and subsistence practices, including hunter-gatherers, nomadic pastoralists, and traditional agricultural groups. Samples from 120 rural Africans were extracted, sequenced, and compared with a dataset of 102 European and African American samples, using identical methods and the Roche 454 platform. The impact of diet and ethnicity was characterized by changes in relative abundance of bacterial taxa. Comparison of the rural African to the urban Philadelphia samples confirms previous studies that show marked differences between African and non-African gut flora. In contrast, within Africa there are relatively subtle differences between populations despite highly distinct diets and high levels of genetic differentiation. Among African populations we find that differences in the gut microbiome are not entirely diet dependent, but rather, are correlated also with geography and ethnicity. This represents the largest microbiome study to date of ethnically diverse Africans living indigenous lifestyles and provides novel data from previously uncharacterized African groups.
V. cholerae-derived modulation of epithelial cell ROS production and the implications in host intestinal colonization
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania
Cholera is marked by a rapid onset, involving profuse watery diarrhea and short duration. This project has implications for scientists and people in developing countries who continually succumb to cholera and its impending social consequences. Significant research has been done on the regulatory network of virulence genes for V. cholera. However, there is limited research focused on modulation of host cells by V. cholera at the site of infection. Recently, our laboratory acquired RNAseq data from the intestinal cells of a V. cholera-infected mouse and it was found that there were dozens of host genes altered for expression upon exposure to the pathogen. In particular, there was noticeably increased expression of Duox2 and DuoxA2, an epithelial NADPH oxidase that produces hydrogen peroxide and the chaperone which localizes it to the membrane, respectively. Furthermore, quantitative real time PCR (qRTPCR) analysis revealed a significant increase in Duox2 mRNA production in Caco2 intestinal epithelial cells and HeLa cervical epithelial cells following V. cholera infection. Lastly, V. cholera infection caused a significant increase in reactive oxygen species (ROS) production from the Caco2 monolayer and a modest increase from the HeLa monolayer, underscoring a potential link between increased Duox2 expression and the ROS burst. These data strongly suggest that the pathogen is inducing a host response through interaction with intestinal cells through an unknown receptor/signaling pathway. My research goals are to elucidate the role that Duox2 expression plays in the host response to V. cholera infection, to discover the pathogen factors that induce this response, and locate the receptor/pathway upon which these signals trigger the response.
T cell egress from the skin via the afferent lymph modulates the course of local tissue inflammation
Daniela Gomez Atria, Malissa C. Diehl, Erika Crosby, and Gudrun F. Debes
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania
T cells recirculate through extralymphoid tissues by entering from blood and exiting via afferent lymph. While it is well established that T cell recruitment into tissue is essential to the local inflammatory response, the influence of T cell egress on the course of inflammation is not known. The chemokine receptor CCR7 mediates T cell egress from extralymphoid sites in the steady state and inflammation. Using mouse models to investigate the consequences of effector T cell activation on tissue exit, we found that antigen recognition at the effector site down-regulated the exit capacity of T cells. However, transgenic expression of CCR7 was able to enhance the migration of antigen-sequestered Th1 cells from the inflamed skin to draining lymph node, suggesting that exit receptors can be targeted to limit T cell accumulation at effector sites. Importantly, enhancing the egress of antigen-stimulated Th1 cells through transgenic CCR7 expression ameliorated the local inflammatory response in an adoptive transfer model of cutaneous delayed-type hypersensitivity. Conversely, lack of exit receptor CCR7 led to prolonged accumulation of effector T cells and enhanced inflammation. Furthermore, using a model of lymph cannulation in sheep, we found that Th1 and Th17 cells traveling in the afferent lymph draining inflamed skin, were highly responsive to the CCR7 ligand CCL21, suggesting the CCR7-CCL21 axis as a physiological target in the modulation of inflammation. In conclusion, our findings reveal T cell tissue exit as an important parameter of the pathophysiology of the inflammatory response.
Identifying new cellular factors associated with replicating viral DNA
Emigdio D. Reyes1,2, Daphne C. Avgousti1,2, Christopher McKennan3, Ariella Sasson4, Mansee S. Patel2, Steven H. Seeholzer3,
and Matthew D. Weitzman1,2
1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania; 2Division of Cancer
Pathobiology, 3Protein and Proteomics Core Facility, and 4Center for Biomedical Informatics, The Children’s Hospital of Philadelphia
Adenoviruses promote a permissive cellular environment by manipulating host factors that could support or hamper their replication. Early viral proteins play an important role in recruiting or excluding specific host factors from the viral DNA, and thus facilitating virus gene expression and genomic replication without triggering antiviral defenses. However, the targets of these early adenovirus proteins are largely unknown. Here, we have identified cellular factors enriched at replicating viral or genomic DNA by performing isolation of proteins on nascent DNA (iPOND) coupled to mass spectrometry (MS) on cells uninfected (host) or infected with wild-type adenovirus serotype 5 (Ad5). We show that over 90% of host factors associated with the replicating viral DNA are similarly enriched on replicating cellular genomic DNA. We also identify a subset of factors that are either “more” or “less” enriched on viral DNA compared to the host genome. In order to identify host factors that are targeted by early viral proteins, we compared enriched proteins on wild-type Ad5 and a mutant lacking the E4 region (Ad5∆E4). This comparison revealed factors differentially enriched on viral DNA. Using immunoblotting and immunofluorescence, we confirmed that newly identified host proteins are targets of gene products from the E4 region. We find that cellular proteins are relocalized, degraded, induced or actively recruited during virus infection. We hypothesize that these cellular factors are exploited or inactivated by adenovirus through its early proteins to promote virus replication. Uncovering new Ad5 inactivation targets will provide a better understanding of basic cellular pathways manipulated by adenovirus to induce a cellular environment permissive for virus replication.
Localization of ELAVL1 correlates with Sendai Virus defective viral genome replication and activation of innate immunity
Jennifer T. Grier, Sabrina Tsang, Wenzhi Song, and Carolina B. Lopez
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania
During rapidly replicating RNA virus infections, the viral polymerase becomes error-prone and generates shortened defective viral genomes (DVGs) that have been shown to induce an antiviral immune response. Given the similarity of RNA sequences of the viral genome and the DVGs, it remains unclear why only DVGs trigger the immune response. We hypothesize that host RNA binding proteins (RBPs) may preferentially bind to DVGs and mediate interactions with immune pathways. Thus, an understanding of host factors that mediate the DVG-triggered antiviral immunity could be employed to bolster protective immunity following vaccination against RNA viruses such as Measles or Respiratory Syncytial Virus, which naturally produce DVGs. The mouse pathogen, Sendai Virus (SeV), is a (-)ssRNA virus that serves as a useful model to study mechanisms of virus-host interactions in the presence or absence of DVGs. Immunoprecipitation of SeV nucleoprotein (NP), the viral-encoded RBP, identified a host RBP, ELAVL1, which was only found when DVGs were present. By immunofluorescence, it was observed during SeV infection that ELAVL1 trans-located from the nucleus into the cytoplasm, the site of SeV replication, and at late time points, strongly co-localized with SeV NP. The timing of ELAVL1 re-localization closely matched the induction of the innate immune response triggered by DVG replication and a shift in the ratio of the (+) to (-) stranded DVGs towards the (+) sense, indicating a potential strand-specific relationship. Thus, ELAVL1 appears to interact with SeV NP and/or DVGs during DVG replication, and may play a role in the host antiviral response to SeV infection.
BRD4 regulates Nanog expression in mouse embryonic stem cells and preimplantation embryos
Wei Liu1, Paula Stein2, Xin Cheng3, Wenli Yang4, Ning-Yi Shao5, Edward E. Morrisey4, Richard M. Schultz2, and Jianxin You1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania; 2Department of Biology, University of Pennsylvania; 3State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 4Institute for Regenerative Medicine, University of Pennsylvania; 5Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY
Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass, which gives rise to embryonic stem cells (ESCs). Here, we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos, as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor, small interfering RNAs, or a dominant negative approach suppresses Nanog expression and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 [brahma-related gene 1, aka Smarca4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4)], a key regulator of ESC self-renewal and pluripotency, in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene, providing new insights into the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.
SAMHD1 restricts Herpes viruses in macrophages
Eui Tae Kim1, Tommy E. White2, Alberto Brandariz-Nunez2, Donal M. Coen3, Felipe Diaz-Griffero2, and Matthew D. Weitzman1
1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania and Children’s Hospital of Philadelphia; 2Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY;
3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA
Macrophages play important roles in host immune defense against virus infection. During virus infection, macrophages acquire enhanced antiviral potential. Restriction of virus replication and progeny production is important to prevent viral spread but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular dNTPs. The Herpes viridae are a large family of double-stranded DNA viruses responsible for a number of human diseases. Macrophages play a number of important roles in the herpes viruses infection, but the cellular effectors and mechanisms that modulate virus replication have not been understood. Here, we demonstrate that SAMHD1 restricts replication of both HSV-1 and HCMV in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1, while ectopic expression of SAMHD1 in U937 cells repressed HSV-1 replication. HSV-1 restriction involved the dNTP hydrolase activity of SAMHD1, and was partially overcome by addition of exogenous deoxynucleosides. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Depletion of SAMHD1 also enhanced HCMV replication in differentiated THP-1 cells. However, SAMHD1 impacted major immediate-early (MIE) genes expression of HCMV. Furthermore, we found that viral kinase UL97 of HCMV phosphorylates SAMHD1 in vitro. Our results suggest that SAMHD1 functions more broadly to inhibit replication of DNA viruses through different mechanisms in macrophages.
Salmonella distinctive signatures determine host species
Min Yue1,2, Dieter M. Schifferli1
1Department of Pathobiology School of Veterinary Medicine, 2Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania
Salmonella infection remains a significant medical and veterinary concern worldwide. Among numerous serovars, the most frequently isolated is Typhimurium. Some isolates are exclusively associated with systemic diseases in specific hosts, while others cause gastroenteritis in a broad range of unrelated host species, including humans and livestock. Much effort has been expended to determine the biological basis for Salmonella host-specificity. A major focus has been the identification of host-specific genes. However, little attention has been paid towards the involvement of allelic variation for host specificity. The aim of this study was to detect associations between outer membrane proteins and the host origin. We pooled together 15 genes encoding outer membrane proteins of 580 independent isolates from different hosts. The 15 genes were found to be under different levels of selection (dN/dS: 0.8-1.2). While ompN showed the most variability (129 substitutions and 38 alleles), half of the isolates had a unique frameshift deletion of 10-nucleotides in lpfD, 29% lacked pefA, a plasmid-encoded fimbrial adhesin gene. By using multiple logistic regression, classification and regression trees, and random forest analysis, we detected a list of host-specific single-nucleotide polymorphisms (SNPs). In particular, principal component analysis defined pattern of SNPs from different hosts. Moreover, the nonsynonymous SNPs of fimH showed host preference binding capabilities with epithelial cells of different host species, in supporting with the association results. Our results provide molecular evidence for strain-specific host adaptation among various “broad-host range” serovar Typhimurium isolates by presenting the cause-effect SNPs for bacterial pathoadaptation.
Defective viral genomes stimulate strong antiviral immune responses to respiratory syncytial virus in humans
Yan Sun1, Micah Gilbert1, Deepika Jain1, Karla Tapia1, Cynthia J. Koziol-White2, Reynold A. Panettieri Jr.2, Richard L. Hodinka3,
and Carolina B. López1
1Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania; 2Department of Medicine, University of Pennsylvania; 3Clinical Virology Laboratory, Children’s Hospital of Philadelphia
Human respiratory syncytial virus (RSV) leads to illness in infants, children, and high-risk adults. Effective vaccines and therapeutic methods are currently unavailable. In order to develop strategies to better serve patients, a better understanding of the interactions between virus replication and the host immune response is needed. Our laboratory recently showed that defective viral genomes generated at the time of high virus replication are the primary danger signals for the triggering of the immune response during Sendai virus infection in mice. Therefore, we hypothesized that DVGs generated during RSV infection stimulate the antiviral response and influence virus virulence in humans. To investigate the function of DVGs, we generated virus stocks with a high content of DVGs (HD) and DVG-free virus (LD). HD not only strongly stimulated the expression of antiviral genes, but also potently inhibited virus replication in vitro and ex vivo. Purified defective viral particles (pDPs) from HD were supplemented to LD infection, showing that pDPs phenotypically mimicked RSV-HD. In vivo, mice infected with HD developed a strong and rapid antiviral response as well and showed reduced pathology. To determine whether DVGs are present in human patients, we analyzed pediatric respiratory secretions from RSV-infected children admitted to The Children’s Hospital of Philadelphia. We observed DVGs in more than 45% (20/41) of specimens. Excitingly, DVG-positive samples showed a robust antiviral response, similar to what we observed in mice and in human lung tissue ex vivo. Taken together, our data demonstrate that RSV DVGs are potent triggers of the host antiviral response to RSV in humans and that DVGs may be a good target for preventive and therapeutic treatments.
Mapping the immunostimulatory activity of a Sendai virus defective viral genome
Jie Xu1, Xiomara Mercado-López2, Yan Sun1, Carolina B. López1
1Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania; 2Universidad de Puerto Rico, Campus Arecibo
Introduction: Defective viral genomes (DVGs) generated during Sendai virus infection are the primary triggers of the host antiviral response. DVGs induce the expression type-I interferons (IFN) and other cytokines upon binding through the intracellular viral sensors RIG-I and MDA5. The molecular mechanism behind the superior immunostimulatory activity of DVGs is unknown.
Methods: To identify RNA motifs that provide potent immunostimulatory activity to DVGs, we generated a series of deletion mutants of a prototype DVG derived from Sendai virus. In vitro transcribed RNAs from these mutants were tested for their ability to induce type I IFNs upon transfection.
Results: In silico single strand modeling of RNA folding of the mutants identified an AU-enriched stem loop domain formed by nucleotides 70-114 of the DVG essential for type I IFN induction. Consistent with this prediction, we
demonstrate that mutants lacking this region lose their stimulatory activity, while mutants with this region intact
Conclusion and discussion: Thus, a minimal RNA motif at the 5’ but not the 3’ complimentary sequence is critical for maximal DVG activity and oligonucleotides including such region may represent novel alternatives to be harnessed as potent adjuvants for vaccination.
CD8+ T cell immune responses occur independently of Acetyl CoA Carboxylase 2-dependent regulation of fatty acid oxidation
JangEun Lee1,3, Matthew C. Walsh1,3, Kyle L. Hoehn4, David E. James5, E. John Wherry1,2, and Yongwon Choi1,3
1Department of Pathology and Laboratory Medicine, 2Department of Microbiology, and 3Institute for Immunology, Perelman School of Medicine, University of Pennsylvania; 4Department of Pharmacology, University of Virginia Health System, Charlottesville, VA;
5Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
CD8+ T cell differentiation during infection is closely associated with changes in nutrient and energy metabolism, though the specific regulating mechanisms are unclear. We previously suggested that indirectly enhanced fatty acid oxidation (FAO) regulates memory CD8+ T cell differentiation. Here, we explored the role of CD8+ T cell FAO in immune responses through cell-specific study of acetyl CoA carboxylase 2 (ACC2), a factor that inhibits CPT-1, a rate-limiting enzyme of FAO in mitochondria. We demonstrated that ACC2 deficiency enhanced cellular FAO, but did not influence Ag-specific CD8+ T cell responses during infection with listeria or LCMV. These results suggest that ACC2-dependent FAO may not be a critical determining factor of CD8+ T cell fate. Further studies on the role of FAO in other metabolic processes that are important to CD8+ T cell function will help us to elucidate CD8+ T cell fate decisions.
Drosha inclusions are new components of DPR aggregates in FTLD-TDP and ALS cases with C9orf72 expansion mutation
Sílvia Porta, Linda K. Kwong, John Q. Trojanowski, Virginia M.-Y. Lee
Center for Neurodegenerative Disease Research,Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are two neurodegenerative disorders that share clinical, genetic, and neuropathological features, i.e. accumulations of pathological TDP-43 inclusions. The presence of abnormal expansion of GGGGCC hexanucleotide repeats (G4C2 HRE) in a non-coding region of the C9orf72 gene is the major genetic cause of both FTLD and ALS. These G4C2 HRE can be transcribed to form nuclear RNA foci that recruit RNA-binding proteins inhibiting their normal function. Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 HRE expression leads to dipeptide-repeat protein (DPR) accumulations in the cytoplasm of neurons. Here, we identify the endoribonuclease Drosha protein as a new component of these DPR aggregates associated with abnormal G4C2 HRE. In C9orf72 expansion positive autopsy confirmed cases of FTLD-TDP (c9FTLD-TDP) and ALS (c9ALS), we found that Drosha is mislocalized to the cytoplasm where it forms neuronal cytoplasmic inclusions (NCIs) in the hippocampus, frontal cortex and cerebellum but no Drosha inclusions were seen in FTLD or ALS cases without C9orf72 mutations. Further characterization of the Drosha positive NCIs in the hippocampus revealed co-localization with p62 and ubiquilin-2, two pathognomonic signatures of c9FTLD-TDP and c9ALS cases, but Drosha inclusions did not co-localize with TDP-43 pathology that is present in all c9FTLD-TDP/c9ALS cases analyzed. We conclude that Drosha may play unique pathogenic role in the onset or progression of FTLD-TDP/ ALS in patients with C9orf72 mutations.
Neogenin1 is required for olfactory sensory axon targeting in the zebrafish olfactory bulb
Puneet Dang and Jonathan A. Raper
Department of Neuroscience, Perelman School of Medicine at the University of Pennsylvania
The axonal projection of olfactory sensory neurons (OSNs) from the nasal epithelium to the olfactory bulb is an attractive system to study the development of complex circuitry. OSNs expressing a particular odorant receptor (OR) are intermixed in the epithelium with sensory neurons expressing other ORs, but the axons of OSNs expressing the same OR converge and terminate together to form glomeruli at predictable locations. Here we describe an essential role for the guidance receptor Neogenin1 (neo1) in zebrafish OSN axon guidance. We identified neo1, a close relative of deleted in colorectal cancer (DCC), as a potential guidance receptor in an expression analysis of single isolated OSNs. Neo1 is expressed in OMP-expressing but not in TRPC2-expressing OSNs. In neo1 mutant fish, a subset of OMP expressing OSN axons do not enter the bulb, but take a dorsal-lateral route away from their normal protoglomerular targets. In contrast, OR111-7:IRES:GAL4 transgene-expressing OSN axons that normally target the central zone (CZ) protoglomerulus enter the bulb correctly in neo1 mutants, but then frequently misproject to the more dorsal DZ protoglomerulus. We have previously demonstrated a requirement for netrin and DCC signaling in attracting these same OR111-7 transgene-expressing axons to the CZ (Lakhina et al., 2012). Thus, it is possible that neogenin is working in concert with DCC as a netrin receptor mediating attraction of olfactory axons to the ventral midline of the bulb. Alternatively, neo1 could act as a receptor for RGMa or RGMd, both of which are expressed at the midline. These possibilities can be distinguished by comparing the neo1 phenotype with the phenotypes obtained when each of these candidate ligands are mutated.
Hippocampal excitability after diffuse brain injury in swine
Alexandra V. Ulyanova, Paul F. Koch, Michael R. Grovola, James P. Harris, Victoria E. Johnson, Douglas H. Smith, D. Kacy Cullen,
and John A. Wolf
Center for Brain Injury and Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania
Mild traumatic brain injury (mTBI) or concussion has been associated with long-term cognitive impairment due to axonal pathology. We utilized in vivo electrophysiological recordings to investigate changes in hippocampal excitability and the development of epileptogenesis using a model of closed-head rotational acceleration in swine. Male Yucatan swine (age 6 months) were injured by rotational acceleration of the head (180-260 rad/sec) in the coronal plane. Field potential activity and neuronal firing rates in the dorsal hippocampus were recorded using high-density electrode arrays with simultaneous afferent stimulation in sham (n=5) or injured animals (n=10) up to 14 days post injury. Single and paired pulse stimulations in the Schaffer collateral and a theta burst paradigm in the entorhinal cortex were utilized. There were significant changes in hippocampal baseline activity and in responses to stimulation at all levels of injury at 7 days post injury. At higher injury levels (260 rad/sec), recurrent paired pulse stimuli rendered hippocampal circuitry unresponsive and induced epileptiform activity such as paroxysmal depolarizing shifts and high-frequency oscillations. Theta burst stimulation induced epileptiform activity at higher injury levels but not in sham or lower injury levels. These alterations suggest an increased excitability, or a shift in the excitation-inhibition balance of the local hippocampal circuitry. Our data suggest that single and repetitive diffuse brain injury induces hippocampal axonal dysfunction and hippocampal excitability changes such as baseline oscillatory activity and neuronal firing rate. We are currently developing a chronic implantation method in order to examine changes in hippocampus and to correlate activity to behavior post injury.
Acoustic startle response in women with premenstrual mood disorders compared to controls
Liisa Hantsoo, PhD1,2,3, Carla Golden, BS1,2,3, Dina Appleby, MS1,2,3, Christian Grillon, PhD4, and C. Neill Epperson MD1,2,3
1Department of Psychiatry, 2Penn Center for Women’s Behavioral Wellness, and 3Penn Center for the Study of Sex and Gender in Behavioral Health, Perelman School of Medicine, University of Pennsylvania; 4National Institutes of Health, Section on Neurobiology of Fear & Anxiety, Bethesda, MD
Nearly 1 in 20 women worldwide experience premenstrual dysphoric disorder (PMDD). The progesterone metabolite allopregnanolone (ALLO) is a potential contributor to PMDD pathology. ALLO, a GABAA receptor agonist, is elevated in the luteal (L; premenstrual) phase of the menstrual cycle and typically dampens acoustic startle (ASR), an aspect of stress response. However, in PMDD, ASR is heightened in the L phase. This reversed pattern suggests suboptimal ALLO function. Preclinically, ALLO impacts BNST-mediated anxiety, versus amygdala-mediated fear. This study examines anxiety potentiated startle (APS) and fear potentiated startle (FPS) during the L and follicular (F) phases. The former manipulation would reflect BNST activity, which we hypothesize is dysregulated in PMDD.
Female participants underwent a threat of shock (NPU) task with 3 conditions: no shock (N), predictable shock (P), and unpredictable shock (U); a mild electric shock (100 ms, 1-5 mA) was administered by a current stimulator applied to the wrist. Startle stimuli were 50 ms white noise bursts at 103 dB. ASR was measured via eyeblink reflex, recording activity from the orbicularis oculi muscle. The NPU task was administered during the F and L phases in a within-subject design.
Controls (n=5) and PMDDs (n=4) both had elevated FPS in the L phase compared to the F phase (F Means: Ctl 5.9, PMDD 5.1; L Means: Ctl 8.6, PMDD 9.8). Controls had similar APS in the F and L phases, while PMDDs showed an increase in APS from the F to the L phase (F: 5.0, L: 17.3).
Preliminary findings suggest that BNST-mediated APS is elevated during the luteal phase in women with PMDD, but not controls. This is consistent with the hypothesis that BNST activity is dysregulated in women with PMDD, likely due to suboptimal ALLO function.
Discovery of potentially adaptive translocation of tau from axons to soma following traumatic axonal injury
Jean-Pierre Dolle, Andrew Jaye, Victoria E. Johnson, Douglas H. Smith
Center for Brain Injury and Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania
Both single and repetitive traumatic brain injury (TBI) have shown abnormal aggregation of hyper-phosphorylated tau comprising neurofibrillary tangles (NFTs), one of the hallmark pathologies of chronic traumatic encephalopathy. However, it has remained unknown how the axonal protein, tau, accumulates in the cell soma following TBI. Using a well-characterized in vitro model, we examined the temporal effects of traumatic axonal injury (TAI) on tau phosphorylation and potential redistribution within neuron compartments.The TAI model dynamically stretches micropatterned axon tracts that span two populations of primary cortical neurons. Within 1 hour following injury, the length of the diffusion barrier, the axon initial segment (AIS), became shorter along with decreased total and phospho-tau immunoreactivity levels in the axons. Conversely, corresponding increases in total phospho-tau immunoreactivity levels were observed in the cell-soma that continued to increase over 48hrs post-injury. This included further translocation of phospho-tau into dendrites. Inhibition of the retrograde motor protein dynein using Ciliobrevin-A attenuated the increases in somal tau / phospho-tau levels after trauma. Notably, these observations occur in the acute and sub-acute setting and, therefore, cannot be directly linked with the chronic formation of NFTs after TBI. onetheless, the data hint at potential mechanisms underlying the redistribution of tau due to TAI. In particular, with the risk that hyper-phosporylated tau might form a blockade to axonal transport after TAI, a process may be initiated to reduce the AIS diffusion barrier, thereby permitting retrograde transport of tau from the axon into the soma.
Novel small molecule triazolopyrimidine and phenylpyrimidine derivatives exhibit microtubule-stabilizing activity
Jane Kovalevich1, Kevin Lou2, Yuemang Yao1, Adam Hoye2, Michael James1, Anne-Sophie Cornec2, Edward Hyde1, Bryant Gay2, Alexander Crowe1, Virginia M.-Y. Lee1, John Q. Trojanowski1, Amos B. Smith III2, Carlo Ballatore2 and Kurt R. Brunden1
1Center for Neurodegenerative Disease Research, Institute on Aging, Perelman School of Medicine, University of Pennsylvania;
2Department of Chemistry, School of Arts and Sciences, University of Pennsylvania
Alzheimer’s disease (AD) and related tauopathies are characterized by hyperphosphorylation and aggregation of the microtubule (MT)-associated protein, tau. Hyperphosphorylation of tau induces its dissociation from MTs, where it plays an integral role in MT stability and facilitation of axonal transport in neurons. Loss of tau function leads to MT hyperdynamicity and disrupted transport of critical cellular proteins, leading to neuronal dysfunction. Compensation for tau deficiency via introduction of MT-stabilizing compounds may attenuate neuronal deficits that accompany AD. We previously demonstrated that the MT-stabilizing compound EpoD increases MT mass and stability, rescues fast axonal transport, and reverses cognitive deficits in tau transgenic mice. Yet, EpoD has certain limitations, including intravenous administration and inhibition of the P-glycoprotein (Pgp) transporter, which may present undesirable clinical manifestations. Moreover, EpoD is the only brain-penetrant MT-stabilizing drug that has been assessed in transgenic mouse models of AD and subsequently progressed to testing in AD patients, highlighting the need to identify additional small molecule drug candidates with MT-stabilizing activity. Recently, we have identified novel triazolopyrimidine and phenylpyrimidine derivatives that display MT-stabilizing activity in vitro and in the brains of wild-type mice. Furthermore, these compounds are orally bioavailable, show prolonged brain retention, and do not interfere with Pgp function. Studies to further characterize the molecular mechanisms underlying the effects of these compounds on MT stability and axonal transport as well as the potential for therapeutic use through compensation for tau deficits in AD are currently underway and discussed below.
Anaplastic lymphoma kinase negatively regulates sleep in the mushroom body of Drosophila
Lei Bai1 and Amita Sehgal1,2
1Howard Hughes Medical Institute, University of Pennsylvania; 2Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania
Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. We show Alk mutants have increased sleep time and consolidation indicative of a heightened sleep drive. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. The long sleep phenotype of Alk is suppressed by mutations in Nf1. Thus Alk and Nf1 interact in both learning and sleep regulation highlighting a common pathway in these two processes. These results may also have implications for treating sleep disturbances common in diseases such as cancer.
Enhanced salience network connectivity correlates with increased fat intake after sleep deprivation
Andrea M Spaeth1, Zhuo Fang2, Senhua Zhu2, Ning Ma2, Siyuan Hu2, John A Detre2, David F Dinges3, Hengyi Rao2,3
1Department of Sleep Medicine, Perelman School of Medicine, University of Pennsylvania; 2Center for Functional Neuroimaging, Department of Neurology, Perelman School of Medicine, University of Pennsylvania; 3Division of Sleep and Chronobiology, Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania
Short sleep duration is a significant risk factor for overeating, increased consumption of fat and weight gain. We examined whether or not changes in resting-state salience network functional connectivity related to changes in caloric and macronutrient intake after total sleep deprivation. Compared to the day following baseline sleep, healthy adults exhibited increased fat intake as well as increased functional connectivity between the anterior cingulate cortex (ACC) and bilateral putamen as well as between the ACC and left insula. Functional connectivity between the ACC and these regions positively correlated with subsequent fat intake during the day following total sleep deprivation. When adults are forced to maintain wakefulness for an extended period of time due to work or lifestyle, changes in brain network activation may lead to high-fat, unhealthy food choices that could contribute to weight gain over time.
Regulation of amyloid precursor protein expression by inflammatory eicosanoid receptors
Li Liu, Katie J. Herbst-Robinson, and Kurt R. Brunden
Center for Neurodegenerative Disease Research, Perelman School of Medicine, University of Pennsylvania
Senile plaques, one of the pathological hallmarks of Alzheimer’s disease (AD) brain, are composed of amyloid-beta (Aβ) peptides derived from the proteolytic cleavage of amyloid precursor protein (APP). It is thought that these plaques trigger neuroinflammation and oxidative stress, as evidenced by the presence of various glial-derived inflammatory molecules and lipid oxidation products within the AD brain. Among the reported oxidation products elevated in AD brain is isoprostane F2aIII, an oxidized form of arachidonic acid that has been shown to increase APP and Aβ production through the activation of the thromboxane-prostanoid (TP) receptor on neurons. Here, we have examined the mechanism by which TP receptor activation results in enhanced APP expression and we demonstrate that Gαq and protein kinase C isoforms are essential signaling components for the up-regulation of APP protein expression and Aβ formation triggered by the activation of the TP receptor. Furthermore, we have discovered that additional Gαq-linked receptors that can be activated by glial-derived inflammatory eicosanoids also regulate APP expression. Notably, the cysteinyl leukotriene 1 (CysLT1) receptor and the prostaglandin E2-binding receptors, EP1 and EP3, regulate APP expression and Aβ production when expressed in HEK293 cells. In primary rat neurons, activation of the TP, CysLT1 and EP3 receptors, but not the EP1 receptor, caused increased APP expression. Collectively, these studies suggest that glial-derived inflammatory eicosanoids and oxidative molecules that are likely elevated in the AD brain trigger the activation of multiple
Gαq-linked receptors to increase APP expression and Aβ production. These studies provide new insights into therapeutic strategies for the treatment of AD.
Lysophosphatidic acid signaling is required for Nox2 activation in lung endothelial cells and alveolar macrophages
Jose Pablo Vazquez-Medina, Chandra Dodia, Shampa Chatterjee, and Aron B. Fisher
Institute for Environmental Medicine, Perelman School of Medicine, University of Pennsylvania
The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) is required for NADPH oxidase (Nox2) activation in pulmonary microvascular endothelial cells (PMVEC) and alveolar macrophages (AM). Lysophosphatidylcholine (LPC) is a major product of Prdx6PLA2 that it is readily metabolized to lysophosphatidic acid (LPA) by the lysophospholipase D (lysoPLD) autotaxin. LPA is a bioactive lipid that signals through G protein-coupled receptors. LPA receptor activation stimulates signaling pathways regulated by small GTPases, such as the Nox2 cytosolic activator, Rac1. We propose that Prdx6PLA2-derived LPA signaling activates Nox2 in PMVEC and AM. Superoxide and hydrogen peroxide generation increased in response to angiotensin II (Ang II) or phorbol ester (PMA) in WT PMVEC and AM but not in Nox2 or Prdx6 null cells. Treatment with the LPA receptor antagonist Ki16425 blocked oxidant generation in response to agonist stimulation in WT PMVEC and AM, and Rac1 translocation to the plasma membrane in PMVEC. Exogenous LPA treatment “recovered” oxidant generation in Prdx6 null PMVEC and AM. Inhibition of lysoPLD with HA130 decreased oxidant generation during PMA and LPC stimulation in WT PMVEC while simultaneous treatment with LPA blocked the effects of HA130. Moreover, Ang II increased intravascular oxidant generation in isolated perfused lungs from knockin mice expressing Prdx6PLA2 (Prdx6C47S) but not in lungs from mice lacking such activity (Prdx6D140A; Prdx6 null). Treatment with Ki16425 decreased oxidant generation in Prdx6C47S lungs to the levels observed in Prdx6D140A or Prdx6 null lungs. These results suggest that Prdx6PLA2-derived LPA signaling is required for Nox2 activation and that Prdx6PLA2 likely generates LPC that needs enzymatic conversion to LPA for Nox2 activation.
Quantification of histone post-translational modifications by mass spectrometry
Zuo-Fei Yuan, Shu Lin, and Benjamin A. Garcia
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania
The nucleosome, the basic unit of chromatin, consists of 146bp of DNA wrapped around histone proteins (H3, H4, etc.). Histones play important roles in chromatin, in the forms of various post-translational modifications (PTMs), such as methylation, acetylation (ac), phosphorylation, etc. Histone PTMs have been functionally related to many distinct nuclear events. Moreover, some PTMs cross-talk and function synergistically to regulate gene expression. Therefore, the quantification of histone PTMs has become an important study. Traditionally, antibody-based methods (e.g., western blot) are used to analyze histone PTMs. These methods have multiple disadvantages. Fortunately, all these disadvantages can be overcome by mass spectrometry (MS). MS is a sensitive and efficient way to detect both previously identified and novel PTMs. Thus, MS is the key technology for histone PTMs. To quantify histone PTMs by MS, we need to know several parameters, such as when peptides elute in MS (i.e. retention time or RT), and the area of the chromatographic peaks (i.e. area under curve or AUC). In some cases there are two or more peptides in one chromatographic peak (e.g., H3K9ac and H3K14ac), making it difficult to separate the mixtures solely by chromatography. However, the key fragment ions between the two modification sites can be used to determine the species in the mixtures. Based on this idea, we developed a program, pProfile, to quantify histone PTMs. We tested pProfile on many histone mass spectral files. It can extract RT, AUC, and layouts within 5 minutes for each file. The result was verified by using a library of synthetic histone peptides. In sum, we have created a new software program pProfile that is accurate and fast for quantification of histone PTMs.
CD47-SIRPa interactions mediate MMP expression
Joshua B. Slee1,2, Kathryn A. Clarke1,2, Robert J. Levy1,2, and Stanley J. Stachelek1,2
1The Children’s Hospital of Philadelphia Department of Pediatrics; 2Perelman School of Medicine, University of Pennsylvania
Implanted medical devices and materials trigger an inflammatory reaction resulting in adsorption of blood proteins and platelets, monocyte/macrophage adhesion, and release of pro-inflammatory cytokines, all of which contribute to clinical complications. Our lab has focused on appending CD47 to implanted polymers as a way to prevent inflammation and promote biocompatibility. CD47 is a ubiquitously expressed transmembrane protein with a role in immune evasion and shows promise at conferring biocompatibility to polymeric surfaces. SIRPa, the cognate receptor for CD47, is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing transmembrane protein expressed on cells of myeloid origin. Recently, our laboratory showed that the anti-inflammatory capacity of CD47-SIRPa interactions extend beyond its canonical role as an inhibitor of phagocytosis. Specifically, a microarray analysis of whole blood exposed to immobilized CD47 surfaces showed a 100-fold increase in select matrix metalloproteinase (MMP) transcription. MMPs are involved in degrading the extracellular matrix, a process involved in the later stages of material-induced inflammation. In these studies, we test the hypothesis that CD47-SIRPa interactions increase MMP protein expression. Whole blood exposure to immobilized CD47 surfaces increased MMP7 expression in plasma, which correlated with increased enzymatic activity assessed by zymography. The role of the CD47/SIRPα signaling pathway was validated using THP-1 cells transduced with SIRPα shRNA, which fail to exhibit increased MMP7 expression. Lastly, rat sub-dermal implants showed increased MMP7 staining in tissue surrounding the implant after 7 days. Collectively, these data support the role of MMP7 activation downstream of CD47-SIRPα interactions.