Talk abstracts
Wednesday 09:00-09:15am: Persistent impact of chronic endocrine disruption upon estrogen signaling
Andrea R. Patterson (Department of Molecular Genetics, The James Comprehensive Cancer Center, The Ohio State University), Xiaokui Mo (Center for Bioinformatics, The Ohio State University Wexner Medical Center), Ali Shapiro (Department of Molecular Genetics, The James Comprehensive Cancer Center, The Ohio State University), Karen E. Wernke (Department of Molecular Genetics, The Ohio State University), Trevor K. Archer (4National Institute of Environmental Health Sciences, Laboratory of Molecular Carcinogenesis, Research Triangle Park, NC), Craig J. Burd (Department of Molecular Genetics, The James Comprehensive Cancer Center, The Ohio State University)
Abstract:
The pervasive nature of estrogenic industrial and dietary compounds is a growing health concern linked to cancer, obesity, and neurological disorders. Prior analyses of endocrine disruptor action have focused primarily on the short-term consequences of exposure. However, these studies are unlikely to reflect the consequences of constant exposures common to industrialized countries. Here, we examined the global effects of long-term endocrine disruption on gene transcription and estrogen signaling. Estrogen-dependent breast cancer cell lines were chronically treated with physiologically relevant levels of bisphenol A or genistein for more than 70 passages. Microarray analysis demonstrated global reprogramming of the transcriptome when compared to a similarly cultured control cell line. Estrogen responsive targets showed diminished expression in both the presence and absence of estrogen. ER recruitment, H3K4 monomethylation, and DNase accessibility were reduced at nearby response elements. Based upon these observations, we investigated the potential of long-term EDC exposure to initiate persistent transcriptional reprogramming. Culture of chronically exposed cell lines in the absence of the endocrine disruptors did not reverse many of the signaling defects that accumulated during treatment. Taken together, these data demonstrate that chronic exposure to endocrine disrupting compounds can permanently alter physiological hormone signaling.
Keywords: Estrogen receptor, Epigenetics, Endocrine disruptor
Wednesday 09:15-09:30am: Characterization and Selection of Biophysically Optimized Antibody Fragments for Enhanced in vivo Tumor Imaging
N. Emerson Long (Ohio State Biochemistry Program), Brandon J. Sullivan (Ohio State Biochemistry Program), Thomas J. Magliery (Ohio State Department of Chemistry and Biochemistry)
Abstract:
Antibody fragments have great potential for clinical application as cancer theranostics. Their small size compared to full-length IgG’s allows for faster blood clearance, potentially decreased immunoreactivity, better tumor penetrance, and easier engineering and production. The smallest possible fragment of an IgG that still binds to its antigen, called the single-chain variable fragment (scFv), can be created by fusing the variable light and variable heavy domains together with a peptide linker. Along with switching domain orientations, altering the length and amino acid sequence of the linker can significantly change the biophysical characteristics such as binding, stability, and quaternary structure. Comprehensive studies of these attributes have not been reported in the literature, making design and optimization of antibody fragments challenging. Here we constructed linker and orientation libraries of 3E8, an antibody specific to TAG 72, a mucinous glycoprotein overexpressed in 80% of adenocarcinomas.
Our studies have confirmed dramatic differences based on linker and orientation choices regarding biophysical properties and in vivo imaging. Specifically we have cloned, expressed, and characterized scFVs, diabodies, and higher order multimer constructs with varying linker compositions and sizes and domain orientations. These constructs were characterized by surface plasmon resonance (SPR) to test for antigen binding, by differential scanning fluorimetry (DSF) to test for thermal stability, and by gel chromatography to test for quaternary structure and homogeneity. We then optimized expression and purification of two biophysically favorable constructs, 3E8.scFV and 3E8.G4S. Both constructs were subjected to mouse biodistribution and pharmacokinetic studies. From this analysis, we selected 3E8.G4S as a lead candidate for cancer imaging and detection. This hypothesis was confirmed with successful PET and SPECT imaging of cancer xenograft mice at 24 hours.
Keywords: Antibody fragments, Cancer diagnostics, Protein engineering
Wednesday 09:30-09:45am: DDR1 deficiency in the murine aorta alters collagen fiber structure and platelet adhesion
Jeffrey Tonniges (Biophysics Graduate Program), Benjamin Albert (Biomedical Engineering), Edward Calomeni (Pathology), Gunjan Agarwal (Biomedical Engineering)
Abstract not available online - please check the printed booklet.
Wednesday 09:45-10:00am: Repeated Social Defeat Stress Induces Neuroinflammation and Impairs Hippocampal Neurogenesis That Differentially Regulate Mood and Cognition
Anzela Niraula (Neuroscience Graduate Program), Daniel B. McKim (Neuroscience Graduate Program), Andrew J. Tarr (Oral Biology), Eric S. Wohleb (Neuroscience Graduate Program), John Sheridan (Oral Biology), Jonathan Godbout (Neuroscience Graduate Program)
Abstract:
Repeated social defeat (RSD) is a murine stressor that models several key physiological, immunological, and behavioral alterations observed in humans exposed to psychosocial stress. RSD induces prolonged anxiety-like behavior associated with myeloid cell trafficking into the brain, including the hippocampus - a key area involved in neuroplasticity, behavior, and cognition. Therefore, the goal of this study was to investigate if the stress-induced monocyte trafficking affected hippocampal neurogenesis and cognitive function. Here, we show that RSD increased inflammatory mediators (IL1b, TNFa and IL-6) in the hippocampus, and enhanced microglia activation and monocyte trafficking (CD45hi) specifically in the caudal hippocampus. RSD also impaired spatial memory recall in the Barnes maze independent of anxiety-like behavior. RSD did not affect the number of proliferating neural progenitor cells and developing neurons when examined 14 hours post-RSD. However, there was a significant reduction in the number of young neurons and mature neurons when examined 10 days and 28 days post-RSD respectively. Consistent with region-specific neuroinflammation, reduction in the number of mature neurons was greater in the caudal hippocampus of the RSD mice compared to controls. The RSD-induced spatial deficits, which are rostral hippocampus-mediated, were resolved by 28 days. Social avoidance which is caudal hippocampus-mediated still persisted 28 days after stress. Thus, stress-induced neuroinflammation is associated with reduced neuroplasticity, and the stress-induced affective and cognitive deficits are differentially associated with hippocampal neurogenesis.
Keywords: neurogenesis, macrophages, stress
Wednesday 10:20-10:40am: Life Under Tension: Exceptional Cadherins for Hearing and Balance
Marcos Sotomayor (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract not available online - please check the printed booklet.
Wednesday 10:40-11:00am: Gestational stress effects on the postpartem reward system: implications for mood, motivation and mothering
Benedetta Leuner (Department of Psychology)
Abstract not available online - please check the printed booklet.
Wednesday 11:00-11:20am: Dissecting functional cooperation during protein-aided RNA catalysis in RNase P
Venkat Gopalan (Department of Chemistry and Biochemistry)
Abstract not available online - please check the printed booklet.
Wednesday 11:20-11:40am: Using genomics to direct patient care: Opportunities and Challenges for Precision Cancer Medicine
Sameek Roychowdhury (Department of Internal Medicine)
Abstract not available online - please check the printed booklet.
Wednesday 03:00-03:15pm: Determinants of specific recognition of mischarged Ala-tRNAPro by a bacterial trans-editing domain
Eric M. Danhart (Ohio State Biochemistry Program), Brianne Sanford, Oscar Vargas-Rodriguez, Daniel McGowan, Marina Bakhtina (Department of Chemistry and Biochemistry, The Ohio State University), Lexie Kuzmishin (Ohio State Biochemistry Program), Marija Kosutic, Ronald L. Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Aminoacyl-tRNA synthetases (ARS) catalyze the attachment of specific amino acids to cognate tRNAs. Mistakes in this process lead to errors in protein synthesis that can be deleterious to cells. Prolyl-tRNA synthetase (ProRS) mischarges tRNAPro with Ala; this aberrant product is hydrolyzed by a cis-editing domain (INS) in most bacteria. However, some bacteria lacking the INS domain encode a homologous free-standing trans-editing domain known as ProXp-ala that functions to clear Ala-tRNAPro. Previous studies showed that specific nucleotides in the acceptor stem of tRNAPro (G72 and A73) are critical for ProXp-ala activity, and that a small RNA stem-loop containing these elements, microhelixPro, is a good substrate for Ala deacylation. To define the elements in ProXp-ala that confer acceptor stem specificity, NMR mapping studies were carried out with an uncharged microhelixPro and with a non-hydrolyzable, amide-linked Ala-microhelixPro mimic. We observe similar but significantly stronger chemical shift perturbations in the presence of the charged microhelix, which also displays 5-fold higher affinity for binding to ProXp-ala, as measured by analytical ultracentrifugation (AUC). The largest chemical shift perturbations were mapped to three main regions: helix α2 at the top of the active site pocket (aa 27-30), β-strands β2 (aa 43-49) and β6 (aa 128-134) within the active site, and β-strand β4 (aa 80-84) that we propose is the G72/A73 interacting domain. Site-directed mutagenesis and AUC studies are consistent with the critical nature of residues 80-83 for substrate binding. Mutation of conserved active site residues K45 and N46 also caused severe losses in activity. Additionally, 15N NMR relaxation experiments revealed that the helix α2 exhibits significant dynamics at the ps-ns timescale. These results allow us to propose a mechanism for recognition of Ala-microhelixPro that involves induced-fit binding, specific protein-RNA contacts, and key contributions from the Ala moiety.
Keywords: NMR, tRNA, dynamics
Wednesday 03:00-03:15pm: Rng16 coordinates contractile-ring constriction and septum formation in cytokinesis
Reshma Davidson (MCDB), Josef Pontasch (Molecular Genetics), Dr. Jian-Qiu Wu (Molecular Genetics)
Abstract not available online - please check the printed booklet.
Wednesday 03:15-03:30pm: Viewing human DNA polymerase β faithfully and unfaithfully bypass an oxidative lesion by time-dependent crystallography
Andrew J. Reed (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program), Rajan Vyas (Department of Chemistry and Biochemistry), E. John Tokarsky (Department of Chemistry and Biochemistry, Ohio State Biophysics Graduate Program), Zucai Suo (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, and Ohio State Biophysics Graduate Program)
Abstract:
One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson-Crick base pair with correct deoxycytidine 5′-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5′-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase β (hPolβ). In the 12 solved structures, both Watson-Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg2+ appeared during the process of phosphodiester bond formation and was located between the reacting α- and β-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolβ reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3′-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.
Keywords: X-ray Crystallography, DNA polymerases, oxidative damage
Wednesday 03:15-03:30pm: Identification of an inner nuclear membrane protein that impacts the spatio-temporal content of both the nuclear envelope and nuclear interior
Mahesh Chemudupati (Ohio State Biochemistry Program), Stephen A. Osmani (Department of Molecular Genetics)
Abstract not available online - please check the printed booklet.
Wednesday 03:30-03:45pm: Characterization of nucleosome structure using DNA origami
J.V. Le (Interdisciplinary Biophysics Graduate Program), Yi Luo (Interdisciplinary Biophysics Graduate Program), Christopher Lucas (Department of Mechanical of Engineering and Aerospace), Michael Poirier (Department of Physics), Carlos Castro (Department of Mechanical of Engineering and Aerospace)
Abstract:
While biophysical tools such as single molecule fluorescence and force spectroscopy have been used to study the structural dynamics of individual nucleosomes or large chromatin assemblies, it is challenging to probe conformational dynamics of gene regulation in the critical 10-100nm range. This work aims to develop DNA origami tools to probe the mesoscale structure and dynamics of nucleosomes and chromatin. DNA origami itself is a recently established nanotechnology that enables the self-assembly of precisely designed nanostructures. Here, we implement a DNA origami hinge structure with the length scale of ~100nm as a nano-caliper to study the structure and dynamics of a single nucleosome.
The device was calibrated by integrating DNA linkers to constrain the hinge arms, showing that it is possible to use the hinge angular distribution as a readout for the size of the sample attached between the arms. We further integrated single nucleosomes with varying but symmetric lengths of DNA linkers to verify our ability to detect structural changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to protein binding. Initial experiments using the hinge-nucleosome construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation when bound near the promoter region of the gene , shows that the angular distribution broadens as a result of increasing TF titrations. Our measurements revealed a dissociation constant in the range of 1-10 nM, which agrees with bulk measurements. Furthermore, preliminary results with H1, which is known to cause compaction of nucleosomes and chromatin, demonstrated a physical shift in nucleosome position rather than the angular distribution.
These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm.
References:
Marras, A. E., L. Zhou, H. J. Su and C. E. Castro. "Programmable Motion of DNA Origami Mechanisms." Proc Natl Acad Sci U S A, (2015).
Poirier, M. G., E. Oh, H. S. Tims and J. Widom. "Dynamics and Function of Compact Nucleosome Arrays." Nat Struct Mol Biol 16, no. 9 (2009): 938-44.
Sadowski, I., J. Ma, S. Triezenberg and M. Ptashne. "Gal4-Vp16 Is an Unusually Potent Transcriptional Activator." Nature 335, no. 6190 (1988): 563-4.
Keywords: DNA origami, nucleosomes, structural dynamics
Wednesday 03:30-03:45pm: Analysis of a novel microtubule-associated protein that displays dynamic cell cycle regulated locations.
Nandini Shukla (The Ohio State Biochemistry Program ), Aysha H. Osmani (Department of Molecular Genetics ), Angela B. Davis (Department of Molecular Genetics ), Stephen A. Osmani (Department of Molecular Genetics )
Abstract not available online - please check the printed booklet.
Wednesday 03:45-04:00pm: An exogenous transposable element is recognized by homology and expression dependent methylation
Dalen Fultz (Molecular Genetics, The Ohio State University), R. Keith Slotkin (Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
Wednesday 03:45-04:00pm: Direct assessment of skeletal muscle contractile strength in live wildtype and rbfox morphant zebrafish larvae
Brit L. Martin (Molecular, Cellular, and Developmental Biology, The Ohio State University), Tom L. Gallagher (Department of Molecular Genetics, The Ohio State University), Neha Rastogi (Department of Physiology and Cell Biology, The Ohio State University), Jonathan P. Davis (Department of Physiology and Cell Biology, The Ohio State University), Christine E. Beattie (Department of Neuroscience, The Ohio State University), Sharon L. Amacher (Department of Molecular Genetics, The Ohio State University)
Abstract not available online - please check the printed booklet.
Wednesday 04:15-04:30pm: Using sur-2 as a gateway to understanding EGF pathway interaction differences in nematodes
Karley K. Mahalak (MCDB), Edward Zitnik (Molecular Genetics), Helen Chamberlin (Molecular Genetics, OSU)
Abstract:
The EGF/Ras signaling pathway is essential for the proper development of most multicellular animals. We are studying the function of the EGF pathway in the context of vulval development in the Caenorhabditis genus of nematodes to better understand the evolutionary flexibility and constraints imposed by signaling pathways on the regulatory networks in which they participate. Normal vulval development in the related species C. elegans and C. briggsae is essentially identical. However, the EGF pathway is essential for vulval cell fate in C. elegans, but not C. briggsae. Specifically, when the EGF pathway is disrupted by a MEK inhibitor (U0126), all of the vulval precursor cells (VPCs) of C. elegans fail to become vulval cells, whereas in C. briggsae, some VPCs still differentiate into vulval cells. We have likewise identified differences between the species in the sur-2 gene. sur-2 encodes the Med23 component of the transcriptional Mediator complex, and acts downstream of EGF signaling in C. elegans, mammals, and other animals. In C. elegans vulval development, sur-2 serves as the link between the EGF/Ras and Notch pathways. Cel-sur-2 mutants exhibit reduced vulval development, and frequently lack the cells that comprise the sides of the vulva (the secondary cells) due to a failure to express LAG-2, a ligand for Notch. In contrast, we have identified a Cbr-sur-2 mutant strain, and found that they have normal levels of vulval development, and an overproduction (rather than loss) of secondary fated cells. To reconcile these phenotypic differences, we have used molecular markers to interpret the cell fate choices in C. elegans and C. briggsae sur-2 mutants. We find that mutants for both species are similar in that they are deficient in the development of primary cells (cells that normally form the apex of the vulva). The species differ on whether this defect interferes with the production of the lateral secondary cells. We interpret that the differences in sur-2 mutants uncovers a key distinction between the species with respect to the connection between EGF/Ras and Notch signaling, and in the importance of EGF signaling in promoting the secondary cell fate.
Keywords: EGF pathway, Mediator Complex
Wednesday 04:15-04:30pm: Cyclic rhamnosylated EF-P establishes antibiotic resistance in Pseudomonas PAK
Andrei Rajkovic (MCDB), Sarah Tyler (Microbiology), Annie Kalionski (Molecular Genetics), Owen E Branson (OSBP)
Abstract not available online - please check the printed booklet.
Wednesday 04:30-04:45pm: Intraspinal TLR4 activation promotes iron storage but amplifies acute cytotoxicity
Evan Z. Goldstein (NGP), Jamie Church (NGP), Phillip G. Popovich (Neuroscience), Dana M. McTigue (Neuroscience)
Abstract not available online - please check the printed booklet.
Wednesday 04:30-04:45pm: Mineralocorticoid Receptors in Skeletal Muscle are a Potential Therapeutic Target for Duchenne Muscular Dystrophy
Jessica Chadwick (Dept. of Molecular & Cellular Biochemistry, & Dept. of Physiology & Cell Biology, College of Medicine, The Ohio State University), James Hauck (Dept. of Molecular & Cellular Biochemistry, & Dept. of Physiology & Cell Biology, College of Medicine, The Ohio State University), Jeovanna Lowe (Dept. of Molecular & Cellular Biochemistry, & Dept. of Physiology & Cell Biology, College of Medicine, The Ohio State University), Denis Guttridge (Department of Mol. Virology, Immunology, and Medical Genetics, College of Medicine, The Ohio State University), Jill Rafael-Fortney (Dept. of Molecular & Cellular Biochemistry, & Dept. of Physiology & Cell Biology, College of Medicine, The Ohio State University)
Abstract:
Duchenne muscular dystrophy (DMD) is a progressive and fatal degenerative disease, affecting around 1 in 3,600 boys. Glucocorticoids (glucocorticoid receptor agonists), the current standard-of-care treatment, defer severity of some symptoms and prolong mobility an average of 2 years. However, these drugs do not exhibit long term efficacy and have numerous deleterious side effects. Our lab has identified a potential new treatment for DMD using the mineralocorticoid receptor (MR) antagonist spironolactone and angiotensin converting enzyme inhibitor lisinopril. Preclinical studies using dystrophic Het (utrn+/-; mdx) mice showed a dramatic improvement in both respiratory and limb muscle force and a reduction of ongoing muscle damage, in addition to preventing cardiomyopathy. Both spironolactone and lisinopril target the mineralocorticoid receptor (directly and indirectly, respectively), however very little is known about the role of MR in the context of skeletal muscle. Despite long-standing clinical use of MR antagonists in cardiology, only serpina-3 has recently been validated as a bona-fide downstream target in cardiomyocytes. We show MR is present in a wide variety of normal and dystrophic mouse skeletal muscles. We confirm MR is expressed in a cell autonomous manner in skeletal muscle using the C2C12 mouse cell line and human primary undifferentiated and differentiated myogenic cultures. Several genes with known roles in striated muscle are deferentially expressed between quadriceps muscles from untreated and spironolactone + lisinopril treated het mice. We demonstrate that many of the corresponding proteins vary in abundance or localization between normal and dystrophic muscles. A subset of the in-vivo gene expression changes were identified when comparing normal human primary myogenic cultures treated with an MR agonist or antagonist, including the one known MR target in heart (serpina-3). These genes represent potential direct MR downstream targets, which may help elucidate the mechanism behind the efficacy of these drugs and serve as biomarkers of treatment.
References:
Population frequencies of inherited neuromuscular diseases-a world survey.
A.E. Emery
Prednisone treatment in Duchenne muscular dystrophy. Long-term benefit.
S. DeSilva, D.B. Drachman, D. Mellits and R.W. Kuncl
Contrasting effects of steroids and angiotensin-converting enzyme inhibitors in a mouse model of dystrophin-deficient cardiomyopathy.
R. Bauer, V. Straub, A. Blain, K. Bushby and G.A. MacGowan
Old and new therapeutic developments in steroid treatment in Duchenne muscular dystrophy.
C. Angelini and E. Peterle
Early Treatment with Lisinopril and Spironolactone Preserves Cardiac and Skeletal Muscle in Duchenne Muscular Dystrophy Mice.
J.A Rafael-Fortney et al.
Molecular signature of mineralocorticoid receptor signaling in cardiomyocytes: from cultured cells to mouse heart.
C. Latouche et al.
Keywords: muscular dystrophy , mineralocorticoid receptor , skeletal muscle
Wednesday 04:45-05:00pm: Exon 2 skipping strategies to treat DMD 5’ mutations: Intramuscular delivery of rAAV9.snRNA mediated skipping and antisense morpholino oligomers.
Tabatha Simmons (The Center for Gene Therapy, Nationwide Childrens Hospital, Molecular Cellular, and Developmental Biology, The Ohio State University), Nicolas Wein (The Center for Gene Therapy, Nationwide Childrens Hospital), Adeline Vulin-Chaffiol (The Center for Gene Therapy, Nationwide Childrens Hospital), Kristin Heller (The Center for Gene Therapy, Nationwide Childrens Hospital), Louise Rodino-Klapac (The Center for Gene Therapy, Nationwide Childrens Hospital), Kevin Flanigan (The Center for Gene Therapy, Nationwide Childrens Hospital, Departments of Pediatrics and Neurology, The Ohio State University)
Abstract:
Exon skipping therapies for Duchenne muscular dystrophy (DMD) have to date been directed toward patients with out-of-frame exon deletions, resulting in larger but in-frame internal deletions and translation of an internally truncated but partially functional dystrophin protein. We are developing exon-skipping therapies for duplication mutations, which account for around 6% of all mutations by testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA) and antisense oligomer-induced skipping in the Dup2 mouse. Intramuscular (IM) injections of rAAV9.U7.ACCA at 6 doses between 1x1010 and 1x1012 total vector genomes were delivered to the tibialias anterior (TA) (N=6) and analyzed for exon skipping and dystrophin production. Additionally, peptide-morpholino conjugates (PPMO) containing an antisense to exon 2 was delivered by IM injection to the TA at doses of either 10 or 20 ug total PPMO (N=3 each) and analyzed for exon skipping and dystrophin production. Treatment with either modality results in significant expression of apparently full-length dystrophin. Physiology has been assessed in the AAV-treated mice, in which correction of absolute force deficits in comparison to the background Bl6 strain are seen, along with a partial yet significant correction of eccentric contraction injury in comparison to untreated Dup2 mice. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Complete skipping of exon 2 results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin allowing for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.
Keywords: DMD, exon-skipping, gene therapy
Wednesday 04:45-05:00pm: Sympathetic Release of Splenic Monocytes Promotes Recurring Anxiety Following Repeated Social Defeat
Daniel B. McKim (Neuroscience Graduate Program), Jenna M. Patterson (Neuroscience), Eric S. Wohleb (Yale University), Brant L. Jarrett Biosciences - COD (Brenda Reader Biosciences - COD), Jonathan P. Godbout (Neuroscience), John F. Sheridan (Biosciences - College of Dentistry)
Abstract:
Neuroinflammatory signaling may contribute to the pathophysiology of chronic anxiety disorders. Previous work showed that repeated social defeat (RSD) in mice promoted stress-sensitization that was characterized by the recurrence of anxiety following sub-threshold stress 24 days after RSD. Furthermore, splenectomy following RSD prevented the recurrence of anxiety in stress-sensitized (SS) mice. We hypothesize that the spleen of RSD-exposed mice became a reservoir of primed monocytes that were released following neuroendocrine activation by sub-threshold stress. Sub-threshold stress 24 days after RSD re-established anxiety-like behavior that was associated with egress of Ly6Chi monocytes from the spleen. Moreover, splenectomy prior to RSD blocked monocyte trafficking and prevented the recurrence of anxiety-like behavior in sensitized mice. Splenectomy, however, had no effect on monocyte accumulation or anxiety when determined 14 hours after RSD. In addition, splenocytes cultured 24 days after RSD exhibited a primed inflammatory phenotype. Next, treatment with a peripheral sympathetic inhibitor prior to sub-threshold stress blocked monocyte redistribution and prevented the re-establishment of anxiety in RSD-sensitized mice. Additionally, increased availability of releasable monocyte was associated with monocyte-progenitor proliferation within the spleen. Collectively, these data show that the spleen is capable of producing and storing primed monocytes that promote exaggerated behavioral responses to acute stress, even many days after a sensitizing event.
Keywords: Stress, Monocytes, Neuroimmunology
Wednesday 05:00-05:15pm: Age-related impairments in the dynamic regulation of active microglia by astrocytes
Diana M. Norden (Department of Neuroscience), Paige J. Trojanowski (Department of Neuroscience), Frederick R. Walker (University of Newcastle, Australia), Jonathan P. Godbout (Department of Neuroscience)
Abstract:
Older individuals are at greater risk for infection and concomitant with this they have a higher frequency for developing neurobehavioral complications that negatively affect health and lifespan. Heightened neuroinflammation may be the key mechanism underlying the development of these neuropsychiatric disorders. In aged rodents, the anti-inflammatory cytokine IL-10 is highly expressed in the brain following immune challenge, yet microglial activation is prolonged and behavioral impairments develop. Recently we showed that astrocytes of adult mice express the IL-10 receptor (IL-10R) and that IL-10 re-directs active astrocytes to produce TGFb, which in turn, attenuates the activation of microglia. Therefore, the purpose of this study was to investigate the degree to which these key cytokine interactions between glia are impaired in the aged brain. Morphological analysis of aged microglia indicated a primed baseline inflammatory state, with increased max cell length and cell perimeter. Similarly, morphological analysis of aged astrocytes showed evidence of significant cyotoskeletal re-organization and hypertrophy. Gene arrays confirmed that there was increased expression of the astrocyte inflammatory markers vimentin and GFAP in the hippocampus of aged mice. Moreover, astrocytes from aged mice had reduced surface IL-10R expression compared to adults. Following immune challenge in vivo, adult astrocytes up-regulated IL-10R and TGFb mRNA. Aged astrocytes, however, failed to increase expression of these mediators. This lack of regulation by TGFb was associated with exaggerated expression of pro-inflammatory mediators in aged microglia. Additionally, active microglia cultured ex vivo with adult astrocytes reduced inflammatory markers while those cultured with aged astrocytes did not. In summary, these novel data indicate that astrocytes have an important role in regulating microglia via TGFb signaling and that an impaired IL-10 response in aged astrocytes contributes to age-related deficits in the regulation of active microglia.
Keywords: Neuroinflammation, aging, microglia
Wednesday 05:00-05:15pm: Title not available online - please see the printed booklet.
Jason R. Pitarresi (OSBP and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA), Jnigahi Wu, Xin Liu, Maria C. Cuitio, Raleigh D. Kladney (Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA), Gustavo Leone, Michael C. Ostrowski (Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA)
Abstract not available online - please check the printed booklet.