Lecture Sessions



10:30 A.M.

Parathyroid hormone alleviates radiotherapy-induced bone loss by promoting DNA repair in a β-catenin-dependent pathway

Abhishek Chandra1, Tiao Lin1, Ji Zhu1, Beom K. Huh1, Allison R. Altman1, Sarah Hagan2, Keith Cengel2, Sherry X. Liu1, and Ling Qin1

1Department of Orthopaedic Surgery, and 2Department Radiation Oncology, Perelman School of Medicine, University of Pennsylvania

Loss in structural integrity in tumor-adjacent bones is a well-recognized late effect of radiotherapy. We propose that parathyroid hormone (PTH), an anabolic agent that potently stimulates bone formation, should preserve bone structure and strength against radiation damage. In this study, 3-month-old rats were focally irradiated at the proximal metaphyseal region of the right tibiae by a unique SARRP irradiator that replicates clinical focal radiotherapy. Rats were then administered vehicle or PTH (1-34, 60 μg/kg) daily for 4 weeks. MicroCT (μCT) scans revealed a significant 20% decrease in trabecular bone volume (BV) in irradiated tibiae compared to the contralateral tibiae in the vehicle group, which is mainly due to a 17% decrease in trabecular number (Tb.N). 3D registration of μCT images before and after radiation was performed to assess individual trabeculae. Finite element analysis indicates that this microstructural deterioration resulted in a 57% decrease in trabecular bone stiffness. On the contrary, PTH injections remarkably increased BV, Tb.N, and stiffness in both non-irradiated and irradiated trabecular bone and preserved all trabecular elements in the irradiated bone. Histological analysis indicates that PTH preserved the bone surface osteoblasts from radiation induced apoptosis. Using UMR-106 osteoblastic cells, we demonstrated that a single dose (8 Gy) of X-ray radiation resulted in DNA damage detected by the comet assay, specifically DNA double-stranded breaks (DSBs) detected by the gamma-H2AX foci assay, caspase3 nuclear localization, and induction of apoptosis. PTH abrogated these effects via a β-catenin-dependent pathway. Here we propose a novel role for PTH in DSB repair and survival of radiated osteoblasts, thus providing structural integrity to the bone.

10:45 A.M.

Distinct α-synuclein strains differentially promote tau inclusions in neurons

Jing L. Guo, Dustin J. Covell, Joshua P. Daniels, Michiyo Iba, Anna Stieber, Bin Zhang, Dawn M. Riddle, Linda K. Kwong, Yan Xu, John Q. Trojanowski, and Virginia M.Y. Lee

Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, and Institute on Aging, Perelman School of Medicine, University of Pennsylvania

Many neurodegenerative diseases are characterized by the accumulation of normally soluble proteins into insoluble fibrillar aggregates, including neurofibrillary tangles comprised of tau in Alzheimer’s disease and Lewy bodies composed of α-synuclein in Parkinson’s disease. Recent studies have shown that these protein aggregates may undergo cell-to-cell transmission through “seeded” fibrillization, whereby existing aggregates can move from one cell to the other and recruit their soluble counterparts into growing aggregates in the healthy cells. Furthermore, different pathological proteins frequently co-deposit in the same diseased brains, such as tau and α-synuclein. To test whether a direct “cross-seeding” can occur between two different proteins, we incubated primary neurons with preformed α-synuclein fibrils assembled from recombinant protein, which were previously shown to induce robust aggregation of endogenous α-synuclein. Remarkably, we discovered two distinct “strains” of synthetic α-synuclein fibrils that demonstrated striking differences in the efficiency of cross-seeding tau aggregation in neurons, and similar results were obtained in mice receiving intracerebral injection of these α-synuclein fibrils. Biophysical and biochemical analyses revealed conformational differences between the two synthetic strains, suggesting multiple conformers could exist for fibrillar α-synuclein. We speculate that different variants of pathological protein aggregates may also occur in neurodegenerative disease brains, which could account for not only the frequent while variable co-deposition of different protein aggregates, but also the tremendous heterogeneity among disorders with the same major protein lesions.

11:00 A.M.

Vimentin links cholangiocyte injury and myofibroblast activation

Lorena Loarca, Maryna Perepelyuk, Shannon Tsai, and Rebecca G. Wells

Division of Gastroenterology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania

Biliary fibrosis after cholangiocyte injury is a significant cause of morbidity, although the mechanism is not well understood. We have shown that expression of the intermediate filament vimentin by cholangiocytes is markedly increased in biliary fibrosis. Vimentin is a key indicator of mesenchymalization of epithelial cells and is associated with loss of cell-cell contacts and increased migration. We hypothesized that mesenchymalized, vimentin-expressing cholangiocytes play an important role in mediating biliary fibrosis. Vimentin expression was induced in mouse cholangiocytes in culture by TNF-α/TGF-β treatment and specificity demonstrated using siRNA. Vimentin induced partial mesenchymalization of mouse cholangiocytes in vitro, as assessed in 2-D by an elongated cell shape, increased expression of mesenchymal markers, decreased expression of epithelial markers, and increased motility. Vimentin-expressing cholangiocytes in spheroids in 3-D culture showed increased invasiveness, loss of lumens, and decreased spheroid integrity. Cells in 3-D culture exposed to increased uniaxial pressure (at levels consistent with those in obstructed bile ducts) demonstrated increased vimentin expression with a corresponding decrease in bile duct integrity. Conditioned media from mesenchymalized cholangiocytes induced myofibroblast activation. These findings suggest a model of biliary fibrosis whereby cholangiocyte injury, including from increased pressure, results in cholangiocyte mesenchymalization and this in turn induces myofibroblast activation and fibrogenesis.

11:15 A.M.

Development of transplantable tissue engineered nerve grafts consisting of stretch-grown axons from swine

Mindy I. Ezra, John A. Wolf, Kevin D. Browne, Harry C. Ledebur, D. Kacy Cullen, and Douglas H. Smith

Department of Neurosurgery, Center for Brain Injury and Repair, Perelman School of Medicine, University of Pennsylvania

Peripheral nerve injuries (PNIs) present a serious medical concern, constituting over 100,000 neurosurgical procedures annually. However, only 50% of patients achieve functional restoration following repair. Despite large efforts, PNI repair has not progressed past using nerve guidance tubes (NGTs) or autografts to treat injuries. Our team is developing novel tissue-engineered nerve grafts (TENGs) that have potential to outperform autografts and to repair currently untreatable PNIs. We hypothesize that TENGs will promote axonal regeneration, target reinnervation, and functional restoration in a swine model of PNI at lengths greater than NGTs or autografts can restore. TENGs are lab-grown nervous tissue, comprised of long axonal tracts spanning two populations of dorsal root ganglia neurons. These grafts are generated via continuous mechanical tension or “stretch-growth.” This process is replicated in vitro via the separation of integrated neurons using a proprietary process and elongator device. Dorsal root ganglia explants from embryonic pigs are plated on two membranes, one stationary and one movable “towing” membrane. Axons extend across the membranes and synapse with neurons on the opposing membrane. The towing membrane is then pulled away, creating long tracts of living axons. Three-dimensional TENGS are made by encapsulating these tracts in proteinaceous matrix and transplanting them within an NGT. The mechanisms of action whereby TENGs promote accelerated axon regeneration and maintain the pro-regenerative capacity of the distal nerve stump are key differentiators from current technologies. This approach not only increases the chances of target reinnervation and functional recovery, but also offers the ability to repair currently untreatable PNIs.



1:15 P.M.

T-Cells edited to express CCR5 or CXCR4 fused to the C34 peptide exhibit robust protection from diverse HIV-1 isolates

George J. Leslie1, Jianbin Wang3, Max W. Richardson2, Beth Haggarty1, Andrea Jordon1, Josephine Romano1, Kevin L. Hua3, James L. Riley2, Michael C. Holmes3, and James Hoxie1

1Department of Medicine and 2Department of Microbiology, Perelman School of Medicine, University of Pennsylvania;
Sangamo Bioscience Inc., Richmond, CA

Advances in cell-based approaches to restore immune function and viral control in HIV-1 infected patients include the use of genetically modified autologous CD4+ T-cells that can be expanded ex vivo and adoptively reinfused. However, there is still a need to develop strategies that mutually protect cells from diverse HIV-1 isolates, i.e. R5-, X4-, and R5X4-tropic viruses. In a novel approach to render CD4+ T-cells resistant to HIV-1, we evaluated the ability of a 34 amino acid peptide from the C-terminal heptad repeat-2 domain of gp41 (C34) to inhibit HIV-1 entry when fused to the amino terminus (NT) of either CCR5 (R5) or CXCR4 (X4). C34-R5 or C34-X4 fusion constructs were introduced by lentiviral vector into primary human T-cells, or T cell lines previously modified using zinc finger endonucleases (ZFN), which knocked out endogenous CXCR4 expression, or transiently expressed in reporter cells. C34-R5 or C34-X4 stably expressed in SupT1 cells mediated chemotaxis, demonstrating that physiologic functions were maintained. In contrast, HIV challenge experiments revealed that neither C34-coreceptor could mediate infection in stably or transiently expressing cells. This effect was highly specific, as C34 peptides containing point mutations were fully permissive for HIV entry. Moreover, the C34 peptide conjugated to CD4 failed to inhibit HIV-1 entry, indicating positioning was critical. Remarkably, C34-R5 and C34–X4 were shown to potently inhibit HIV-1 infection and/or entry in a trans-dominant manner irrespective of viral tropism. This broad and potent trans-dominant inhibition of HIV-1 by C34-modified chemokine coreceptors represents a new and powerful approach to engineering HIV-resistant autologous CD4+ T-cells.

1:30 P.M.

Mitochondrial mobility in astrocytes is decreased by neuronal activity and glutamate transport

Joshua G. Jackson1, John C. O’Donnell3, Hajime Takano2, Douglas A. Coulter2,4, and Michael B. Robinson1,3

1Department of Pediatrics and 2Division of Neurology, The Children’s Hospital of Philadelphia; 3Department of Pharmacology and 4Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania

Within neurons, mitochondria are non-uniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na+/K+-ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We tracked the mobility of mitochondria within individual astrocytic and neuronal processes in organotypic hippocampal cultures. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes 3.3-fold. Inhibition of ionotropic receptors had a similar effect. These results indicate that neuronal activity decreases the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. The percentage of mobile mitochondria in astrocytes was not affected by activation or inhibition of mGluR5 glutamate receptors. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate D-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulates the movement of astrocytic mitochondria and suggests a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake.

1:45 P.M.

Ethnic and genetic influences on human body odor production

Katharine Prokop-Prigge1, Erica Thaler2, and George Preti1,3

1Monell Chemical Senses Center; 2Department of Otorhinolaryngology - Head and Neck Surgery, and 3Department of Dermatology, Perelman School of Medicine, University of Pennsylvania

Human axillary (“body”) odor is a complex mixture of volatile organic compounds (VOCs). Although this odor is mainly perceived by today’s society as unpleasant, several studies indicate that axillary odorants contain chemical signals that affect the menstrual cycle or that may be involved in a major histocompatibility complex allele-dependent mate selection, and point to the importance of human body odors. Recent studies link a genetic variation in ABCC11 to different types of axillary odorant and earwax (cerumen) production. Ethnic diversity in ABCC11 exists: The A allele is seen frequently (80-95%) in East Asian populations (e.g., Japanese, Korean, and Chinese) but is quite rare (0-3%) among individuals of European and African descent. The present study addresses the role of ethnicity and genetic variation in axillary and cerumen odor production. Employing organoleptic and analytical chemistry techniques, we have examined differences in axillary odor among individuals of European, East Asian, and African descent. Volatile compounds were analyzed in cerumen samples from the external auditory canal and in axillary secretions collected from donor T-shirts. Our results reveal that odorants produced by East Asians differ markedly from Caucasian or African American individuals. The type and amount of cerumen and axillary odorants were also well correlated, and suggest that cerumen can serve as a surrogate model for investigating human axillary odor type.

2:00 P.M.

A novel p53 target gene, TRIML2, differentially regulates the apoptotic potential of P72R polymorphic variants of p53

Che-Pei (Pat) Kung, Sakina Khaku, and Maureen Murphy

Molecular and Cellular Oncogenesis Program, The Wistar Institute

The p53 tumor suppressor gene is mutated in over 50% of human cancers. Several genetic polymorphisms of p53 have been found to alter its functions, including Proline (P) and Arginine (R) variants at residue 72. P72 is more common in African Americans and induces a higher level of cell cycle arrest and senescence. Meanwhile, R72 is more prevalent in Caucasians and has a stronger ability to induce apoptosis, partially due to its enhanced ability to translocate to the mitochondria. However, the role of p53-mediated transcription in these functional differences is unclear. To address this question, we performed gene expression microarray analysis with normal human fibroblast cells (NHFs) homozygous for P72 or R72 variants, and identified several p53-target genes that were differentially regulated by these variants, including tripartite motif family-like 2 (TRIML2). Stronger induction of TRIML2 in R72 cells was confirmed in multiple cell lines as well as mouse embryonic fibroblasts (MEFs) extracted from Human p53 knock-in (Hupki) mice possessing either human p53 variant. DNA damage-mediated TRIML2 induction is p53-dependent, and chromatin immunoprecipitation (ChIP) analysis was performed to confirm that TRIML2 is a bona fide p53 target gene. Overexpression and shRNA-knockdown experiments indicate that TRIML2 contributes to p53-mediated apoptosis by regulating p53’s stability and transcriptional function. Interestingly, TRIML2 regulates a specific subset of p53 target genes, including metabolism-associated genes, to affect p53’s ability to induce apoptosis. Overall, we have identified TRIML2 as the first known p53 target gene to influence the differential apoptotic potential associated with P72R polymorphism.