Medicine and Health Sciences Commons^™

Acute Acat1/Soat1 Blockade Increases Mam Cholesterol And Strengthens Er-Mitochondria Connectivity., Taylor C Harned, Radu V Stan, Ze Cao, Rajarshi Chakrabarti, Henry N Higgs, Catherine C Y Chang, Ta Yuan Chang

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain …

A Mathematical Model Of Glut1 Modulation In Rods And Rpe And Its Differential Impact In Cell Metabolism, Andrea Aparicio, Erika T Camacho, Nancy J. Philp, Stephen A Wirkus

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

We present a mathematical model of key glucose metabolic pathways in two cells of the human retina: the rods and the retinal pigmented epithelium (RPE). Computational simulations of glucose transporter 1 (GLUT1) inhibition in the model accurately reproduce experimental data from conditional knockout mice and reveal that modification of GLUT1 expression levels of both cells differentially impacts their metabolism. We hypothesize that, under glucose scarcity, the RPE’s energy producing pathways are altered in order to preserve its functionality, impacting the photoreceptors’ outer segment renewal. On the other hand, when glucose is limited in the rods, aerobic glycolysis is preserved, which …

Uncontrolled Mitochondrial Calcium Uptake Underlies The Pathogenesis Of Neurodegeneration In Micu1-Deficient Mice And Patients, Raghavendra Singh, Adam Bartok, Melanie Paillard, Ashley L. Tyburski, Melanie B Elliott, György Hajnóczky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Dysregulation of mitochondrial Ca2+ homeostasis has been linked to neurodegenerative diseases. Mitochondrial Ca2+ uptake is mediated via the calcium uniporter complex that is primarily regulated by MICU1, a Ca2+-sensing gatekeeper. Recently, human patients with MICU1 loss-of-function mutations were diagnosed with neuromuscular and cognitive impairments. While studies in patient-derived cells revealed altered mitochondrial calcium signaling, the neuronal pathogenesis was difficult to study. To fill this void, we created a neuron-specific MICU1-KO mouse model. These animals show progressive, abnormal motor and cognitive phenotypes likely caused by the degeneration of motor neurons in the spinal cord and the cortex. We found increased susceptibility …

Uveitis-Mediated Immune Cell Invasion Through The Extracellular Matrix Of The Lens Capsule, Jodirae Dedreu, Sonali Pal-Ghosh, Mary J Mattapallil, Rachel R Caspi, Mary Ann Stepp, A Sue Menko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune …

Targeting Fibroblast Activation Protein In Tumor Stroma With Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth And Augment Host Immunity Without Severe Toxicity., Liang-Chuan S. Wang, Albert Lo, John Scholler, Jing Sun, Rajrupa S. Majumdar, Veena Kapoor, Michael Antzis, Cody E. Cotner, Laura A. Johnson, Amy C. Durham, Charalambos C. Solomides, Md, Carl H. June, Ellen Puré, Steven M. Albelda

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAP(hi) stromal cells and inhibited the …

Decorin Protein Core Affects The Global Gene Expression Profile Of The Tumor Microenvironment In A Triple-Negative Orthotopic Breast Carcinoma Xenograft Model., Simone Buraschi, Thomas Neill, Rick T Owens, Leonardo A Iniguez, George Purkins, Rajanikanth Vadigepalli, Barry Evans, Liliana Schaefer, Stephen C Peiper, Zi-Xuan Wang, Renato V Iozzo

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal) and Homo sapiens (epithelial) tissue origins. We found that …

Rip1-Dependent And Independent Effects Of Necrostatin-1 In Necrosis And T Cell Activation., Youngsik Cho, Thomas Mcquade, Haibing Zhang, Jianke Zhang, Francis Ka-Ming Chan

Department of Microbiology and Immunology Faculty Papers

BACKGROUND: Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function.

METHODOLOGY/PRINCIPAL FINDINGS: We compared the effect of …

Mitostatin Is Down-Regulated In Human Prostate Cancer And Suppresses The Invasive Phenotype Of Prostate Cancer Cells., Matteo Fassan, Domenico D'Arca, Juraj Letko, Andrea Vecchione, Marina P Gardiman, Peter Mccue, Bernadette Wildemore, Massimo Rugge, Dolores Shupp-Byrne, Leonard G Gomella, Andrea Morrione, Renato V Iozzo, Raffaele Baffa

Kimmel Cancer Center Faculty Papers

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in …

Tdp-43 Potentiates Alpha-Synuclein Toxicity To Dopaminergic Neurons In Transgenic Mice., Tian Tian, Cao Huang, Jianbin Tong, Ming Yang, Hongxia Zhou, Xugang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

TDP-43 and α-synuclein are two disease proteins involved in a wide range of neurodegenerative diseases. While TDP-43 proteinopathy is considered a pathologic hallmark of sporadic amyotrophic lateral sclerosis and frontotemporal lobe degeneration, α-synuclein is a major component of Lewy body characteristic of Parkinson's disease. Intriguingly, TDP-43 proteinopathy also coexists with Lewy body and with synucleinopathy in certain disease conditions. Here we reported the effects of TDP-43 on α-synuclein neurotoxicity in transgenic mice. Overexpression of mutant TDP-43 (M337V substitution) in mice caused early death in transgenic founders, but overexpression of normal TDP-43 only induced a moderate loss of cortical neurons in …

Imaging Spontaneous Mmtvneu Transgenic Murine Mammary Tumors: Targeting Metabolic Activity Versus Genetic Products., Mathew L Thakur, Devakumar Devadhas, Kaijun Zhang, Richard G Pestell, Chenguang Wang, Peter Mccue, Eric Wickstrom

Department of Radiology Faculty Papers

INTRODUCTION: Despite the great strides made in imaging breast cancer (BC) in humans, the current imaging modalities miss up to 30% of BC, do not distinguish malignant lesions from benign ones, and require histologic examinations for which invasive biopsy must be performed. Annually in the United States, approximately 5.6 million biopsies find benign lesions. More than 50% of human BCs overexpress cyclin D1, and all BCs exhibit VPAC1 oncogene products. Together, these gene products may provide an excellent biomarker for the early and accurate detection of BC. We have evaluated 4 biologically active peptide analogs that have high affinity for …

Sustained Expression Of Tdp-43 And Fus In Motor Neurons In Rodent's Lifetime., Cao Huang, Pedro Yuxing Xia, Hongxia Zhou

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) are two highly conserved ribonucleoproteins. Pathogenic mutations of the TDP-43 or the FUS gene are all linked to amyotrophic lateral sclerosis (ALS) that is characterized by progressive degeneration of motor neurons. To better understand the correlation of ALS disease genes with the selectivity of chronic motor neuron degeneration, we examined the longitudinal expression of the TDP-43 and the FUS genes in C57BL6 mice and in Sprague-Dawley rats. TDP-43 and FUS were robustly and ubiquitously expressed in the postnatal mice and rats, but were markedly decreased in the adult rodents. In adulthood, …

Silencing Of The Pink1 Gene Expression By Conditional Rnai Does Not Induce Dopaminergic Neuron Death In Mice., Hongxia Zhou, Björn H Falkenburger, Jörg B Schulz, Kim Tieu, Zuoshang Xu, Xu Gang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA …

Selective Role For Superoxide In Insp3 Receptor-Mediated Mitochondrial Dysfunction And Endothelial Apoptosis., Muniswamy Madesh, Brian J Hawkins, Tatyana Milovanova, Cunnigaiper D Bhanumathy, Suresh K Joseph, Satish P Ramachandrarao, Kumar Sharma, Tomohiro Kurosaki, Aron B Fisher

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane …

Functionally Active T1-T1 Interfaces Revealed By The Accessibility Of Intracellular Thiolate Groups In Kv4 Channels., Guangyu Wang, Mohammad Shahidullah, Carmen A Rocha, Candace Strang, Paul J Pfaffinger, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is …

Stretch-Induced Calcium Release In Smooth Muscle., Guangju Ji, Robert J Barsotti, Morris E Feldman, Michael I Kotlikoff

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) …