Molecular and Cellular Biology Lab
Dhirendra P. Singh, Ph.D.
Lab: DRC1 Room 4054; 402-559-8807
Office: DRC1 Room 4051; 402-559-8805
Unveiling causes and mechanisms
of age-related diseases and therapeutic interventions
The general interests of Singh's lab include the causes and molecular mechanism(s) responsible for cell survival, ways in which these survival processes go awry in various diseases, and identification of the downstream deleterious cellular signaling involved in initiation and progression of aging and age-related degenerative disorders, with the goal of developing mechanism-based therapy.
The eye lens and cataractogenesis are used as a model system for aging diseases because of their unique and simple structure and metabolic features. Investigators at the laboratory make extensive use of state-of art modern techniques and biochemical analysis applicable to the research projects.Much of the research is based on the hypothesis that, as lens epithelial cells (LECs) survive throughout life,such cells may contain a survival molecule.
Through studying the mechanism and regulation of cell survival, the research team have discovered three molecules: i) Lens Epithelium-Derived Growth Factor (LEDGF), which acts as a transcription factor; ii) lenticular Peroxiredoxin 6 (Prdx6), a multifunctional protective protein, and iii) Lens Epithelium-Derived Protein (LEDP), which maintains endoplasmic reticulum-Golgi homeostasis and blocks LEC death from unfolded protein response and endoplasmic reticulum (ER) stress inducers.
Research has been devoted to characterizing LEDGF, Prdx6 and LEDP; determining how these molecules are regulated during aging, and identifying ways in which their expression and functions are crucial for cell survival and cell homeostasis. The lab’s research has anticipated many major trends and contributed substantially to knowledge in several areas: i) the molecular mechanisms of pathological signaling involved in etiopathogenesis of age-related disorders, particularly blinding diseases; ii) the role and characterization of oxidative stress induced by external and physiological factors in disrupting cell homeostasis; iii) application of the moonlighting protein Prdx6 to sick cells, and enhancing Prdx6 transcription by means of naturally occurring compounds to postpone pathogenic signaling that causes disease; and iv) identification of LEDGF and LEDP expression level as crucial to maintenance of cell physiology. (Aberrant expression of these molecules lead to etiopathogenesis, while physiological levels maintain cell homeostasis.)
The lab’s forward-looking efforts have provided insight into basic molecular mechanisms of initiation and progression of age-related cataract, glaucoma and other degenerative disorders, and have suggested plausible approaches to control or postpone them.
Born in India, Dr. Singh came to the United States in 1992, as a Fogarty International Research Associate at the National Institutes of Health, Bethesda, MD for two years. He moved to the Harvard Medical School as a junior faculty member in 1994, and joined the Center for Ophthalmic Research, Department of Surgery, Brigham and Women’s Hospital, Boston, MA, as assistant director of Molecular Biological Research. In 2002, Singh Joined University of Nebraska Medical Center, and currently is Professor of Ophthalmology and Visual Sciences, Truhlsen Eye Institute (TEI), Nebraska Medicine at University of Nebraska medical Center.
Dr. Singh received his Ph.D. degree in molecular immunobiology in 1988 from Central Drug Research Institute, Lucknow, and Kanpur University in India. He received post-doctoral training in immunology and molecular biology at this same Institute and National Eye Institute, NIH, Bethesda, MD. In 1986 he was awarded a travel grant from the World Health Organization to attend an International Conference (FAOB) as well as to get training on molecular biological training at the University of Singapore. He has made several significant contributions in the fields of molecular immunology and cell biology, and in 1987 the Indian Society for Parasitology honored him as one of the best researchers in the country on the basis of his published work (Babar Mirza Award).
Dr. Singh received the John Mannick Research Award in the Department of Surgery at the Brigham and Women’s Hospital, Harvard Medical School, for his research on causes of aging disease-cataractogenesis. His efforts have also included research on the molecular mechanisms of many autoimmune ocular diseases. He has contributed much to understanding of the major immunological determinants of the classical retinal protein S-antigen (S-Ag). In this work he found a remarkable similarity between the S-Ag determinants and specific sequences in several proteins extracted from microorganisms that also cause experimental autoimmune uveitis (EAU). His discovery of this cross-reactivity has helped re-define the immunological ideas of “self” and “non-self” determinants in ocular autoimmune diseases. He also found that a single amino acid substitution in the sequence of S-Ag can prevent the induction of the uveitis. This work constitutes a first-rate contribution to knowledge of the basic immunology of these potentially blinding diseases. Dr. Singh is well-published and pursuing innovative research and serves as principal investigator on many research projects. Based on his contributions to research, the University of Nebraska Medical Center recognized Dr. Singh as Distinguished Professor in 2008. His research team provides novel research applicable to understanding blinding disorders, and possibly opening the way to postponement of eye disorders and age-related degenerative disorders in general.
Discovery of Lens Epithelium-Derived Growth Factor (LEDGF)-In 1994 at the Center for Ophthalmic Research, Brigham and Women’s Hospital, Harvard Medical School, Dr. Singh and colleagues began with the hypothesis that LECs might contain a survival molecule. Drs. Shinohara, Chylack Jr. and Singh discovered a novel survival factor, Lens Epithelium-Derived Growth Factor (LEDGF; US patent: 67500520), that acts as a transcription factor. Dr. Singh has established that LEDGF, which stimulates the survival of cells including LECs, has the potential for enhancing cellular survival of LECs, the metabolic “engine” of the lens. His laboratory demonstrated that LEDGF is posttranslationally modified by Sumo1 (Small ubiquitin-like Modifier), and contains activating and repressing DNA binding modules which regulate survival gene(s) expression, including small heat shock proteins (hsps) and antioxidants genes depending upon cellular microenvironment and thus modify the physiology of cells to maintain cellular homeostasis. This is an important clue to the mechanisms by which expression or regulation of LEDGF is important for cell survival, and its aberrant expression may increase the risk of age-related cataract or other pathogenic disorders like cancer. Collectively, these works show great promise for both understanding the fundamental mechanisms of age-associated degenerative diseases including cataract, and application of LEDGF, a survival molecule, to the prevention of such disorders.
Discovery of lenticular Peroxiredoxin 6 (Prdx6), a moonlighting protein, in eye lens-Using targeted inactivation of Prdx6gene in mice, Singh and his research team have shown that Prdx6-depleted lenses and/or LECS contain elevated levels of reactive oxygen species (ROS), and bioactive TGF b1 and its inducible genes. TGFb1 inducible genes, such as a-SM-actin and big-h3, are implicated in the pathophysiology of cataractogenesis. These findings show that Prdx6 plays a rheostat role in regulating gene expression by controlling the ROS level. This led to development of a model system, Prdx6 knockout,for studying the role of ROS-driven oxidative stress-induced deleterious signaling in aging.
Recent innovative research has studied delivery to the eye lens of Prdx6 linked to TAT transduction domain. Using the Shumiya cataract rat (SCR), a model for hereditary cataract, Singh and colleagues have shown for the first time that recombinant Prdx6 linked to TAT, when injected subconjunctivally, can reach the lens, internalize into LECs, and delay cataractogenesis. Singh has expanded this research to diabetic complications and glaucoma. In collaborative studies, his group found that Prdx6 protected LECs against hyperglycemia-induced toxicity, and that a supply of Prdx6 attenuated environmental and cellular stresses that evoke cellular abnormalities, thereby protecting ganglion and trabecular-meshwork cells. This research holds great promise for treatments of blinding diseases, particularly age-related cataract and diabetes, possibly suggesting nonsurgical means of treating these conditions, which are the leading causes of blindness in the world today.
Recently, Singh and his group demonstrated that Prdx6 is multifunctional protective protein and saves many cell types from cellular stressors. More recently Singh team has become interested in more mechanistic studies of Prdx6’s functions and its regulation at the transcription level to develop transcription-based therapy for inducing natural cellular defenses by means of supplying dietary supplements. Considering the difficulty of protein delivery, this would be a novel approach to identify dietary compound(s) that may enhance the body’s antioxidant defenses. Along this line, he and his team are determining downstream signaling involved in regulation of Prdx6 transcription by using dietary supplements as well as FDA-approved drugs. His group has identified such compounds, and are now examining their efficacy in vivo. The strategy of using dietary supplements or existing drugs, if successful, could be employed widely to attenuate oxidative stress-associated degenerative diseases including ocular disorders.
The current research is interesting in its extension of the idea of developing transcription-based stimulation of an endogenous molecule to protect cells against stressors, through the use of dietary supplements (inductive therapy)