The principal focus area of the Gene Expression Laboratory, under the direction of Dominic Cosgrove, Ph.D., is molecular mechanisms underlying progressive pathologies resulting from mutations in basement membrane proteins. Current funded projects are renal and inner ear pathogenesis in Alport syndrome and retinal and inner ear pathology in Usher syndrome type IIa. The role(s) of cell adhesion and integrin signaling, cytokines, and chemokines are central to the experimental approach used to investigate these diseases. Gene knockout mouse models for both Alport syndrome and Usher syndrome type IIa have been created and characterized in the laboratory, and are the principal model systems for studying the molecular nature of pathogenesis. In addition, immortalized cell lines have been derived from the retina, the stria vascularis, and the renal glomerulus of both normal and mutant mice. These cell lines allow experiments to be conducted that question the cellular response to the microenvironment of cell matrix and cytokines. Further, cell culture systems are amenable to biochemical and molecular manipulations that are not possible in the whole animal. Biochemical and molecular approaches towards understanding mechanisms of pathogenesis have a high probability of yielding novel therapeutic strategies. Research performed in this laboratory has resulted in a number of novel approaches that ameliorate the renal pathogenesis in Alport syndrome, and two of these approaches have resulted in the identification of novel drugs that are in the pipeline at Biogen and Novartis.
The laboratory consists of about 2300 sq. ft. and is fully equipped for research in molecular biology and protein chemistry with workstations for up to fourteen individuals. The fully equipped molecular biology wet lab contains all of the necessary equipment to accomplish laboratory objectives including agarose and acrylamide gel apparatus and dryers, a spectrophotometer (Hitachi U-2000 Spectrophotomer), ultraviolet transilluminator (UVP EpiChemieII imager which is capable of recording chemiluminscence), PCR machines (3 MJ PCT-200 Peltier Thermal Cyclers), coarse and fine balances, hybridization incubators (2 Lab Line Rotary Hybridization Incubators), shaking and stationary bacteriologic incubators (2 LABCO orbital shakers), microfuges (1 Eppendorf 5415C and 1 Eppendorf 5417C) and a medium speed and table top ultracentrifuge, refrigerators (3 standard and two sliding glass door type), freezers (3 –20C), ultralow freezers (2 Revco –80C and 1 Cryostar –150C), a cryopreservation unit capable of holding 12,000 vials of cells, and 8 PC’s. Two independent cell culture suites (approximately 120 sq. ft. each), which are dedicated to this laboratory, include a hepa-filtered positive-pressure room with wrapped ceiling tiles and germicidal lamps. Equipment includes a doublewide laminar flow hood, four stacked double-chamber CO2 incubators, low speed centrifuges, an Olympus CK-2 inverted microscope, and a refrigerator freezer. The lab also has a micromanipulation room containing two stereo dissecting microscopes, fiber optic light source, a Nikon Diaphot with a dual fluorescence cube interfaced with both a camera and a CCD video camera and high resolution monitor. A Microm cryostat and a Leica paraffin microtome are also housed in this facility. There is a 250 square foot space for desks and computers. In a separate room we have an Olympus BH-2 immunofluorescence microscope with PlanApo 10x, 20x, and 40x objectives and 6 fluorescence cubes interfaced with a SPOT-RT 12 bit digital camera, and image analysis system. Adjacent to the Gene Expression wet laboratory is a shared electron microscopy facility with a Phillips CM10 electron microscope that is readily accessible and routinely used. Newly acquired equipment includes an ABI Prism 7000 real time PCR system, and a DAKO auto staining system for standardizing histochemical and immunohistochemical staining procedures. Much of the equipment is shared with the Gene Marker laboratory.
Marisa Zallocchi, Ph.D. (Post-Doc) Dr. Zallocchi is an expert in protein biochemistry and molecular biology. Her primary focus is on the mechanism of retinal and inner ear pathogenesis associated with Usher syndrome type IIa. Dr. Zallocchi’s work on characterizing usherin function depends heavily on protein interaction studies.
Daniel T. Meehan, B.S. (Technologist V) Mr. Meehan has expertise in embryonic stem cell culture, primary animal cell culture, molecular biology, and protein biochemistry. With over 20 years experience and training in molecular and cellular biology, and 12 years in the Cosgrove laboratory, Mr. Meehan adds a dimension of stability and continuity to the varied experimental design strategies employed to address complex biological questions.
Duane Delimont, M.S. (Technologist IV) Mr. Delimont has expertise in cell culture and molecular biology. Mr. Delimont’s primary responsibilities include the management of the transgenic and gene knockout mouse colonies and molecular cloning and vector construction projects.
Cosgrove, D., Meehan, D.T., Pozzi, A., Chen, X., Rodgers, K.D., Tempero, R.M., Delimont, D., Zallocchi, M., and Rao, V.H. (2008) Integrin α1β1 regulates MMPs via p38 MAPkinase in mesangial cells: implications for Alport syndrome. Am. J. Pathol. 172:761-773.
Peng, Y-W, Zallocchi, M., Meehan, D.T., Delimont, D., Chang, B., Hawes, N., Wang, W., and Cosgrove, D. (2008) Progressive morphological and functional defects in retinas from α1 integrin-null mice. IOVS 49:447-454.
Boosani CS, Nalabothula N, Munugalavadia V, Cosgrove D., Keshamouni VG, Shebani N., and Akulapalli S. (2009) FAK and p38 MAP kinase-dependent activation of apoptosis and caspase-3 in retinal endothelial cells by α1(IV)NC1. IOVS 50: 4567-4575.
Zallocchi, M., Meehan, D.T., Delimont, D., Askew, C., Garrige, S., Gratton, M.A., Rothermund-Franklin, C., and Cosgrove, D. (2009) Localization and expression of Clarin-1, the CLRN1 gene product, in auditory hair cells and photoreceptors. Hearing Res. 255: 109-120.
Meehan,D.T., Delimont, D., Cheung, L., Zallocchi, M., Sansom, S.C., Holzclaw, J.D., Rao, V., and Cosgrove, D. (2009) Biomechanical strain mediated maladaptive gene regulation as a contributing factor in Alport glomerular disease. Kidney Int. 76: 968-976.
Zallocchi M, Sisson JH, and Cosgrove, D (2010) Biochemical characterization of native usher protein complexes from a vesicular subfraction of tracheal epithelial cells. Biochemistry 49: 1236-1247.
Cosgrove D. and Zallocchi M (2010) Clarin-1 expression in photoreceptors. Hearing Res. 259: 117.
Dennis, J., Meehan, D.T., Delimont, D., Zallocchi, M., Perry, GA, O’brien, S. and Cosgrove, D. (2010) Collagen XIII mediates VLA-1 dependent monocyte transendothelial migration in renal fibrosis. Am J. Pathol 177: 2527-2540.
Peng, Y-W, Zallocchi, M., Wang, W-M, Chen, C-K, Delimont, D., and Cosgrove, D. (2010) Delayed rod transducin translocation and light induced rod degeneration in Usher syndrome type 1B mice. IOVS. (under revision)
Summary of Research Program
For Physicians and Scientists
The principal focus of the laboratory is aimed at understanding the molecular mechanisms underlying genetic diseases caused by mutations in genes encoding basement membrane proteins. We study two different, but related diseases; Alport syndrome (resulting from mutations in type IV collagen genes) and Usher syndrome type IIa (resulting from mutations in the gene encoding usherin, a novel basement membrane protein). Both of these diseases result in delayed onset, progressive pathologies. Alport syndrome is characterized by a childhood onset progressive glomerulonephritis associated with high frequency-specific sensorineural hearing loss. Since no drug treatments exist, patients undergo dialysis and kidney transplant with limited success. Usher syndrome type IIa is the most common single genetic cause of deafness and blindness. The blindness phenotype is a delayed onset progressive retinitis pigmentosa. The cellular and molecular nature of the pathogenic processes for these two diseases appear similar, in that the absence of the basement membrane protein results in progressive changes in cell behavior that culminate in structural and functional changes underlying the pathology. We aim to understand how these changes come about, and why there is a delayed onset of recognizable symptoms.
Both Alport syndrome and Usher syndrome type IIa are caused by mutations in genes encoding basement membrane proteins. Basement membranes are sheet-like structures that lie outside of cells and provide a matrix for cell adhesion, migration, and communication. Both Alport syndrome and Usher syndrome type IIa are delayed onset, progressive diseases. This characteristic affords a window of opportunity for therapeutic intervention. The challenge facing researchers is to understand the biology of the disease in sufficient detail to allow the design of therapeutic strategies aimed at the specific disease mechanisms. We have designed and produced mouse models for both of these diseases using a strategy called gene targeting. The mice provide a means of studying the onset and progression of the disease in great detail. Studies of Alport kidney disease have yielded important clues that are the basis for two different therapeutic strategies developed in this laboratory that are at different stages of drug development for eventual use in humans. Research aimed at defining the mechanism of Usher IIa pathology is less developed at this point in time. Importantly, information gained from the Alport syndrome studies can be directly applied to the Usher syndrome type IIa studies, since both processes involve cell communication with basement membranes. We therefore expect that Usher syndrome type IIa research will advance at a rapid pace.