The Bhattacharya laboratory uses a multidisciplinary approach which includes mass spectrometric proteomic and lipidomic approaches to study neurodegenerative diseases. The major focus is a group of progressive irreversible blinding diseases collectively called glaucoma and also a group of demyelinating diseases termed multiple sclerosis that frequently is associated with visual impairment preceding neurological deficits. The lab also has an interest in basic visual processes.
Current Research
Major focus of the laboratory is ocular neurodegenerative diseases collectively called glaucoma. Our proteomic mass spectrometric analysis differentially identified cochlin and peptidyl arginine deiminase2 associated with diseased human trabecular meshwork and optic nerve tissue respectively. Current projects involve understanding the role that these proteins play in glaucoma pathology.
Cochlin and trabecular meshwork
Overexpression of cochlin causes morphological changes in TM cells and cochlin and TREK-1 interaction has functional implications. Immunocytochemical analysis of cells expressing cochlin and green fluorescence protein (GFP). The cells were imaged at 23.5, 24.5 and 29 hours post-transfection as indicated and probed for TREK-1 (red), GFP (green) and cochlin (pink). Arrows show localization of cochlin and TREK-1 in the filopodia. Scale bar, 5µm (top panel), 50 µm (middle panel), 25µm (bottom panel).
Primary open angle glaucoma (POAG)is often associated with elevated intraocular pressure (IOP). The IOP is attributed to resistance to aqueous outflow in the anterior eye segment. Trabecular meshwork in the anterior eye segment is the region thought to offer most resistance to aqueous outflow. Primary glaucomas are late onset and results in progressive irreversible vision loss. Diseases such as glaucoma, nonsyndromic progressive hearing disorders and progressive brain damage with elevated intracranial pressure have similarities: they proceed with increased fluid flow, are late onset and progressive in nature. Using proteomic approaches, we have analyzed trabecular meshwork ™ from human donor eyes resulting in identification of cochlin exclusively in glaucomatous TM. We have found cochlin deposits in glaucomatous TM. In DBA/2J mouse model of glaucoma we have observed similar deposits and we are now investigating role of cochlin in glaucoma pathogenesis. Cochlin is deposited in extracellular matrix space along with mucopolysaccharides in glaucomatous TM tissue but not in controls. Cochlin has von Willebrand factor A like domains, which are involved in late onset diseases associated with changes in fluid flow regimes. My laboratory has recently established that cochlin serves as mechanosensing molecule. Cochlin interacts with transmembrane channel TREK-1 for transduction of shear stress signal resulting in modulation of cytoskeleton of TM cells. Our research has brought the new concept that the mechanosensing at extracellular matrix is an integral part of the dynamic TM remodeling. Continued and aberrant shear force transduction results in failure of cellular remodeling at the TM level which is contributed by mechanotransducing proteins. In order to continuously monitor cochlin and determining cochlin-protein interactions we utilized magnetic nanoparticles and magnetomotive optical coherence tomography. We have recently improved this method for live detection of a target protein and its quantification enabling us to correlate the amount of cochlin expression with IOP changes.
Lipid composition of Trabecular meshwork
Mass spectrometric analysis of lipids in the TM. The lipids of the TM or TM cells are analyzed using daughter ion directed parent ion scan. Phosphocholine (PC) shown here to generate a daughter ion (184) for two PC molecule with a parent mass of 594.40 and 622.40 amu.
Lipids are the active constituents of all cell membranes. They are also potent signaling molecules and have other biological functions. However, overall lipids are a less well studied class of compounds compared to proteins. In POAG, the increased intraocular pressure due to impeded aqueous humor outflow is contributory to pathologic process of neuronal cell death. There is no widely-accepted definition of lipid, despite a firm understanding of what represents the term “lipid” among experts in the field. Different lipid classes due to differences in inherent chemistry pose an insurmountable challenge with respect to analyses. The mass spectrometry and in particular triple quadrupole mass spectrometers have significantly advanced analyses of class specific identification of known and unknown lipids based on available purified standards using methods such as precursor ion or neutral loss scans. Our laboratory has spearheaded quantitative analyses of TM lipid using mass spectrometry. We have identified and characterized new hitherto unknown lipids in the TM. We have found that mechanotransducing channels such as TREK-1 is greatly modulated by lipids. It is no surprise that prostaglandin lipid analogs are one of the most widely used drugs in glaucoma. Our combined proteomic and lipidomics approach is now providing rare mechanistic insights into biology of TM.
Proteomic analysis of optic nerve and peptidyl arginine deiminase2
A proteomic ion trap mass spectrometer (LCQ Deca) in the laboratory being used for identification of proteins by mass spectrometric protein sequencing.
Glaucomas are diseases characterized by degeneration of optic nerve. The mechanism by which the optic nerve undergoes structural and functional damage in glaucoma is poorly understood. The goal of our laboratory is to determine the differential protein profile between normal and glaucomatous optic nerves using mass spectrometry. In our lab, protein changes studied using human cadaver donor eyes, mass spectrometry and Western analysis have identified peptidyl arginine deiminase2 (PAD2) as one such target. PAD2 converts protein bound arginine residues into citrulline, a posttranslational modification process termed as deimination. Our recent work using a glaucomatous mouse model and a transgenic mouse model of multiple sclerosis has provided important insight about the role that PAD2 and deimination play in pathogenesis. We utilize a transgenic proteolipid protein (PLP) in CD1 background and also in B6 background enabling dissection of genetic and immunological components. We have established deimination as regulator of neurite outgrowth in the genetic model of PLP transgenic animal. We use non-invasive methods such as magnetic resonance imaging (MRI) and pattern electroretinogram (PERG) to determine loss of visual function together with assessment of neurological deficits. Endpoint proteomic and lipidomics analyses enables us to identify key protein and lipid molecules whose role is then tested in vitro using neuron and astrocyte culture which is further evaluated using the rodent disease models as mentioned above. In vitro and in vivo models also enable us to determine entities that may serve as therapeutic intervention strategies.
Vision, Learning and Behavior
Fluorescence microscopic images of Zebra finch retina sections Retina sections (8-10 µm) were imaged with a confocal microscope. Antibody against Recoverin, with secondary coupled to Alexa 596 and control (preimmune serum) was used for detection as indicated. Nuclear staining was performed using DAPI. RPE: retinal pigment epithelium, PR: photoreceptors, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL, ganglion cell layer.
Visual deprivation is expected to have a profound effect on building of the memory. Even short term visual deprivation alters neural processing of tactile form. Congenitally blind individuals usually perform auditory tasks better than sighted persons or individuals who have become progressively blind. Congenitally blind individuals are able to build memory based on auditory and other perceptions. Transcranial magnetic stimulation of the occipital pole interferes with verbal processing in blind subjects and early visual cortex activation correlates with superior verbal memory performance in the blind. A model system where vocalization is learned may provide clues about the effect of visual deprivation on learning and memory. The avian model, Zebra finch (Taeniopygia guttata) when deprived of vision allows detection of song learning as a measurable behavioral parameter to compare with sighted controls. It has been established that songbirds subjected to tutoring in their pre-adolescent stage sing well. The lab has embarked on a systematic study of the effect of visual deprivation on song learning with aim to correlate changes in learned vocalization with vision deprivation in Zebra finches. Protein changes have been detected in primary visual pathway as well as in the other sensory systems due to visual deprivation. Quantitative and qualitative detection of proteomic and lipidomics changes in primary visual pathway as well as other brain regions of zebra finches are under progress. Interestingly some of these changes are also associated with epigenetic changes. Eventually we expect this model to allow determination of brain activity and development in visually deprived and partially-deprived subjects that will provide insight into the auditory and tactile learning process and role of vision in learning and memory.
Comparative ocular anatomy
A pilot project in our laboratory is to build an educational resource, that is, a comparative ocular fact database. This database will be web based and allow (visual) anatomical and biochemical curated facts to be deposited and retrieved. This database will provide background to readily decide the organism of choice and will also act as a learning tool in classroom setting.
Analyses of ocular injuries and regeneration
May protein and lipids serve as predictors of severity and duration of exposure to foreign agents such as alkali, thermal and metallic impaction injuries? Quiescent corneas (deficient in protein and DNA synthesis) and mass spectrometry is being used in addressing these questions. We have identified lipids that are generated by injured corneas as early response to alkali injuries which greatly promotes repair and regeneration. We are actively involved in identification and characterization of repair and regeneration inducing/promoting lipids.
