Summary: The Bhattacharya laboratory uses a multidisciplinary approach which includes proteomic mass spectrometry to study neurodegenerative diseases of the eye. The major focus is a group of progressive irreversible blinding diseases collectively called glaucoma. The lab also has an interest in basic visual processes.

Sanjoy K. Bhattacharya, Ph. D.
Assistant Professor of Ophthalmology

View published research articles by this doctor in the National Library of Medicine.

Current Projects:A major focus of the laboratory is ocular neurodegenerative diseases, especially glaucoma. Our proteomic mass spectrometric analysis differentially identified cochlin and peptidyl arginine deiminase 2 as associated with diseased human trabecular meshwork and optic nerve tissue respectively. Current projects involve understanding the role that these proteins play in glaucoma pathology.

1. Cochlin and trabecular meshwork. Aqueous humor is actively produced in the ciliary epithelium, traverses through the pupil. After bathing the cornea where is provides nutrition and removes excretory materials, the aqueous then leaves through structures in the anterior eye chamber. An imbalance between aqueous production and outflow causes elevation in the intraocular pressure (IOP). Glaucomas are late onset and cause progressive irreversible vision loss. A glaucoma is termed primary when no known cause can be attributed and termed secondary is an illness or injury leads to the onset.  Primary open angle glaucoma is often associated with elevated IOP.  Aqueous outflow experiences most resistance at the trabecular meshwork (TM) in the anterior eye segment, resistance is further increased in glaucoma. Our proteomic analyses of TM from surgical or cadaver human donor eyes revealed presence of cochlin exclusively in glaucomatous TM. Presence of cochlin was confirmed using Western and immunohistochemical analysis. Cochlin deposits were also found in the DBA/2J mice model of glaucoma. In my laboratory this model is being used to investigate the role of cochlin in glaucoma pathogenesis. Cochlin is deposited in the extracellular matrix (ECM) space along with mucopolysaccharides in glaucomatous TM tissue, but not in controls.

   Normal Glaucomatous Microscopic images of trabecular meshwork (TM) sections showing cochlin and mucopolysaccharide deposits in glaucomatous TM

   Cochlin has three domains, one with homology to coagulation factor C of the horseshoe crab, and two related to von Willebrand factor A. Proteins with the von Willebrand factor A-like domain are involved in late onset diseases associated with changes in fluid flow regimes. Cochlin deposits in cochlea have been found associated with autosomal progressive hearing loss and other vestibular diseases such as Ménière's disease and presbycusis. My laboratory is attempting to understand how cochlin is deposited and the role it plays in TM using various techniques including proteomic mass spectrometry. Although a great deal is known about regulatory circuits in cytoplasm, relatively little is known about regulation of proteins and other biomolecules in the ECM. For example, is deposition of cochlin in the ECM of the glaucomatous TM due to over-expression or decreased degradation? In addition to seeking answers to these questions, the laboratory will attempt to achieve a better understanding of TM-ECM interactions. Novel and emerging proteomic analysis tools will be applied in the laboratory to address relatively complex ECM problems as well.
   
   
2. Proteomic analysis of optic nerve and peptidyl arginine deiminase2. 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 are studied using human cadaver donor eyes, mass spectrometry and Western analysis. Using mass spectrometry, we have identified peptidyl arginine deiminase2 (PAD2) as one such target. PAD2converts protein bound arginine residues into citrulline, a posttranslational modification process termed as deimination. Currently our laboratory has identified about 45 proteins as substrate for PAD2 that undergoes deimination. These target proteins of PAD2 upon posttranslational modification by this enzyme undergoes modulation in their normal functions. The modifications are currently considered pathogenic. PAD2 appears to over-express in human glaucomatous optic nerve, in vivo in DBA/2J mice optic nerve and in cultured astrocytes when subjected to elevated pressure. Elevated levels of PAD2 level or protein deimination can not be restored to normal levels by reduction in pressure alone. In cultured astrocytes, simultaneous pressure reduction and treatment with an inhibitory small RNA molecule have been shown to reduce the PAD2 level and consequent posttranslational modification. This is relevant from the standpoint of therapeutics. PAD2 catalyzed deimination may weaken the optic nerve head matrix due to loss of anchorage of ECM proteins. The investigator has shown elevated level of deimination for several myelin proteins including myelin basic protein (MBP) in the myelinated region of the glaucomatous optic nerve. In other neuronal systems, deimination of MBP has been shown to result in mislocalization and is expected to affect insulation. Thus elevated PAD2 in glaucomatous optic nerve may exacerbate damage due to other factors, and lowering PAD2 level and deimination is expected to be beneficial. This is being tested using the DBA/2J model where PAD2 elevation in the optic nerve parallels elevation is intraocular pressure. Mass spectrometrically identified disease associated proteins and protein modifications retain the potential to serve as markers for individuals predisposed to an increased risk of developing glaucoma. Such information procured from a large number of donors should provide insight into the pathologic mechanisms associated with glaucoma, and could help identify possible new drug targets for glaucoma therapy.
   
   
3. Vision, Learning and Behavior. 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. Convergence of visual and tactile shape processing has been found to occur in the human lateral occipital complex. An avian model system may provide clues about the effect of visual deprivation on learning and memory. The avian model, Zebra finch (Taeniopygia guttata) allows for detection of a measurable behavioral parameter, their song. Currently, the expressed sequence tag information is available and sequencing of the entire songbird genome is part of the NIH roadmap initiative. Neurogenomic and informatics approaches encompassing several reputed centers are underway as a concerted effort. 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 or song as a measurable parameter with vision deprivation in Zebra finches. We expect protein changes in primary visual pathway as well as in the other sensory systems due to visual deprivation. We have embarked on capture of protein changes using quantitative and qualitative proteomic approaches in primary visual pathway as well as other brain regions of zebra finches. Our preliminary results are extraordinarily encouraging. 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.
   
   

   Fluorescence microscopic images of Zebra finch eye (retina) sections (8-10 µm) obtained with a Carl Zeiss Axiovert 200 M inverted microscope equipped with Axiovision 4.3 software. Antibody detection for recoverin, with secondary coupled to Alexa 596 The controls is a preimmune serum 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.

   
   
   
4. 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 deposition and retrieval. This database will provide background to readily decide the organism of choice and will also act as a learning tool in classroom setting.