David Krizaj - US grants

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand the cellular mechanisms that underlie vision. The premise underlying this application is that the remarkable adaptability of vision is based on the plasticity of intracellular signaling pathways in retinal neurons. The focus of the work will be to study the mechanisms that regulate the intracellular Ca2+ concentration and transmitter release in retinal rod and cone photoreceptor neurons. The proposed experiments combine electrophysiological, optical and immunocytochemical methods. Results obtained in these studies will help us select cellular targets for therapeutic interventions during retinal disease and blindness. Three specific aims are proposed: (1) to characterize Ca2+ regulation in photoreceptor inner segments; (2) to identify differences in Ca2+ homeostasis between rod and cone photoreceptor inner segments; and (3) to determine the relationship between [Ca2+]і and exocytosis in photoreceptors.

DESCRIPTION (provided by applicant): Glaucoma is complex of devastating blinding diseases that together represent the primary cause of irreversible blindness in the U.S. There is substantial evidence that pathological mechanical stimulation mediated by an increase in intraocular pressure (IOP) plays a causal role in the etiology of glaucoma. The present proposal is to characterize the molecular mechanisms which might underlie the cellular response in this disease but could also play a key function in other diseases that involve mechanical stress in the retina, such as diabetic retinopathy, ischemia and macular edema. We found that a mechanosensitive cation channel, TRPV4, is selectively localized to retinal ganglion cells (RGCs) and in Muller glial cells. Because these are the two cell types that are specifically targeted in glaucoma, we hypothesize that mechanosensitive channels mediate the effects of pathological increases in IOP. The central focus of the proposal is to characterize this transduction mechanism in RGCs by combining biophysical, cellular and translational approaches. Studies proposed in Aim 1 will establish the molecular mechanism of mechanosensitive channel activation and desensitization, their role in calcium transport, cellular physiology and RGC survival. We will test conditions that mimic RGC injury in low-tension pathologies and test a number of models under which mechanosensitive channels might contribute to excitotoxic RGC injury. The proposed studies will also capitalize on preliminary work which shows remarkable effectiveness of non toxic small molecule antagonists in blocking pressure-stimulated loss of RGCs in vitro and in vivo glaucoma models. Aim 2 of the proposed research builds on the characterization of pressure-sensitive channels in Aim 1 to study how these mechanisms regulate the swelling response of RGCs and retinal astroglia. Although cells typically swell in response to normal light-evoked neuronal activity, swelling is exacerbated in pathological conditions such as ischemia and diabetes, and can be highly neurotoxic. The proposed studies will explore the molecular complexes that involve swelling-activated calcium channels, water channels, calcium waves and regulatory volume decrease mechanisms. Thus, the goal of proposed research is to establish an intuitive conceptual and experimental framework that helps unify our understanding of retinal IOP transduction, cell swelling, and volume sensing and calcium homeostasis in retinal cells. By doing so, it will help predict the effects of mechanical forces that act through direct hydrostatic compression of cellular membranes as well as determine molecular mechanisms that are activated by tensile stretching, pulling and swelling. We will then test these predictions using mouse models of inducible and chronic glaucoma. This may help to refine our understanding of mechanical injury in vision disorders such as glaucoma, diabetic retinopathy and ischemia and contribute to developing effective neuroprotective treatments.

Project Summary-Physiology Core The OVERALL aims of this Vision Research Core (VRC) are to provide: · access to resources outside the scope of individual R01 awards · access to technical expertise outside the scope a single laboratory · staff training to remove barriers to efficient translational research and collaboration · collaboration initiatives among VRC labs The research areas supported by the VRC span the analysis and treatments of retinal degenerations, developmental disorders, glaucoma and other disorders, as well as and a range of cutting-edge basic science initiatives. We have implemented four resource modules that continue the natural evolution of how this research group works together, serving 17 investigators holding 22 NEI R01 awards. The Physiology Module is a powerful set of tools that VRC faculty use for animal model validation and disease profiling: ERGs, OptoMotry behavioral testing, and a range of ocular imaging tools (OCT, Micron). Our new module includes in vivo / in vitro 2-photon imaging. Specifically the Physiology Module provides: · Three UTAS ERG systems (two within the JMEC vivarium) · An OptoMotry system within the JMEC vivarium · Two Micron mouse imaging systems (one within the JMEC vivarium) · An additional new Micron IV mouse imaging system with high resolution OCT, image-guided ERG function, slit lamp and focal laser injury submodules, recently acquired with funds from the Department of Ophthalmology and an Administrative supplement of the NEI (P30EY014800-14S1) · Rotarod for locomotor and vestibular phenotyping within the JMEC vivarium · Dual Spectralis OCT systems (one within the JMEC vivarium) · A Bruker Ultima 2-photon microscope (in core on floor 5) · Technical support for all these systems

PROJECT SUMMARY/ABSTRACT There is substantial evidence that pathological increases in intraocular pressure (IOP) play a causal role in the pathological remodeling of trabecular meshwork cells which regulate the drainage of aqueous humor from the anterior eye however the identity and function of the mechanosensing mechanisms remains largely unknown. The present proposal aims to characterize these mechanisms at biophysical, molecular and cellular levels as well as in bioengineered models of conventional outflow. Aim 1 will establish the mechanical thresholds of human TM cells obtained from non-symptomatic and glaucomatous patients, characterize effect of mechanical stress (pressure, stretch and swelling) on intrinsic mechanosensitive channels and establish its time- dependent properties (acute & chronic adaptation). This is expected to lead to a novel model of tensile homeostasis in the TM based on balanced activation of opposing types of mechanosensitive channels. Aim 2 links pressure-sensitive channels to the remodeling of actin cytoskeleton and focal adhesion contacts with the extracellular matrix, uses innovative strain-sensitive optical cytoskeleton probes and defines the function of mechanical coupling in the regulation of the conventional outflow resistance using biomimetic nanoscaffolds populated with healthy and glaucomatous human TM cells. The proposed research thus aims to establish novel conceptual, experimental and translational frameworks that will unify our understanding of retinal IOP regulation within the context of mechanotransduction, cell swelling, volume sensing and calcium homeostasis, with the aim to lead towards the development of effective, first-in-kind treatments for glaucoma.

Retinal ganglion cells and Müller glia are particularly susceptible to mechanical forces which drive inflammatory activation and RGC degeneration in diseases such as glaucoma, but the pressure transduction mechanisms are not well understood. Earlier studies have been limited to phenotyping the genetic, molecular, cellular and behavioral consequences of RGC injury and glial activation induced by elevated pressure. While many biochemical pathways were shown to be altered in hypertensive eyes, the molecular sensors that transduce mechanical forces remain obscure, confounding interpretations of time-dependence of pressure-induced remodeling changes within the retina. The dominant hypotheses about pressure injury in glaucoma focus on the role of forces on the stretch of the lamina cribrosa yet mice develop the disease but do not have the collagenous lamina. The axocentric hypotheses also cannot explain how mild pressure elevations induce early changes in dendritic architecture and synaptic function, or activate glia without visible changes in axonal transport. It is also not known how physiological levels of intraocular pressure might inform RGC physiology and whether they are sufficient to integrate with the synaptic (light) responses. Finally, although glia are often the earliest responder to mechanical stress, the mechanisms that impel mechanosensitivity to these cells and how they impact RGC physiology remain largely unknown. The proposed work addresses these confounds by identifying the mechanotransducers and elucidating their role in RGC and Müller glial calcium homeostasis and polymodal integration of pressure into the (patho)physiological retinal response. The project tests the central hypothesis that pressure sensitivity of dendrites, somata and axons of RGCs and glia is governed by mechanosensitive ion channels, which maintain tensile homeostasis and modulate calcium homeostasis, excitability and gliotransmitter release in response to changes in ocular pressure or strain. Leveraging the recently derived data and using novel mechanobiological tools, Aim 1 will identify and characterize mechanosensing ion channels in the RGC plasma membrane, quantify their activation by pressure and matrix stretch, and test the hypothesis that mechanical strains are transmitted from the plasma membrane into the cell interior through the cytoskeleton. In Aim 2 we propose to characterize the polymodal mechanism through which mechanical stimuli are integrated with the effects of temperature and synaptic (light) responses, and to test a novel hypothesis regarding the regulation of RGC tensile homeostasis. Aim 3 will characterize the molecular mechanisms whereby mechanically induced glial activation influences RGC physiology, thus providing insight into the early inflammatory mechanisms in diseases such as glaucoma. Taken together, the proposed studies may deepen our understanding of retinal function by uncovering new mechanisms that respond to acute and chronic mechanical forces and by reconciling currently disparate hypotheses about retinal pressure transduction. In addition, these studies will aid in the understanding of neurodegeneration that is required to optimize early diagnosis and neuroprotective treatment, which are currently lacking in glaucoma. During the last few years, mutations in putative mechanosensing ion channels have been shown to cause many human diseases and disorders, including severe dysplasias, gliovascular abnormalities and axonal neuropathies but their impact on visual signaling is unknown due to the absence of basic studies. The information provided by these studies may thus contribute insights into mechanosensitive mechanisms that underlie retinal disease as well as transduction of mechanical stress within the CNS.

PROJECT SUMMARY/ABSTRACT Mechanosensitive ion channels were shown to drive mechanically induced inflammatory signaling in the central and peripheral nervous systems and to exacerbate pathology in neuropathies, skeletal diseases, traumatic brain injury and other neurodegenerative conditions. Suppressing their activation with gene knockdown and pharmacology reduces inflammatory neuropathy and neuronal injury yet despite this knowledge their role in neuroinflammation is little understood, and their contribution to pressure-associated retinal diseases such as glaucoma has never been investigated. The goal of the proposed project is to resolve this major gap in knowledge by defining the molecular targets of intraocular pressure in the principal retinal macroglia, the Müller cell, and establish the significance of Müller pressure sensing for neuroinflammation, glia-neuronal interactions and neurodegeneration. The overall hypothesis of this project is that mechanosensitive TRP and piezo channels trigger and drive inflammatory activation in the presence of mechanical stressors such as intraocular pressure and strain and that this process can be targeted by pharmacological/genetic methods to alleviate neuronal injury. We will address this hypothesis in two specific aims. Aim 1 focuses on the characterization of properties of mechanoactivated ion channels that mediate pressure signaling in Müller cells. In Aim 2 we propose to take advantage of new conditional mouse models to elucidate how pressure elevations induce reactive gliosis, the role of mechanotransduction in glia-glia and glia-neuronal circuits, and significance of this mechanism for ganglion cell stress and survival. Successful completion of these multidisciplinary, thematically related yet independent approaches may help define new diagnostic and treatment strategies in hypertensive glaucoma.