Valery I. Shestopalov, Ph.D.

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Current Research

Retinal Disease Biology

What are the common cellular pathways driving retinal ganglion cells (RGCs) degeneration in glaucoma and other optic neuropathies? We used network analysis of transcriptomic and proteomic changes in human tissues affected by glaucoma to identify glial-specific and neuron-specific pathways activated in glaucoma. These pathology-induced changes have been later confirmed to cause RGC dysfunction and degeneration, and, therefore, represent new drug targets for these blinding diseases.

Inflammasome drives retinal dysfunction and degeneration

My lab has focused on the role of the inflammasome after we have detected activation of this innate immune complex in retinal glia and ganglion cells. We discovered that several inflammasome complexes contribute to neuronal dysfunction and are currently investigating how they mechanistically facilitate pyroptotic and apoptotic signaling in affected retinas. We pioneered the field of retinal inflammasome research by reporting Casp1 activation and IL-1β release in the retina in PLosOne in 2012 and discovered active gasderminD and pyroptotic death of RGCs in acute OHT injury, which we reported in 2019 in Front. Mol.Neurosci. We demonstrated that pathological overactivation of the inflammasome results in cleavage-activation of gasderminD pores that drive neuronal dysfunction and death in the hypertension-affected retinas. We have identified several new therapeutic targets mechanistically linked to mechanosensory regulation and assembly of the inflammasome.

Inflammasome complexes labeled with bioindicator ASC-citrine (green) colocalizes to RGCs (white) and astroglia (red) in the retina and optic nerve after ocular hypertension injuries

Relevant publications

• Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury (2012) PloS ONE, 7(2):e31991
• Pannexin1 sustains the electrophysiological responsiveness of retinal ganglion cells. (2018) Sci. Rep. 
• Inflammasome activation induces pyroptosis in the retina exposed to ocular hypertension injury. (2019) Front. Mol. Neurosci.
• Transgenic inhibition of astroglial NF-kB improves neurological outcome following EAE by suppressing chronic CNS inflammation. J. Immunol. (2009) 182:2628-40
• Effect of g-synuclein silencing on apoptotic pathways activation in retinal ganglion cells. J.Biol.Chem (2008), 283(52):36377-85.

Modulation of the disease-causing signaling

Provided that inactivation of inflammasome activity protected the functionality and viability of retinal ganglion neurons, we started testing if blocking inflammasomes and downstream toxicity pathways can suppress glaucomatous degeneration and provide a new therapeutic strategy in glaucoma and mechanical optic nerve injuries. Here, we also focus on identifying the key upstream regulator and downstream effector pathways, mechanosensor and signaling molecules that activate neuronal and glial inflammasomes and induce gasderminD pore formation. We have designed and started testing several classes of pathway-modulating compounds: sdRNAi, peptide-based drugs, and AAV2 vector-driven gene modulation. Our preliminary data showed that these interventions preserved vision in animal models of glaucoma, retinal ischemia, and traumatic optic nerve injury.

The Lab is actively testing a trans-corneal Coulomb-Controlled Iontophoresis (CCI) delivery method, invented by Dr. Jean-Marie Parel at BPEI. This non-invasive alternative to intraocular injections is actively driving small molecule drugs, nanoparticles, and RNAi compounds into the back of the eye, to the retina, and optic nerve. Our pilot studies showed that our therapeutic RNAi compounds suppress the inflammasome, providing structural and functional protection to the retina when delivered non-invasively by the intraocular CCI. The feasibility was demonstrated in different disease models, including lacrimal gland degeneration (1) and two models of human glaucoma. Our therapeutic RNAi/iontophoresis platform targets key components of pro-inflammatory pathways and can be applied as a part of a combination treatment of glaucoma or as a new stand-alone therapy.

Relevant Publications

• Manipulation of Panx1 Activity Increases The Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells. (2017) IOVS, 1;58(13):5654-5665. doi: 10.1167/iovs.17-22071
• Pannexin 1 enhances Axonal Growth in Mouse Peripheral Nerves. (2017) Front. Cell. Neurosci. 22;11:365. doi: 10.3389/fncel.2017.00365
• Topical RNAi Drug Delivery to the Posterior of the Eye. Abstr. Dept. of Defense Research Conference, MEEI/Harvard, Boston, MA, March 2017.

Systems biology analysis of glaucoma-induced changes in the retina and optic nerve

My Lab successfully applied network analysis, a systems biology approach, to screen multi-omics data from patient tissues, affected by glaucoma and identify pathways mechanistically implicated in disease initiation and progression.

Relevant Publications

• Transgenic inhibition of astroglial NF-kB protects from optic nerve damage and retinal ganglion cell loss in experimental optic neuritis. (2012) J. Neuroinflammation 10;9:213
• Preliminary Quantitative Proteomic Characterization of   Glaucomatous in vivo Rat Retinal Ganglion Cells, (2010) Exp. Eye Res. 91:107-10.
• Network Analysis of Primary Optic Nerves Astrocytes in Human Glaucoma. (2009) BMC Med. Genomics, 2:24
• Effect of g-synuclein silencing on apoptotic pathways activation in retinal ganglion cells. J.Biol.Chem (2008), 283(52):36377-85.
• Differential gene expression profiling of large and small retinal ganglion cells. J. Neurometh. (2008) 174:10-7.
• Quantifying Retinal Nerve Fiber Layer Thickness in Whole-mounted Retina. (2006) Exp. Eye Res. 83:1096-10
• Microarray analysis of gene expression in adult retinal ganglion cells. (2006) FEBS Letts, 580:331-335 

Surface receptors and channels in transcriptional regulation of innate immune responses in the retina

Cell-cell signaling via surface receptors and channels is essential for coordinated responses of retinal neurons to changes in activity, metabolism, and the environment. The onset of glaucomatous pathology, most commonly associated with an increase of IOP,  perturbs this signaling and metabolite/ionic balance in RGCs to trigger various stress responses, including activation of an innate immune complex of the inflammasome. Pannexin1, TRPV, and purinergic P2X channels at the cell surface comprise the key integral part of neuronal signalosome that senses changes in IOP and danger signals.

Relevant Publications

• Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways. (2016) J. Neuroinflamm. Apr 28;13(1):93. doi: 10.1186/s12974-016-0558-y.
• Mechanosensitive release of ATP through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for chronic extracellular ATP elevation with sustained mechanical strain. (2014) Glia 62(9):1486-501
• Membrane receptors, channels, and mechanosensitivity: a role in facilitating RGC death in optic neuropathies and retinal ischemia. (2013) Curr. Eye Res. 39(2):105-19.
• b1 integrin-focal adhesion kinase (FAK) signaling modulates RGC survival. (2012) PloS ONE, 7(10):e48332.
• Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury (2012) PloS ONE, 7(2):e31991.
• Toll-Like Receptor 4 contributes to retinal ischemia/reperfusion injury. (2010) Mol. Vis. 16:1907-12.
• Inactivation of astroglial NF-κB promotes survival of retinal neurons following ischemic injury. (2009) Eur. J. Neurosci. 30:175-85
• Transgenic inhibition of astroglial NF-κB improves neurological outcome following EAE by suppressing chronic CNS inflammation. J. Immunol. (2009) 182:2628-40
• Network Analysis of Primary Optic Nerves Astrocytes in Human Glaucoma. (2009) BMC Med. Genomics, 2:24
• Expression of pannexins in the retina. (2006) FEBS Letts, 580:2178-2182

Pannexin 1 in neuronal remodeling and regeneration

These new pathogenic functions of Panx1 came into the focus of my research in 2006, when we discovered a significant enrichment of these channels in the brain and retina, particularly in CNS and PNS neurons and in retinal ganglion cells. Activation of these channels is implicated in electrophysiological activity of RGCs, synaptic physiology of motor neurons and taste cells, neuronal precursor differentiation and recruitment, and axonal post-injury remodeling.

Relevant Publications

• Mechanism of p2x7 receptor-dependent enhancement of neuromuscular transmission in pannexin 1 knockout mice (2018). Purinergic Signal. 14(4):459-469; doi: 10.1007/s11302-018-9630-7;
• The two faces of pannexin: new roles in inflammation and repair. (2018) J. Inflam. Res. 11:273-288. doi: 10.2147/JIR.S128401
• Pannexin 1 plays dual roles in the neural precursor cell response following photothrombotic stroke. (2016) J.Neurosci. 36(4):1203-1210; 
• Role of Pannexin1 in purinergic regulation of synaptic transmission in mouse motor synapses. (2017) Biomembranes (Rus) 34:48-57
• Pannexin 1 enhances Axonal Growth in Mouse Peripheral Nerves. (2017) Front. Cell. Neurosci. 11:365. doi: 10.3389/fncel.2017.00365
• Pannexins are potential new players in the regulation of cerebral homeostasis during the sleep-wake cycle. (2017) Front. Cell. Neurosci. 11:210. doi 10.3389/fncel.2017.00210.
• Sleep-wakefulness cycle and behavior  in pannexin-1 knockout mice. (2017) Behavior. Brain Res. 1;318:24-27, DOI: 10.1016/j.bbr.2016.10.015 
• Dispensable ATP permeability of Pannexin 1 channels in a heterologous system and in mammalian taste cells. (2012)  J. Cell. Sci. 125: 5514-23   (PMCID: PMC3561859)
• Unified patch clamp protocol for the characterization of Pannexin 1 channels in isolated cells and acute brain slices. (2011) J. Neurosci. Meth. 199(1):15-25
• Panx1 activity is dispensable for olfactory function. (2014) Frontiers in Cellular Neuroscience, 8:266 
  The Role of Pannexin 1 Hemichannels in Inflammation and Regeneration. Frontiers in Physiology, (2014), 25;5:63.
• Pannexin1 stabilizes synaptic plasticity and is needed for learning. (2012) PloS ONE, 7(12):e5176

Pannexin1 in vascular endothelial physiology

Panx1 is abundant in the smooth muscle cells in smaller arteries and arterioles. In arteries, the release of ATP via Panx1 hemichannels was shown to be involved in the incremental contractile response during adrenoceptor activation. Unexpectedly, we discovered that the pattern of Pannexin1 expression and its impact on function depends on the size of blood vessels. In this collaborative project, we have shown that in the saphenous artery (a larger resistance-type vessel) Panx1 is expressed predominantly in the endothelium, and Panx1-/- mice lacking this channel have significantly impaired endothelial function. This project demonstrated that that Panx1 facilitates endothelium-dependent relaxations via regulation of one or several endothelium-derived hyperpolarization mechanisms.

Relevant Publications

• Regulation in Mesenteric Arteries of Pannexin-1-Knockout Mice. (2017) Biomembranes (Rus) 34:137-146
• Pannexin 1 facilitates arterial relaxation via an endothelium-derived hyperpolarization mechanism. 2015 FEBS Letts. 28;589(10):1164-70. doi: 10.1016/j.febslet.2015.03.018
• Endothelial function is impaired in conduit arteries of Panx1 knockout mice. (2014) Biology Direct, 9: 8, (PMCID:PMC4046076)
• Pannexin 1 facilitates arterial relaxation via an endothelium-derived hyperpolarization mechanism. 2015 FEBS Letts. 28;589(10):1164-70. doi: 10.1016/j.febslet.2015.03.018.
• Alterations of Purinergic Regulation in Mesenteric Arteries of Pannexin-1-Knockout Mice. (2017) Biomembranes (Rus) 34:137-146
  

Regulation of astroglial toxicity in retinal disorders

Glial-induced neuroinflammation and neurotoxicity is the most common response to stress and injury, with distinct molecular and cellular mechanisms contributing to disease initiation and progression. The understanding of molecular and cellular mechanisms underlying disease initiation and progression involved systems analysis of astrocyte responses to glaucoma and acute ocular hypertension. Activation of the pro-inflammatory network was detected by systems analysis of astrocyte responses to glaucoma, EAE and acute ocular hypertension. By using pathways informatics, we correlateed transcriptomic changes with the neurotoxicity of activated glia. In silico metadata validation in conditional knockout models identified neuro-inflammatory pathways regulated by NF-kappaB factor, complement system, and Panx1 as the key pathways of astroglial toxicity.

Relevant Publications

• Network Analysis of Primary Optic Nerves Astrocytes in Human Glaucoma. (2009) BMC Med. Genomics, 2009 9;2:24. (PMCID: 2705386)
• Inactivation of astroglial NF-κB promotes survival of retinal neurons following ischemic injury. (2009) Eur. J. Neurosci. 30:175-85 (PMCID: PMC2778328)
• Transgenic inhibition of astroglial NF-kB protects from optic nerve damage and retinal ganglion cell loss in experimental optic neuritis. (2012) J. Neuroinflammation 10;9:213 (PMCID:PMC3490907)
• Retinal glial responses to optic nerve crush are attenuated in Bax-deficient mice and modulated by purinergic signaling pathways. (2016) J. Neuroinflamm.  28;13(1):93. (PMCID:  PMC4850653)

Molecular pathways of cell-cell communication in the lens 

The lens of the eye is a marvel of biological engineering. It has the ability to change shape or “accommodate” in order to focus light, making it possible to see both near and far objects clearly. This is possible because the lens is made up of living cells, which provide flexibility and elasticity. A problem, however, is that most cells are not transparent. To also ensure their crystal clarity, lens cells must sacrifice the light-scattering organelles interconnect to maintain the ability to feed themselves. These connections are mediated by gap junctions, hemichannels, and fusion pores, which we discovered in 2002. More recently, we were the first to characterize pannexin hemichannels in the retina and lens. We investigated the role of these pathways in the context of ocular tissue homeostasis and in pathology. 

Relevant Publications

• Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core. J. Cell Sci. v 113, 1913-1921 *reviewed by Nature News,  http://www.nature.com/news/2000/000525/pf/000525-2_pf.html  (PMID: 10806102, free access)
• Development of a macromolecular diffusion pathway in the lens. (2003) J Cell Sci. 15, p.4191-9  (PMID:12953070, free access)
• Lens Connexins α3/Cx46 and α8/Cx50 interact with Zonula Occludens Protein-1 (ZO-1). Mol. Biol. Cell, v. 14, 2470-81  (PMCID: PMC194895)
  

Ocular surface microbiome

This project was initiated in 2009 as a collaboration between the Bascom Palmer Eye Institute and the Argonne National Laboratory. We sought to characterize normal ocular surface microbiota across different genders and age groups. The project received funding from the NIH NEI in 2010-2015; a multi-PI R01 research grant in collaboration with Dr. R. Van Gelder, Department of Ophthalmology at the University of Washington, Seattle. This study has characterized low-biomass viral and microbial metagenomes at the human ocular surface as well as changes induced by ocular surface infections and diseases.

 

Relevant Publications

• Diversity of bacteria at healthy human conjunctiva. (2011) IOVS. 52(8):5408-13 (PMCID: PMC3176057)
• TUIT, a novel BLASN-based algorithm for 16S rRNA sequencing data analysis. (2013) BioTechniques, 56:78–84 (PMCID: PMC4186660)
• Paucibacterial microbiome and resident DNA virome of the healthy conjunctiva. Invest Ophthalmol Vis Sci. 2016 Oct 1;57(13):5116-5126.