Research

Eye Research

Overview: Visual information is conveyed from the eye to the brain via the optic nerve which contains the axons of the retinal ganglion cells (RGCs). Damage to RGCs and the optic nerve causes permanent vision loss, for example in glaucoma, ischemic optic neuropathies, and traumatic injury. Notably, glaucoma is expected to affect ~80 million people worldwide by 2020, of whom ~10% are predicted to go blind. There are currently no approved medicines or therapies to restore vision after RGC and optic nerve damage, as unlike axons in the peripheral nervous system, RGC axons in the adult mammal do not spontaneously regenerate when damaged. In addition, damage to the optic nerve can result in retrograde degeneration and apoptotic death of the associated RGCs. We are studying the basic cell biology of the RGC in order to discover signaling pathways that regulate RGC survival and optic nerve healing and regeneration in disease. We expect to derive novel insights into RGC biology and develop novel therapeutics to prevent loss of or restore vision. RGCs are central nervous system neurons and share cellular mechanisms with neurons in the brain. Our research should have relevance not just in the field of ophthalmology, but to neurology in general. For example, axon regeneration is a critical issue in human stroke which often involves the white matter.

cAMP Signaling in the RGC

One intracellular signaling pathway whose upregulation can promote RGC neuroprotection and axonal regeneration is that mediated by the second messenger cAMP and its effector Protein Kinase A (PKA). Numerous studies have shown that treatment with cAMP elevating agents promotes RGC survival and restoration of optic nerve structure and function after optic nerve crush, a relevant surgery model for traumatic optic neuropathy. While cAMP is by itself not a strong inducer of optic nerve regeneration, cAMP potentiates the effects of other regenerative agents, including neurotrophic factors like brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF). A major interest of the Kapiloff lab is to discover the mechanisms how cAMP signaling promotes neuroprotection and axonal regeneration in the RGC, including how cAMP is synergistic with neurotrophic factors. Recent work suggests the scaffold protein mAKAPα is a nodal regulator of cAMP signaling in the RGC relevant to these processes. Using live cell imaging and cAMP and PKA activity FRET based reporters targeted to mAKAPα complexes, we are exploring how adenylyl cyclase and phosphodiesterase anchored by mAKAPα specifically regulate cAMP fluxes at mAKAPα signalosomes in cultured neurons. In parallel, we are investigating how cAMP at mAKAPα signalosomes specifically contributes to neuroprotection and axonal regeneration both in vitro in hippocampal neurons and in vivo after optic nerve crush, a model of retrograde RGC degeneration.

RGC Gene Expression

Several transcription factors have been implicated in the regulation of neuroprotection and axonal regeneration, including MEF2. In addition, we have found that class IIa histone deacetylases regulate retinal ganglion cell survival after injury. Using state-of-the-art transcriptomics approaches, we are studying how gene expression in the retinal ganglion cell is regulated by signal-dependent transcription factors and histone deacetylases and how this gene expression can be modulated by gene therapy to preserve vision in disease.

Cardiac Research

Overview: Left ventricular hypertrophy is a major risk factor for the development of heart failure, a syndrome of great public heath significance, contributing to 300,000 deaths each year in the U.S. alone. At the cellular level, myocyte hypertrophy is the primary response of the heart to chronic stress. In disease, this non-mitotic growth is accompanied by changes in gene expression, ion fluxes and metabolism that can affect cardiac contractility and induce myocyte apoptosis and interstitial myocardial fibrosis. This pathologic remodeling is the proximate cause of heart failure and the associated morbidity and mortality. Basic questions being asked in the Kapiloff lab include how pathological concentric and eccentric hypertrophy are differentially regulated and whether myocyte hypertrophy can be regulated independently of interstitial fibrosis in disease. We are interested in identifying signaling molecules that might be therapeutically targeted to prevent the pathological cardiac hypertrophy that leads to heart failure.

Concentric vs. Eccentric Hypertrophy

Cardiac hypertrophy at the whole-organ level reflects non- mitotic growth of the cardiac myocytes. The roughly cylindrical adult myocyte can grow either in width (diameter) or length, resulting in thickened ventricular walls or chamber dilation, respectively. In theory, concentric myocyte growth increases the width of cardiomyocytes, inducing parallel assembly of sarcomeres and thereby reducing ventricular wall stress (Law of LaPlace). In contrast, eccentric myocyte growth increases cardiomyocyte length, inducing serial addition of sarcomeres to accommodate greater ventricular volumes without stretching individual sarcomeres beyond the optimum length for contraction (Frank-Starling Law). In pressure overload diseases, such as aortic stenosis or hypertension, there is increased systolic wall stress, and concentric hypertrophy initially predominates. In volume overload diseases, such as following a myocardial infarction or dilated cardiomyopathy, eccentric hypertrophy predominates, presumably in response to increased diastolic wall stress. A fundamental question being addressed in the Kapiloff lab is what confers the differential growth of the myocyte in width and length. We are identifying myocyte signaling pathways and transcription factors that are central to this phenotypic switch.

mAKAP

A major focus of our laboratory has been the characterization of multimolecular signaling complexes organized by the scaffold protein mAKAPβ in cardiac myocytes. We have shown that at the nuclear envelope, mAKAPβ “signalosomes” transduce cAMP, mitogen-activated protein kinase, Ca2+, and hypoxic signals regulating the transcription factors NFATc, MEF2 and HIF-1α. In Vitro and in vivo data have revealed that mAKAPβ signalosomes convey signals required for pathological myocyte hypertrophy and the development of heart failure. We are continuing to study signaling by mAKAPβ complexes, including the ongoing characterization of both past and newly identified mAKAP binding partners, in particular signaling related to RSK3 and calcineurin and the regulation of MEF2 and type IIa HDACs.

Calcineurin

Anchoring by scaffold proteins can confer isoform-specific enzyme regulation, dedicating specific signaling enzyme isoforms to individual cellular processes. The Ca2+/calmodulin-dependent, serine/threonine phosphatase calcineurin (CaN) plays a central role in the regulation of pathological myocyte hypertrophy. In cardiac myocytes, there are two different isoforms of the CaN catalytic subunit, Aα and Aβ, and yet, as shown originally by the laboratory of Jeffrey Molkentin, only Aβ is required for myocyte hypertrophy. Because CaN isoforms share a similar substrate specificity and common binding partners, it is unclear why there would be functional differences between the isoenzymes. Through a screen for CaNAβ-specific scaffold proteins, we have identified a new mode of CaNAβ-specific anchoring. Ongoing work in the Kapiloff lab is revealing how some of these scaffolds are relevant to the regulation of myocyte growth.

Heart Failure Therapeutics

Our research into mAKAPβ complexes has led to the identification of novel targets for the treatment of heart failure. For example, we discovered that type 3 p90 ribosomal S6 kinase (RSK3) is required for pathological remodeling in vivo. RSK3’s unique N-terminal domain confers high affinity, regulated binding to mAKAP, defining a novel protein-protein interaction that explains the selective binding of that kinase isoform to the scaffold. We are currently developing the use of adeno-associated virus gene therapy vectors to block pathological hypertrophy and heart failure in vivo via expression of RSK3 anchoring disruptor peptides. In parallel, we are developing additional gene therapy vectors to directly target the mAKAPβ scaffold. Together these studies are revealing new basic signal transduction mechanisms, while at the same time being part of a translational pipeline aimed at developing new clinically effective therapies for heart failure. (Disclosure: Dr. Kapiloff owns equity in Cardiac RSK3 Inhibitors, LLC, a company interested in developing mAKAPβ /RSK3-targeted therapeutics.)

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The Kapiloff lab performs basic and translational research in the eye and the heart.

Eye Research

Cardiac Research