BAX to basics: How the BCL2 gene family controls the death of retinal ganglion cells
Introduction
Retinal ganglion cells (RGCs) are long projection neurons that carry visual information from the retina to the brain. They are also the principal cell type affected in optic neuropathies, like glaucoma. Glaucoma is a major blinding disease, with an estimated 60 million people affected worldwide, or approximately 1 person in 40 over the age of 40 years old (Quigley, 2011, Quigley and Broman, 2006). The most relevant risk factor for this disease is an increase in intraocular pressure (IOP), which is suspected to increase strain on the optic nerve head, leading to changes that ultimately cause damage to the RGC axons as they exit through this structure. As a result, the only current treatment for glaucoma is to lower IOP, and while this is effective at slowing the progressive loss of RGCs, it is neither a cure, nor is it successful in a significant proportion of individuals. Research strategies in glaucoma have begun to focus on developing therapies that directly target the health of the affected RGCs. An effective RGC-directed therapy, combined with conventional IOP-lowering treatments, could provide substantially greater beneficial effects to preventing the progression of the disease. Two decades ago, several groups reported that RGCs die using a programmed cell death pathway called apoptosis, in response to both acute (i.e., axotomy) and chronic (experimental glaucoma) damage to the optic nerve (Berkelaar et al., 1994, Garcia-Valenzuela et al., 1994, Garcia-Valenzuela et al., 1995, Quigley et al., 1995). Subsequent to this, studies using genetically engineered mice demonstrated that this form of RGC apoptosis was executed principally using the intrinsic pathway, which involves mitochondrial dysfunction (Li et al., 2000). The realization that RGCs died using an intrinsic genetic program created the opportunity to directly target the biochemical pathways involved in the cell death process, and prevent RGC death (Almasieh et al., 2012). This initiated a flurry of studies aimed at providing protection to the RGCs (termed neuroprotection), which have ranged from blocking extracellular ion channels to the sustained application of neurotrophic factors (Galindo-Romero et al., 2013, Koeberle et al., 2010). While many of these studies have shown promise, the protective effects are universally transient. To date, only a single manipulation of RGCs, the deletion of the pro-apoptotic gene Bax in mice, has provided a virtually permanent blockade of the apoptotic pathway in RGCs after optic nerve damage (Li et al., 2000, Libby et al., 2005b, Semaan et al., 2010). The reason for this may stem from the role of the BAX protein acting as the principal regulator of mitochondrial involvement in the intrinsic apoptotic pathway, an event that is generally considered as the “point of no return” in the cell death process (Chang et al., 2002). As a consequence, the function of this protein acts as a bottle neck through which many different apoptotic pathways must pass through. Since it is likely that the activation of RGC death is a complex and redundant process in glaucoma, we have considered that therapeutic intervention targeting BAX function is the most provocative and effective strategy for neuroprotection.
Successfully targeting any biochemical process requires a detailed understanding of both the molecular mechanisms of the central protein, along with its upstream and downstream molecular players. BAX is a member of a larger gene family based on amino acid domain homologies found in the prototypical member, BCL2. In this review, the BCL2 gene family will be discussed in detail, including the history of its discovery, each of the three protein family members and how these members interact to regulate mitochondrial changes as part of the intrinsic apoptotic program. The mechanisms of BAX function will be discussed, including its activation, dimerization and oligomerization steps, and the consequences of this process on mitochondria and downstream apoptotic events. In this context, we will also discuss the pathophysiology of RGC death from time of injury until cellular phagocytosis. Mouse models used to study optic nerve damage will be used to highlight the BCL2 gene family functions in RGCs, including new data from our lab discussing BAX localization and timeline after optic nerve crush. Finally, we will discuss some of the quintessential findings from Bax-deficient mouse models, and their value as a tool to study RGC pathology.
Section snippets
The BCL2 gene family
A cell line derived from a patient with acute lymphoblastic leukemia was found to contain a t(14:18) chromosomal translocation (Pegoraro et al., 1984), which moved an immunoglobulin enhancer next to a gene located on chromosome 18 that was subsequently named BCL2 (B-cell lymphoma 2). This gene was quite unlike other oncogenes of the time because rather than promote cell proliferation (Tsujimoto et al., 1984), it promoted cell survival (Vaux et al., 1988). A series of studies showed that
The switch to cell death: BAX mechanisms
The initial steps of the intrinsic apoptotic pathway converge at BAX activity, whose function controls the cell's commitment to the pathway by altering the physiology of resident mitochondria. BAX undergoes a series of ordered events leading to pore formation in the MOM, facilitating the release of signaling molecules such as cytochrome c. This definitive event is commonly considered the irreversible step in the apoptotic pathway, or the “switch” from life to death. As the central activator of
Mouse models of optic nerve damage
Virtually everything we know about Bcl2 gene family function in RGCs has been obtained using genetically modified mice. It is relevant to begin this section with a discussion of mouse models of optic nerve damage because Bax-deficient mice have helped inform the temporal sequence of events leading to both the overall pathology of glaucoma, and the more granular activation of the RGC apoptotic program. Retinal ganglion cell degeneration is a process characterized by a series of molecular events
Intrinsic vs. extrinsic apoptosis and the phenomenon of secondary degeneration
The involvement of mitochondria as a primary step in the activation of cell death is the principal distinguishing feature separating intrinsic from extrinsic apoptosis. Extrinsic apoptosis is distinguished by the activity of the tumor necrosis factor (TNF) family of ligands, such as TNFα, FasL, and the TNF-related apoptosis-inducing ligand (TRAIL). These ligands can bind to specialized receptors on cells containing “death domains”. Activated receptors then coalesce into death-inducing signaling
Perspectives and future directions
The study of BAX activation and function has become one of the most important areas of research in the field of apoptosis. While early theories suggested that the pro-apoptotic BAX protein and its anti-apoptotic counterparts were involved in a stoichiometric ballet for survival, two decades of study now show that activation of this protein is a more complicated process involving a third class of proteins carrying the all-important BH3 domain. In neurons, BAX is critically required to execute
Acknowledgements
This work was supported by grants from the National Eye Institute R01 EY012223, P30 EY016665, National Institutes of Health grants T32 GM081061 and S10OD018221. Some of this work was also supported by an unrestricted research support grant and a Senior Scientist Award from Research to Prevent Blindness, Inc. The authors would like to thank Mr. Joel Dietz for help with the Bax-deficient mouse colony, Dr. Gillian McLellan and Ms. Carol Rasmussen for help with the Spectralis OCT imaging, Drs.
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Percentage of work contributed by each author in the production of the manuscript is as follows: Margaret Maes - 50%; Robert Nickells - 50%; Cassandra Schlamp - Conducted some analysis of experimental data and contributed all the graphic work and image processsing of the figures. Dr. Schlamp also edited the manuscript.