Applying proteomics to research for optic nerve regeneration in glaucoma: what’s on the horizon?

KEYWORDS:

• Optic nerve
• proteomics
• neurodegeneration
• neuroregeneration
• glaucoma

Glaucoma, a leading cause of blindness, is a progressive neurodegenerative disease affecting the optic nerve. Multiple pathogenic mechanisms for glaucomatous neurodegeneration have been linked to ocular hypertension-generated mechanical or vascular stress, aging, and genetic/epigenetic risk factors. Following the initial injury to optic nerve axons in glaucomatous eyes, retrograde signals to nerve somas triggers retinal ganglion cell (RGC) apoptosis, distal axon segments separated from cell body and disconnected from synaptic terminals continue degeneration through autonomous processes, and glial cells respond by inflammatory activation that contributes to secondary injury processes at different neuronal compartments from retina to brain. The injury-triggered stress responses, including reactive gliosis that leads to extracellular matrix remodeling and glial scar formation, result in creating an inhibitory environment for neuroregeneration. Accumulating evidence from experimental studies over the past few decades have recognized the reversibility of these processes. A major goal of the glaucoma research today, therefore, includes to uncover the underlying molecular pathways to thereby develop new treatments for neuroprotection, neurorescue, and immunomodulation, while another active direction of research in the field includes optic nerve regeneration [1,2]. Despite increasing molecular information, progressing toward effective treatment of glaucoma remains highly challenging. With respect to complexity of the big picture, combination strategies targeting multiple sites of injury, multiple cell types, and multiple pathways are warranted. Besides reducing the preventable stress stimuli (such as elevated intraocular pressure) and protecting the stressed neurons against injury, stimulation of neuronal repair programs is needed for functional recovery. Undoubtedly, the efficiency of different treatment strategies relies on each other. While RGC axon and soma health depend on each other, axon dysfunction and synapse loss precede overt RGC injury, thereby providing a therapeutic window to stimulate these neurons to regenerate. Obviously, improving the RGC survival is the first critical step toward neuroregeneration, albeit a sustaining need to also defend newly regenerating neurons in glaucomatous eyes.

The glaucoma research exploring the neurodegenerative and neuroinflammatory mechanisms have successfully utilized the quantitative mass spectrometry–based techniques of proteomics that offer a high throughput discovery tool. The large-scale proteomics data collected from human donor samples and animal models have provided a hypothesis-generating framework guiding toward key molecular processes involved in early [3] and advanced stages of this blinding disease [4,5]. Although many studies of glaucoma have focused on the analysis of gene expression and supplied useful information, the proteomics approach presents the advantage to provide posttranslational information directly about gene function and downstream mechanisms operating under injurious conditions. Comparison of proteomics data sets between glaucomatous and control samples have delivered important information about glaucoma-related alterations in protein expression and pinpointed molecular targets for new treatments. Besides shotgun proteomics to establish protein databases and determine increased or decreased protein expression with glaucoma, targeted approaches of proteomics have analyzed posttranslational modifications and interactions of proteins toward specific pathogenic pathways [4,5]. Through the gain- and loss-of-function studies ongoing to test specific molecular outcomes, translation of the proteomics information into better understanding and treatment of glaucomatous neurodegeneration is expected to grow even further. A similar proteomics approach also promises for valuable applications in the neuroregeneration research. The optic nerve regeneration brings about an exciting possibility for glaucoma treatment; however, to regenerate the optic nerve and resume its function seem as challenging as effective neuroprotection in glaucoma. Many experimental studies have used cultures of purified RGCs, organotypic retinal cultures, or in vivo injury models for analysis of molecular events, and gene expression analysis at the site of injury or within RGCs has indicated various molecules to be targeted for promoting the nerve growth [6,7]. Despite some success in yielding the axonal outgrowth from the eye through the optic nerve and across the optic chiasm, the number of regenerating neurons and elicited postsynaptic responses have remained limited [8,9]. Recent advances in optic nerve regeneration hold promise [10]; however, many issues (related to short and long distance growth of axons, axonal projection to appropriate brain targets, and synapse formation for circuit integration to restore visual topography and function) persist. A recent study has also showed that even if the regenerating axons after optic nerve transection formed functional synapses, they might fail to restore visual behavior due to poor conduction associated with the lack of myelination [11]. Thus, the current status of optic nerve regeneration signifies a substantial need to identify additional molecules and pathways for further success. The proteomics approach, when combined with other strategies, has the power to identify previously unrecognized proteins and pathways key to move the optic nerve regeneration research forward.

Proteomics analysis techniques have been used in other fields of neuroregeneration; however, only a few studies have applied the proteomics in optic nerve regeneration research. These studies have analyzed retinal explants for proteomics profiling by a 2D gel-based approach coupled with mass spectrometry for peptide fingerprinting and have discovered some molecules controlling the axon regeneration [12–15]. A more recent study has employed quantitative mass spectrometry to characterize the molecular responses of RGCs to optic nerve crush injury. This study has described a network of injury-responsive pathways that included both previously known players of the regenerative mechanisms and new molecules that might represent novel targets for neuronal repair strategies [16]. It is promising that comparative proteomics analysis in appropriate experimental models can further improve the molecular understanding of why injured optic nerve exhibits regeneration failure in mammals (relative to some lower species, like fish, in which injured optic nerve can regenerate; or relative to peripheral nervous system neurons that can also repair themselves after injury, unlike central nervous system neurons); which molecular pathways should be chosen to activate axonal outgrowth; what are the specific molecules to guide the growing axons along the visual pathways; which molecules should be targeted to establish functional synapses. Indeed, these are the key questions included in the National Eye Institute Audacious Goals Initiative Report covering the optic nerve regeneration [17].

Since the analysis of disease-causing molecular mechanisms ideally requires isolation of specific cell types, proteomics studies of neurodegenerative and neuroinflammatory processes in glaucoma have analyzed the isolated samples of RGCs and glial cells [18,19]. The cell type-specific proteomics data sets have truly supported distinct molecular responses of RGCs and glia in glaucoma. As opposed to prominent stress response and up-regulation of various molecules linked to cell death signaling in RGCs, glial cells have predominantly exhibited cellular activation and inflammatory responses in the glaucomatous retina. Although sample pooling helps obtain sufficient amount of protein sample for quantitative mass spectrometry, currently available instruments enable analysis of the cell type-specific samples isolated from a single eye. Unceasingly advancing technologies should even allow single-cell proteomics in the near future.

Thus, proteomics offers a systematic and cell type-specific approach to portray the molecular pathways that orchestrate optic nerve survival and regeneration. Evidently, cell type-specific molecular information is essential to address multiple obstacles for progress in optic nerve regeneration research. First of all, since RGC subtype specificity is crucial to innervate the right central targets to restore visual function [20], collecting additional information about specific subtypes of RGCs is an obvious need. Expanding the cell type-specific information seems similarly imperative to better understand how glial cells influence the regeneration of injured neurons. Effective manipulation of the inhibitory signals from local glia, and related vascular and inflammatory responses, is as important as targeting the neurons themselves for neuroregeneration [1]. Not only astroglia but also microglia (that similarly play an array of pivotal roles from supplementing the neurosupportive and neurotrophic factors, or promoting the neuronal repair by debris clearance, to stimulation of neurodegenerative inflammation) require specific attention. Moreover, cell type-specific analysis should discover new molecular cues to promote remyelination of the regenerating axons for functional recovery. Because inducing the regenerative ability of neurons and recovering the visual function require manipulation of both neuronal and glial factors simultaneously, proteomics analysis of individual cell types of interest should be particularly useful to gain cell type-specific molecular information for accomplishing this goal.

Further advancing the molecular understanding and effectively translating the proteomics data from animal models to human disease should open up new ways to optic nerve regeneration in glaucoma. As supported by recent studies, combination treatments enhancing several regenerative pathways, such as supplementing the growth stimulators, delivering the pathway and target guidance factors, suppressing the local inhibitory signals, and enhancing the axon conduction, should assure the functional outcome. By manipulating the specific molecules in targeted and temporary fashion, it can be possible to stimulate axonal regrowth and guide the growing axons by attracting or repelling the molecular gradients to select proper targets for functional connections. Local inhibitory signals, as well as aberrant growth and sprouting of the regenerating neurons, may also be suppressed by similar cell type-targeted strategies. Increasingly approaching new delivery systems to target pertinent cell types, such as RGC-specific or glia-specific targeting vectors, along with the recent success of human retinal gene therapy, should shortly enable direct translation of the new data into feasible treatments. Stem cell-based strategies and advancement of nanotechnology-engineered gradient scaffolds should also greatly benefit from the new proteomics information for axon guidance, target selection, and forming functional synapses to restore vision.

Declaration of interest

G. Tezel’s work is supported in part by National Eye Institute, Bethesda, MD (1R21EY024105), and Glaucoma Research Foundation, San Francisco, CA. G. Tezel is also the recipient of the Homer McK. Rees Scholarship in Glaucoma Research, and an awardee of the Peacock Trusts. In addition, the Research to Prevent Blindness Inc., New York, NY provides an unrestricted grant to the Columbia University, Department of Ophthalmology. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

This paper was not funded.