Glaucoma and the brain: a novel approach towards retinal ganglion cell protection

Summary PhD thesis Eline Dekeyster Glaucoma refers to a group of optic neuropathies, all characterized by progressive degeneration of retinal ganglion cells (RGCs) - the cells that send visual information from the eye to the brain - resulting in a gradual loss of vision. Ocular hypertension (OHT) is considered one of the major risk factors for development and progression of glaucoma, and thus lowering eye pressure through topical application of hypotensive eye drops or surgery, is the cornerstone of glaucoma therapy today. Unfortunately, a number of patients do not benefit from such treatments, and although it generally slows down disease progression, controlling eye pressure does not really halt RGC degeneration. This stresses the need for development of new therapies aimed at long‑term retinal neuroprotection and preservation of vision. Within this context, this PhD project intends to contribute to the knowledge on the pathological mechanisms underlying glaucomatous RGC death and to highlight possible novel strategies towards RGC protection. The first part of this thesis was dedicated to the optimization and characterization of two mouse models of glaucoma: the OHT model and the normotensive optic nerve crush (ONC) model, each focusing on different aspects of the disease. Next, a closer look was taken at the link between glaucoma and the brain. Although long considered purely an eye disease, glaucoma is increasingly becoming recognized as a disease of the entire visual system. Using the OHT model, the effect of elevated eye pressure on the central visual brain areas was evaluated. Reduced RGC synapse density, retinotopically correlated to degeneration of RGC soma, and intensive but transient astroglial reactivity were observed in the main subcortical RGC projection area, which for mice is the superior colliculus (SC). Furthermore, diminished neuronal activity in the primary visual cortex extending to specific extrastriate areas was seen early after OHT induction. Complete recovery of cortical activity over time demonstrated the capacity of the adult visual cortex to functionally reorganize in an attempt to adapt to glaucomatous RGC degeneration. Our results uncovered for the first time effects on visual cortex activity patterns in a murine OHT model and, yet again, highlight the importance of including the brain in glaucoma research. For their survival, RGCs depend on neurotrophic factors, e.g. neurotrophins, produced locally in the eye as well as in the projection areas in the brain, from where they are retrogradely transported along the RGC axons towards the cell bodies in the retina. According to the neurotrophin deprivation hypothesis, diminished retrograde delivery of neurotrophic support during an early stage of glaucoma pathogenesis is one of the main triggers that induce apoptotic signaling in RGCs. Therefore, interfering with neurotrophic signaling seems an attractive tool to achieve neuroprotection. In this PhD project, the well-known neurotrophin brain-derived neurotrophic factor (BDNF) was chosen as the lead molecule to study the role of neurotrophic factors in glaucoma. The levels of BDNF and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), were examined in the retina and SC of mice subjected to OHT or ONC. Both models differentially influenced BDNF and TrkB levels. Defining a specific role for BDNF signaling within glaucoma pathology remains difficult as various studies using a variety of animal models of glaucoma have yielded unique results, including those presented within this manuscript. In line with the hypothesis of deprived neurotrophic support being an important contributor to glaucomatous RGC death, exogenous neurotrophin administration to the eye has been shown to reduce loss of RGCs; however, the neuroprotective effect was mostly transient and insufficient for sustained RGC survival. Therefore, we hypothesized that treatment at the level of extraretinal neurotrophin sources in the brain might be beneficial, as target-derived neurotrophins are likely to induce signaling pathways that diverge from local neurotrophin signaling. Brain-directed treatment was approached in two ways: 1) viral vector-mediated upregulation of BDNF in the SC was used to boost retrograde delivery of BDNF to the retina; 2) a more broad strategy to optogenetically increase neuronal activity in the SC was implemented to enhance production of a whole spectrum of endogenous neurotrophic factors. In light of these hypotheses, viral vector technology was optimized for use in the mouse SC. Although the previously reported temporary neuroprotective effect of intravitreally-delivered recombinant BDNF was confirmed, viral vector-induced overexpression of BDNF in the SC did not lead to protection of the RGCs in our glaucoma models. This unfortunate result most likely resulted from decreased neurotrophin responsiveness upon vector mediated BDNF overexpression. Regarding the second strategy employing SC manipulation to aim for RGC protection, optogenetic methods were introduced and validated. The basic principle underlying optogenetics is the introduction of genes, e.g. through viral vector technology, encoding light-sensitive ion channels or opsins into cells, rendering them responsive to light of a specific wavelength. For the first time, the application of an opsin with specifically slow kinetic properties, the stabilized step-function opsin (SSFO), was validated for use in the mouse SC. A setup for chronic optogenetic stimulation in awake, freely moving animals, was developed, and shown to effectively stimulate collicular activity. Two consecutive behavioral responses were observed upon unilateral SC stimulation, a short escape response, followed by prolonged pursuit-like behavior. In conclusion, a large component of this thesis was dedicated to method optimization, such as the two mouse models of glaucoma, viral vector technology, and a setup for in vivo optogenetic stimulation. These methods will importantly contribute to future research within the research group. Furthermore, new findings on the role of higher order visual brain centers in glaucoma pathogenesis were discovered. Although we were not able to deliver a proof-of-concept for the neurotrophin deprivation hypothesis, important insights concerning the complexity of neurotrophic factor treatments were highlighted.1 General introduction; 2 Mouse models for glaucoma research; 3 Effects of ocular hypertension on the superior colliculus and visual cortex of mice; 4 Characterization of viral vectors for transgene expression in the mouse superior colliculus; 5 A role for brain-derived neurotrophic factor in glaucoma; 6 Optogenetic stimulation in the mouse superior colliculus; 7 General conclusions and future perspectivesnrpages: 217status: publishe

Automated neuronal counting in retinal whole mounts: an ImageJ-based solution

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Glaucoma from eye to brain: are MMP-2 and MMP-14 involved?

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The zebrafish as a gerontology model in nervous system aging, disease, and repair

Considering the increasing number of elderly in the world’s population today, developing effective treatments for age-related pathologies is one of the biggest challenges in modern medical research. Age-related neurodegeneration, in particular, significantly impacts important sensory, motor, and cognitive functions, seriously constraining life quality of many patients. Although our understanding of the causal mechanisms of aging has greatly improved in recent years, animal model systems still have much to tell us about this complex process. Zebrafish (Danio rerio) have gained enormous popularity for this research topic over the past decade, since their life span is relatively short but, like humans, they are still subject to gradual aging. In addition, the extensive characterization of its well-conserved molecular and cellular physiology makes the zebrafish an excellent model to unravel the underlying mechanisms of aging, disease, and repair. This review provides a comprehensive overview of the progress made in zebrafish gerontology, with special emphasis on nervous system aging. We review the evidence that classic hallmarks of aging can also be recognized within this small vertebrate, both at the molecular and cellular level. Moreover, we illustrate the high level of similarity with age-associated human pathologies through a survey of the functional deficits that arise as zebrafish age.publisher: Elsevier articletitle: The zebrafish as a gerontology model in nervous system aging, disease, and repair journaltitle: Ageing Research Reviews articlelink: http://dx.doi.org/10.1016/j.arr.2015.10.004 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe

MMPs in the trabecular meshwork: promising targets for future glaucoma therapies?

Glaucoma is one of the world's most common blinding diseases, affecting more than 60 million people worldwide. Although the disease presents as a neurodegenerative disorder affecting retinal ganglion cell axons in the optic nerve and their somata in the retina, the elicitors of this optic neuropathy are often located outside the neuroretina. Disturbances in aqueous humor outflow, leading to ocular hypertension, are considered to be the major risk factor for the development of glaucoma. Although an amplitude of pharmacological and surgical measures is available to lower IOP in glaucoma patients, these are not always sufficient to halt the disease. Multiple surveys in glaucoma patients, as well as in vitro studies in anterior segment explant or cell cultures, reported changes in the expression and activity of several matrix metalloproteinases (MMPs) in the aqueous humor and trabecular meshwork, in response to elevated IOP. In this review, we describe MMPs as important modulators of aqueous humor outflow, functioning in a feedback mechanism that continuously remodels the trabecular meshwork extracellular matrix composition in order to maintain a stable outflow resistance and IOP. We review the evidence for the involvement of MMPs in glaucoma disease onset and investigate their potential as therapeutic targets for the development of future glaucoma therapies.status: publishe

Prolonged optogenetic stimulation of the mouse superior colliculus elicits escaping and orienting behaviour

Aims: Electrical and pharmacological stimulation of the superior colliculus (SC) has been described to mediate a variety of behaviours ranging from freezing, escape and orientation. Here, we investigated the effect of prolonged optogenetic stimulation of the SC. Methods: We used the stable step-function opsin (SSFO), a modified ChR2 channel, which stays active for 20-30 minutes. The right SC of C57Bl6J mice was stereotactically injected with an AAV2/7-CaMKII-SSFO vector. Stimulation consisted of a 2s blue light pulse of 50 mW/mm2on day 1, 3 and 5 and a different batch of animals was stimulated with light powers ranging from 0,05 mW/mm2 to 50 mW/mm2. Results: Upon stimulation with the highest light power, all mice displayed strong escape behaviour at first, running around in their cage. After about 30 seconds they quieted down and showed a counter-clockwise turning behaviour for up to 30 minutes. Using lower light power, there was no escape response , leaving only the long-lasting contralateral orientation turning. Conclusions: At high light powers, SSFO-mediated SC stimulation resulted in a short escape response, despite the prolonged nature of the stimulation that is indicated by the long-lasting turning behaviour. These findings suggest that the circuit for escape behaviour might respond mainly to stimulus onset and loses its responsiveness upon prolonged stimulation. Furthermore, as lower light powers only elicited turning behaviour, stimulus size appears to be an important factor in triggering escape but not orientation. In summary, our data indicate differential response properties of the collicular circuits mediating escape and orientation.status: publishe

MMPs in the Neuroretina and Optic Nerve: Modulators of Glaucoma Pathogenesis and Repair?

Multiple studies in glaucoma patients and in animal models of spontaneous and experimentally-induced glaucoma, reported changes in the expression and activity of several matrix metalloproteinases (MMPs) in the retina, optic nerve, aqueous humor, and trabecular meshwork. These data have led to the hypothesis that MMPs might be involved in glaucoma onset and/or disease progression. However, reports are conflicting and research aiming at providing a clear definition of their causative role is lacking. In glaucoma, MMPs are thought to act at two different levels. In the trabecular meshwork, they fine-tune the aqueous humor outflow rate and intraocular pressure, in the neuroretina and optic nerve, however, their role during glaucoma disease progression is much less clear. This review provides a comprehensive overview of the research conducted on the expression and function of MMPs in the retina and optic nerve, and on the elucidation of their potential involvement during glaucoma pathogenesis. Additionally, we describe the insecure balance between detrimental and potential beneficial MMP activities during central nervous system recovery and how MMP-based therapies could help to overcome the current pitfalls in the development of retinal ganglion cell neuroprotection and axon regeneration approaches for the treatment of glaucoma.status: publishe

Unravelling mouse behaviour upon optogenetic activation of the superior colliculus

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SSFO-mediated optogenetic activation of the mouse superior colliculus results in a repeatable behavioral response

The superior colliculus (SC) is a conserved midbrain structure responsible for reflexive behavioral responses to external stimuli, such as escaping predators and pursuing objects of interest. Unilateral electrical stimulation of the SC has been shown to elicit ipsilateral (escaping) and contralateral (pursuing) movements. To further investigate the response properties of these circuits, we revisited these experiments using an optogenetic approach. In particular, we used the stabilized step function opsin SSFO (ChR2 C128S/D156A), which has slow channel kinetics, allowing it to remain in an open state for 20 – 30 minutes. As such, a short pulse of blue light results in prolonged depolarization of SSFO-expressing cells. SSFO was introduced via viral vector injection in the right SC of C57Bl6J mice, incorporating it in neurons of both the superficial and deeper layers of the SC. Provided with blue light stimulation, mice repeatedly showed a strong behavioral response. At first, they ran around in their cage for 20 – 30 seconds as if escaping from a non-existing threat. After quieting down, mice displayed characteristic movements consisting of large counterclockwise body turns, which became shorter and less frequent as time progressed. Nevertheless, a preference for counterclockwise turning was detectable up to 30 minutes after stimulation of the right SC. Notably, repeating these stimulations yielded a similar result, indicating a reproducible activation. In summary, SSFO-mediated optogenetic stimulation can directly elicit a complex and prolonged behavioral response, including escape- and pursuit-like movements, corresponding to what has been observed upon electric stimulation. Moreover, the timing of this behavioral response closely mimics the duration of the SSFO ‘open state’ conformation. Taken together, we demonstrated the potential of optogenetics to repeatedly activate the natural functions of neuronal circuits for a prolonged period of time. Future experiments will investigate the effect of SC layer- and cell type-specific stimulation to further elucidate the way orienting behavior is governed.status: publishe

Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains

Mouse disease models have proven indispensable in glaucoma research, yet the complexity of the vast number of models and mouse strains has also led to confusing findings. In this study, we evaluated baseline intraocular pressure, retinal histology, and retinofugal projections in three mouse strains commonly used in glaucoma research, i.e. C57Bl/6, C57Bl/6-Tyr(c), and CD-1 mice. We found that the mouse strains under study do not only display moderate variations in their intraocular pressure, retinal architecture, and retinal ganglion cell density, also the retinofugal projections to the dorsal lateral geniculate nucleus and the superior colliculus revealed striking differences, potentially underlying diverging optokinetic tracking responses and visual acuity. Next, we reviewed the success rate of three models of (glaucomatous) optic neuropathies (intravitreal N-methyl-d-aspartic acid injection, optic nerve crush, and laser photocoagulation-induced ocular hypertension), looking for differences in disease susceptibility between these mouse strains. Different genetic backgrounds and albinism led to differential susceptibility to experimentally induced retinal ganglion cell death among these three mouse strains. Overall, CD-1 mice appeared to have the highest sensitivity to retinal ganglion cell damage, while the C57Bl/6 background was more resistant in the three models used.publisher: Elsevier articletitle: Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains journaltitle: Experimental Eye Research articlelink: http://dx.doi.org/10.1016/j.exer.2016.01.006 content_type: article copyright: © 2016 Elsevier Ltd. All rights reserved.status: publishe