Ilse Bollaerts

Neuroinflammation as Fuel for Axonal Regeneration in the Injured Vertebrate Central Nervous System

Damage to the central nervous system (CNS) is one of the leading causes of morbidity and mortality in elderly, as repair after lesions or neurodegenerative disease usually fails because of the limited capacity of CNS regeneration. The causes underlying this limited regenerative potential are multifactorial, but one critical aspect is neuroinflammation. Although classically considered as harmful, it is now becoming increasingly clear that inflammation can also promote regeneration, if the appropriate context is provided. Here, we review the current knowledge on how acute inflammation is intertwined with axonal regeneration, an important component of CNS repair. After optic nerve or spinal cord injury, inflammatory stimulation and/or modification greatly improve the regenerative outcome in rodents. Moreover, the hypothesis of a beneficial role of inflammation is further supported by evidence from adult zebrafish, which possess the remarkable capability to repair CNS lesions and even restore functionality. Lastly, we shed light on the impact of aging processes on the regenerative capacity in the CNS of mammals and zebrafish. As aging not only affects the CNS, but also the immune system, the regeneration potential is expected to further decline in aged individuals, an element that should definitely be considered in the search for novel therapeutic strategies.

Matrix metalloproteinases as promising regulators of axonal regrowth in the injured adult zebrafish retinotectal system

Overcoming the failure of axon regeneration in the mammalian central nervous system (CNS) after injury remains a major challenge, which makes the search for proregenerative molecules essential. Matrix metalloproteinases (MMPs) have been implicated in axonal outgrowth during CNS development and show increased expression levels during vertebrate CNS repair. In mammals, MMPs are believed to alter the suppressive extracellular matrix to become more permissive for axon regrowth. We investigated the role of MMPs in axonal regeneration following optic nerve crush (ONC) in adult zebrafish, which fully recover from such injuries due to a high intrinsic axon growth capacity and a less inhibitory environment. Lowering general retinal MMP activity through intravitreal injections of GM6001 after ONC strongly reduced retinal ganglion cell (RGC) axonal regrowth, without influencing RGC survival. Based on a recently performed transcriptome profiling study, the expression pattern of four MMPs after ONC was determined via combined use of western blotting and immunostainings. Mmp‐2 and ‐13a were increasingly present in RGC somata during axonal regrowth. Moreover, Mmp‐2 and ‐9 became upregulated in regrowing RGC axons and inner plexiform layer (IPL) synapses, respectively. In contrast, after an initial rise in IPL neurites and RGC axons during the injury response, Mmp‐14 expression decreased during regeneration. Altogether, a phase‐dependent expression pattern for each specific MMP was observed, implicating them in axonal regrowth and inner retina remodeling after injury. In conclusion, these data suggest a novel, neuron‐intrinsic function for multiple MMPs in axon regrowth that is distinct from breaking down environmental barriers. J. Comp. Neurol. 524:1472–1493, 2016.

Aberrant Collagen Composition of the Trabecular Meshwork Results in Reduced Aqueous Humor Drainage and Elevated IOP in MMP-9 Null Mice

Purpose Homeostatic turnover of the trabecular meshwork extracellular matrix (ECM) is essential to regulate aqueous humor outflow and to maintain intraocular pressure homeostasis. In this study, we evaluated aqueous humor turnover, intraocular pressure, and trabecular meshwork organization in MMP-9 null mice. Methods Intraocular pressure and aqueous humor turnover were measured in MMP-9 null versus wild-type mice. Morphology of the anterior segment of the eye, with special attention to the structural organization of the trabecular meshwork, was investigated by means of optical coherence tomography, light microscopy, and transmission electron microscopy. Furthermore, using quantitative real-time polymerase chain reaction and immunostainings, we evaluated the ECM composition of the trabecular meshwork. Finally, the integrity and function of the retina and optic nerve were assessed, via optical coherence tomography, histologic techniques, and optomotor testing. Results MMP-9 null mice displayed early-onset ocular hypertension and reduced aqueous humor turnover. While transmission electron microscopic analysis did not reveal any abnormalities in the cellular organization of the trabecular meshwork, detailed investigation of collagen expression indicated that there is an aberrant trabecular meshwork ECM composition in MMP-9 null mice. Notably, at the age of 13 months, no glaucomatous neurodegeneration was seen in MMP-9 null mice. Conclusions Our observations corroborate MMP-9 as an important remodeler of the collagenous composition of the trabecular meshwork and provide evidence for a causal link between MMP-9 deficiency, trabecular meshwork ultrastructure, and ocular hypertension.

Successful optic nerve regeneration in the senescent zebrafish despite age-related decline of cell intrinsic and extrinsic response processes

Dysfunction of the central nervous system (CNS) in neurodegenerative diseases or after brain lesions seriously affects life quality of a growing number of elderly, since the adult CNS lacks the capacity to replace or repair damaged neurons. Despite intensive research efforts, full functional recovery after CNS disease and/or injury remains challenging, especially in an aging environment. As such, there is a rising need for an aging model in which the impact of aging on successful regeneration can be studied. Here, we introduce the senescent zebrafish retinotectal system as a valuable model to elucidate the cellular and molecular processes underlying age-related decline in axonal regeneration capacities. We found both intrinsic and extrinsic response processes to be altered in aged fish. Indeed, expression levels of growth-associated genes are reduced in naive and crushed retinas, and the injury-associated increase in innate immune cell density appears delayed, suggesting retinal inflammaging in old fish. Strikingly, however, despite a clear deceleration in regeneration onset and early axon outgrowth leading to an overall slowing of optic nerve regeneration, reinnervation of the optic tectum and recovery of visual function occurs successfully in the aged zebrafish retinotectal system.

Complementary research models and methods to study axonal regeneration in the vertebrate retinofugal system

Due to the lack of axonal regeneration, age-related deterioration in the central nervous system (CNS) poses a significant burden on the wellbeing of a growing number of elderly. To overcome this regenerative failure and to improve the patient’s life quality, the search for novel regenerative treatment strategies requires valuable (animal) models and techniques. As an extension of the CNS, the retinofugal system, consisting of retinal ganglion cells that send their axons along the optic nerve to the visual brain areas, has importantly contributed to the current knowledge on mechanisms underlying the restricted regenerative capacities and to the development of novel strategies to enhance axonal regeneration. It provides an extensively used research tool, not only in amniote vertebrates including rodents, but also in anamniote vertebrates, such as zebrafish. Indeed, the latter show robust regeneration capacities, thereby providing insights into the factors that contribute to axonal regrowth and proper guidance, complementing studies in mammals. This review provides an integrative and critical overview of the classical and state-of-the-art models and methods that have been employed in the retinofugal system to advance our knowledge on the signaling pathways underlying the restricted versus robust axonal regeneration in rodents and zebrafish, respectively. In vitro, ex vivo and in vivo models and techniques to improve the visualization and analysis of regenerating axons are summarized. As such, the retinofugal system is presented as a valuable model to further facilitate research on axonal regeneration and to open novel therapeutic avenues for CNS pathologies.

An Antagonistic Axon-Dendrite Interplay Enables Efficient Neuronal Repair in the Adult Zebrafish Central Nervous System

Neural insults and neurodegenerative diseases typically result in permanent functional deficits, making the identification of novel pro-regenerative molecules and mechanisms a primary research topic. Nowadays, neuroregenerative research largely focuses on improving axonal regrowth, leaving the regenerative properties of dendrites largely unstudied. Moreover, whereas developmental studies indicate a strict temporal separation of axogenesis and dendritogenesis and thus suggest a potential interdependency of axonal and dendritic outgrowth, a possible axon-dendrite interaction during regeneration remains unexplored. To unravel the inherent dendritic response of vertebrate neurons undergoing successful axonal regeneration, regeneration-competent adult zebrafish of either sex, subjected to optic nerve crush (ONC), were used. A longitudinal study in which retinal ganglion cell (RGC) dendritic remodeling and axonal regrowth were assessed side-by-side after ONC, revealed that—as during development—RGC axogenesis precedes dendritogenesis during central nervous system (CNS) repair. Moreover, dendrites majorly shrank before the start of axonal regrowth and were only triggered to regrow upon RGC target contact initiation, altogether suggestive for a counteractive interplay between axons and dendrites after neuronal injury. Strikingly, both retinal mechanistic target of rapamycin (mTOR) and broad-spectrum matrix metalloproteinase (MMP) inhibition after ONC consecutively inhibited RGC synapto-dendritic deterioration and axonal regrowth, thus invigorating an antagonistic interplay wherein mature dendrites restrain axonal regrowth. Altogether, this work launches dendritic shrinkage as a prerequisite for efficient axonal regrowth of adult vertebrate neurons, and indicates that molecular/mechanistic analysis of dendritic responses after damage might represent a powerful target-discovery platform for neural repair.

Modelling aging and age-associated pathology in zebrafish

Abstract With the increasing number of active elderly in our society, healthy aging has become a main priority for policy makers, clinicians, and researchers. Although research of the pathophysiological mechanisms underlying aging has taken leaps forward in recent years, developing effective treatments to ensure quality of life up to advanced age remains one of the biggest challenges in biomedical research today. Animal research models are indispensable in solving this complex puzzle, and research using the zebrafish has definitely gained popularity over the past decade. Owing to their relatively short life span, processes of gradual aging can be studied in a reasonable time window, while the extensive characterization of its well-conserved molecular and cellular physiology has delivered a set of most valuable tools and techniques. In this manuscript, we review the current findings from zebrafish gerontology research and point out the striking similarities—and differences—between zebrafish and mammals, which could eventually lead to a better understanding of mechanisms of aging, disease, and repair.