Author:

Keywords:

zebrafish, optic nerve regeneration, retina, MMPs, loss-of-function methods, axons, dendrites

Abstract:

Dysfunction of the central nervous system (CNS) after injury or in neurodegenerative diseases increasingly impairs life quality in our aging society. Also optic neuropathies, like glaucoma, are becoming increasingly prevalent in our elderly population, affecting over 60 million people worldwide. They are characterized by retinal ganglion cell (RGC) axonal degeneration and death, ultimately resulting in irreversible blindness because adult mammals lack the capacity to repair/regrow damaged axons in the CNS. Although combinatorial approaches indicate that it might be possible to repair the injured mammalian optic nerve (ON), they have not yet induced substantial visual recovery, nor are they, up to now, suitable for clinical applications. Therefore, comparative studies focus on the spontaneously regenerating adult CNS of zebrafish, which proves to be a valuable research model to investigate regenerative strategies of damaged neurons and to identify novel pro-regenerative molecules. Notably, a recent transcriptome profiling study performed on regenerating adult zebrafish eyes at various time-points after optic nerve crush (ONC), pinpointed several potential pro-regenerative molecules, under which four matrix metalloproteinases (mmp-2, -9, -13a, -14). While MMPs have been implicated in axonal outgrowth during CNS development and are known to be upregulated during vertebrate CNS repair, their impact on in vivo CNS regeneration remains undocumented. Within this context, this PhD project then also intended to unravel the role of Mmp-2, -9, -13a and -14 in ON regeneration in adult zebrafish subjected to ONC. The first part of this thesis was dedicated to an in-depth characterization of RGC survival, axonal regrowth/extension and primary visual recovery in the zebrafish ONC model. Next, we obtained first in vivo insights in a contributory role for MMPs in RGC axonal regrowth as retinal broad-spectrum MMP inhibition seriously attenuated optic tectum reinnervation by regenerating RGC axons without influencing RGC survival. Moreover, combined use of Western Blotting and immunostainings revealed a phase-dependent expression pattern for each MMP in the retina after ONC, primarily implying Mmp-2 and -13a in RGC axonal regrowth. As such, via utilization of several loss-of-function approaches to interfere with the functioning of one specific MMP, e.g. pharmaceutical MMP inhibition, morpholino and genome editing technology, we were able to identify Mmp-2 and -13a as in vivo regulators of RGC axonal regeneration in adult zebrafish after ONC. Notably, since zebrafish lack an inhibitory environment, these data suggest a novel, neuron intrinsic, function for multiple MMPs in RGC axonal regrowth in addition to their role in breaking down environmental barriers as deduced from mammalian studies. The future identification of the underlying MMP substrates by means of quantitative proteomics and subsequent validation of promising candidate molecules in fish and mice might then also unravel new pro-regenerative molecules for the injured mammalian CNS. While being an important part of the neuronal circuitry, the response of dendrites upon induction of axonal regeneration in mammals is almost unattended. Notably, the few publications that do discuss this issue, indicate that dendritic morphology is seriously influenced by the pro-regenerative molecule used to trigger axonal regrowth, ranging from a reduction to an increase in size and complexity. As the eventual recovery of sight after ON injury will equally depend on the proper restoration of dendritic structure, we then also characterized the inherent response of dendrites during successful RGC axonal regeneration in adult zebrafish. Remarkably, both temporal expression analyses of synaptic and dendritic markers, as well as morphometric analysis of inner plexiform layer thickness, indicated that regeneration-competent injured neurons repeat the developmental order of neurite growth, where axogenesis is primary to dendritogenesis. Finally, a last part of this work focused on the elucidation of a potential role of MMPs in retinal dendritic remodeling in adult zebrafish after ONC. Both retinal broad- and narrow-spectrum Mmp(-2) inhibition seemingly prevented RGC dendrites from shrinking upon ON injury. Intriguingly, in both conditions, a disturbance of dendritic retraction occurred concurrently with a reduced RGC axonal regrowth, thus indicative of a potential antagonistic interplay between dendritic remodeling and axonal regrowth after ONC. In addition, due the consecutive progress of axonal and dendritic growth during development and regeneration, and based on a publication discussing TRAK1/2 driven polarized mitochondrial transport in neurons respectively regulating axonal and dendritic outgrowth, we hypothesized that an energetic trade-off might lie at the base of this apparent inter-dependency. As such, future experiments should interfere with mitochondrial trafficking in zebrafish RGC dendrites and analyze the effect on RGC axonal regeneration. If these data would result in a proof-of-concept, i.e. dendritic remodeling serving as fuel for axonal regeneration, we envision a major shift in the research focus of the neuroregenerative research field and the potential uncovering of various novel therapeutic targets.