Intravitreal Administration of rhNGF Enhances Regenerative Processes in a Zebrafish Model of Retinal Degeneration (2025)

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish

Experimental Eye Research

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgfdependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways.

Fgf Signaling is Required for Photoreceptor Maintenance in the Adult Zebrafish Retina

PLoS ONE, 2012

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.

Inhibition of the TGFβ Pathway Enhances Retinal Regeneration in Adult Zebrafish

PLOS ONE, 2016

In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous studies have shown that the transforming growth factor beta (TGFβ) signaling pathway is activated during the regeneration of different tissues in the zebrafish and is needed for regeneration in the heart and the fin. In this study, we have investigated the role of the TGFβ pathway in the Nmethyl-N-nitrosourea (MNU)-induced chemical model of rod photoreceptor de-and regeneration in adult zebrafish. Immunohistochemical staining for phosphorylated Smad3 was elevated during retinal regeneration, and phosphorylated Smad3 co-localized with proliferating cell nuclear antigen and glutamine synthetase, indicating TGFβ pathway activation in proliferating Müller glia. Inhibiting the TGFβ signaling pathway using a small molecule inhibitor (SB431542) resulted in accelerated recovery from retinal degeneration. Accordingly, we observed increased cell proliferation in the outer nuclear layer at days 3 to 8 after MNU treatment. In contrast to the observations in the heart and the fin, the inhibition of the TGFβ signaling pathway resulted in increased proliferation after the induction of retinal degeneration. A better understanding of the underlying pathways with the possibility to boost retinal regeneration in adult zebrafish may potentially help to stimulate such proliferation also in other species.

Glial cell line-derived neurotrophic factor expression in the retina of adult zebrafish (Danio rerio)

Neuroscience …, 2007

The glial cell line-derived neurotrophic factor (GDNF) is a well-known growth factor acting on many neuronal populations of central, peripheral and autonomous nervous system. This factor was also previously detected in the retina of developing rat and chicken while no data are available for the zebrafish. In this study transcripts of GDNF mRNA were observed in adult retina extracts by RT-PCR. The presence of the GDNF protein was confirmed by SDS-PAGE and Western blotting analysis in adult retina homogenates. In situ hybridization and immunohistochemical experiments demonstrated that GDNF mRNA and protein localized in the photoreceptors, in the outer nuclear layer, in the inner plexiform layer and in the ganglion cell layer. These results showed that the expression of GDNF is not probably restricted during development but it might be involved in the physiology of adult zebrafish retina.

Tumor Necrosis Factor-Alpha Is Produced by Dying Retinal Neurons and Is Required for Muller Glia Proliferation during Zebrafish Retinal Regeneration

Journal of Neuroscience, 2013

Intense light exposure causes photoreceptor apoptosis in dark-adapted adult albino zebrafish (Danio rerio). Subsequently, Müller glia increase expression of the Achaete-scute complex-like 1a (Ascl1a) and Signal transducer and activator of transcription 3 (Stat3) transcription factors and re-enter the cell cycle to yield undifferentiated neuronal progenitors that continue to proliferate, migrate to the outer nuclear layer, and differentiate into photoreceptors. A proteomic analysis of light-damaged retinal homogenates, which induced Müller glia proliferation when injected into an undamaged eye, revealed increased expression of tumor necrosis factor ␣ (TNF␣) signaling proteins relative to undamaged retinal homogenates. TNF␣ expression initially increased in apoptotic photoreceptors and later in Müller glia. Morpholino-mediated knockdown of TNF␣ expression before light damage diminished the expression of both Ascl1a and Stat3 in Müller glia and significantly reduced the number of proliferating Müller glia without affecting photoreceptor cell death. Knockdown of TNF␣ expression in the Müller glia resulted in fewer proliferating Müller glia, suggesting that Müller glial-derived TNF␣ recruited additional Müller glia to re-enter the cell cycle. While TNF␣ is required for increased Ascl1a and Stat3 expression, Ascl1a and Stat3 are both necessary for TNF␣ expression in Müller glia. Apoptotic inner retinal neurons, resulting from intravitreal injection of ouabain, also exhibited increased TNF␣ expression that was required for Müller glia proliferation. Thus, TNF␣ is the first molecule identified that is produced by dying retinal neurons and is necessary to induce Müller glia to proliferate in the zebrafish retinal regeneration response.

Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development: BDNF and TrkB in the retina of zebrafish

Journal of Anatomy, 2010

Brain-derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age-dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post-fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full-length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.

Clcf1/Crlf1a-mediated signaling is neuroprotective and required for Müller glia proliferation in the light-damaged zebrafish retina

Frontiers in Cell and Developmental Biology

Zebrafish possess the innate ability to fully regenerate any neurons lost following a retinal injury. This response is mediated by Müller glia that reprogram and divide asymmetrically to produce neuronal precursor cells that differentiate into the lost neurons. However, little is understood about the early signals that induce this response. Ciliary neurotrophic factor (CNTF) was previously shown to be both neuroprotective and pro-proliferative within the zebrafish retina, however CNTF is not expressed following injury. Here we demonstrate that alternative ligands of the Ciliary neurotrophic factor receptor (CNTFR), such as Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine receptor-like factor 1a (Crlf1a), are expressed within Müller glia of the light-damaged retina. We found that CNTFR, Clcf1, and Crlf1a are required for Müller glia proliferation in the light-damaged retina. Furthermore, intravitreal injection of CLCF1/CRLF1 protected against rod photoreceptor cell death in ...

Up-regulation of brain-derived neurotrophic factor by application of fibroblast growth factor-2 to the cut optic nerve is important for long-term survival of retinal ganglion cells

Journal of Neuroscience Research, 2008

Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes the survival of retinal ganglion cells (RGCs) in the frog, Rana pipiens, and results in a rapid up-regulation of BDNF and TrkB synthesis by the RGCs. Here we investigate whether this upregulation is maintained in the long term, and whether it is required for FGF-2's survival effect. At 6 weeks after axotomy and FGF-2 treatment we found more RGCs immunopositive for BDNF protein and higher intensity of BDNF and TrkB immunostaining, accompanied by increases in BDNF and TrkB mRNA in RGCs. Application of fluorescently-labeled siRNA targeted against BDNF to the cut RGC axons showed that it was transported to the cell bodies. Axonal siRNA treatment eliminated the increases in BDNF immunostaining and mRNA that were induced by FGF-2, and had no effect on TrkB mRNA. This reduction in BDNF synthesis by siRNA greatly reduced the long term survival effect of FGF-2 on RGCs. This, taken together with previous results, suggests that, while FGF-2 may initially activate survival pathways via ERK signaling, its main long-term survival effects are mediated via its upregulation of BDNF synthesis by the RGCs.

Basic fibroblast growth factor applied to the optic nerve after injury increases long‐term cell survival in the frog retina

Journal of Comparative Neurology, 2000

The neuroprotective effects of basic fibroblast growth factor (bFGF) on the long-term survival of axotomized retinal ganglion cells (RGCs) were studied in the frog Rana pipiens. Cell loss was quantified in different regions of the ganglion cell layer using Nissl staining and tetramethylrhodamine dextran amine backfilling. All regions of the retina showed a significant decrease (32-66%) in RGC numbers between 4 and 16 weeks after axotomy. Some cells showed morphological and biochemical signs of apoptosis. A single application of bFGF to the optic nerve stump at the time of axotomy protected many of the cells 6 weeks after the injury, but this effect was lost by 12 weeks. A second application of bFGF, 6 weeks after the injury, rescued many RGCs at 12 weeks. In contrast, single or double injections of bFGF into the eyeball had no effect on RGC survival. Axotomized RGCs were significantly enlarged and elongated after axotomy, and these morphological changes were increased by bFGF treatment. In the normal retina and optic nerve, immunocytochemical staining showed bFGF-like immunoreactivity (-LI) in the pigment epithelial layer, in the outer segments of photoreceptors, and in occasional RGCs. Strong bFGF-LI was present in Mü ller cells and in optic nerve astrocytes and oligodendrocytes. FGF receptor-LI was present in photoreceptors, outer plexiform layer, retinal ganglion cell axons, and Mü ller cells. FGF receptor-LI was also observed in optic nerve glia.

Basic fibroblast growth factor-induced protection from light damage in the mouse retina in vivo

Journal of Neurochemistry, 2008

Basic fibroblast growth factor (bFGF) has proven neuroprotective efficacy in the rodent retina against a diverse array of injurious stimuli. However, there is no consensus to date as to the molecular mechanisms underlying this neuroprotection. The study presented herein demonstrates increased expression of endogenous bFGF in the albino mouse retina in response to acute exposure to sublethal levels of light stress. The increased expression correlates with significant photoreceptor protection from light damage. The neuroprotection is likely to be mediated by bFGF as we demonstrate that a shorter exposure to bright light stress that does not up-regulate bFGF fails to protect photoreceptors from light damage. Furthermore, intravitreal bFGF injection into the retina of mice 3 h prior to light damage affords almost complete photore-ceptor protection from light-induced degeneration. In addition, injected bFGF induces the activation of protein kinase B and extracellular signal-regulated kinase 1/2 signalling which correlate directly with the pathways we find to be activated in response to light stress and up-regulated bFGF. Moreover, we demonstrate that both bright light pre-conditioning and intravitreal bFGF injection result in dramatic increases in levels of inactive glycogen synthase kinase 3b and cyclic AMP response element binding protein phosphorylation indicating a potential mechanism by which bFGF promotes survival of photoreceptors in vivo.