Gap-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat

Retinal ganglion cells (RGCs) in rats were retrogradely labeled with the fluorescent tracer Fluorogold (FG) and subjected to GAP-43 and c-JUN immunocytochemistry to identify those RGSs that are capable of regenerating an axon. After optic nerve section (ONS) and simultaneous application of FG to the nerve stump (group 1 experiments), GAP-43 immunoreactive RGCs (between 2 and 21 days after ONS) always represented a subfraction of both FG-labeled (i.e., surviving) RGCs and RGCs exhibiting c-JUN. GAP-43 immunoreactive RGCs represented 22% of RGCs normally present in rat retinae and 25% of surviving RGCs at 5 days after ONS but were reduced to 2% and 1%, which is 6% and 5% of survivors at 14 and 21 days, respectively. In animals that received a peripheral nerve (PN) graft after ONS (group 2 experiments), RGCs with regenerating axons were identified by FG application to the graft at 14 and 21 days. When examined at 21 and 28 days, all FG-labeled RGCs exhibited GAP-43 immunoreactivity, and FG/GAP-43-labeled RGCs were 3% and 2% of those resent in normal rat retinae. In relation to surviving. RGCs GAP-43 immunoreactive RGCs represented 10% at both time points. FG-/GAP-43 labeled RGCs also exhibited c-JUN, but c-JUN immunoreactive RGCs were at both time points at least twice as numerous a FG-/GAP-43-labeled RGCs. These data suggest that regenerating axons in PN grafts derive specifically from GAP-43 reexpressing RGCs. Appearance of GAP-43 immunoreactivity may therefore identify those RGCs that are capable of axonal regeneration or sprouting. 1994 John Wiley & Sons, Inc.     

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo

Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders.     

Osteopontin drives retinal ganglion cell resiliency in glaucomatous optic neuropathy

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Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats

To investigate the short-and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent market diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or in intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an inital abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A. persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons. © 1993 John Wiley & Sons, Inc.     

A Chrnb3‐Cre BAC transgenic mouse line for manipulation of gene expression in retinal ganglion cells

The mechanisms by which retinal ganglion cells (RGCs) make specific connections during development is an intense area of research and have served as a model for understanding the general principles of circuit wiring. As such, genetic tools allowing for specific recombination in RGCs are critical to further our understanding of the cell‐specific roles of different genes during these processes. However, many RGC‐specific Cre lines have drawbacks, due to their broad expression in other cell types and/or retinorecipient regions or lack of expression in broad swaths of the retina. Here, we characterize a Cre BAC transgenic line driven by elements of the cholinergic receptor nicotinic beta 3 subunit (Chrnb3). We show that Cre expression is restricted to RGCs in the retina and sparsely expressed in the brain, importantly excluding retinorecipient regions. Furthermore, Chrnb3‐Cre mice label a wide variety of RGCs distributed throughout the retina and Cre activity is detected embryonically, shortly following RGC differentiation. Finally, we find that Chrnb3‐Cre‐labeled RGCs innervate multiple retinorecipient areas that serve both image‐forming and nonimage forming functions. Thus, this genetic tool will be of broad use to investigators studying the RGC‐specific contributions of genes to visual circuit development.     

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

Our recent studies implicate the transient receptor potential vanilloid-1 (TRPV1) channel as a mediator of retinal ganglion cell (RGC) function and survival. With elevated pressure in the eye, TRPV1 increases in RGCs, supporting enhanced excitability, while Trpv1 -/- accelerates RGC degeneration in mice. Here we find TRPV1 localized in monkey and human RGCs, similar to rodents. Expression increases in RGCs exposed to acute changes in pressure. In retinal explants, contrary to our animal studies, both Trpv1 -/- and pharmacological antagonism of the channel prevented pressure-induced RGC apoptosis, as did chelation of extracellular Ca²⁺. Finally, while TRPV1 and TRPV4 co-localize in some RGC bodies and form a protein complex in the retina, expression of their mRNA is inversely related with increasing ocular pressure. We propose that TRPV1 activation by pressure-related insult in the eye initiates changes in expression that contribute to a Ca²⁺-dependent adaptive response to maintain excitatory signaling in RGCs.     

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

Our recent studies implicate the transient receptor potential vanilloid-1 (TRPV1) channel as a mediator of retinal ganglion cell (RGC) function and survival. With elevated pressure in the eye, TRPV1 increases in RGCs, supporting enhanced excitability, while Trpv1 -/- accelerates RGC degeneration in mice. Here we find TRPV1 localized in monkey and human RGCs, similar to rodents. Expression increases in RGCs exposed to acute changes in pressure. In retinal explants, contrary to our animal studies, both Trpv1 -/- and pharmacological antagonism of the channel prevented pressure-induced RGC apoptosis, as did chelation of extracellular Ca²⁺. Finally, while TRPV1 and TRPV4 co-localize in some RGC bodies and form a protein complex in the retina, expression of their mRNA is inversely related with increasing ocular pressure. We propose that TRPV1 activation by pressure-related insult in the eye initiates changes in expression that contribute to a Ca²⁺-dependent adaptive response to maintain excitatory signaling in RGCs.     

Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.

     

Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons

Olfactory bulb ensheathing glia (OEG) have attracted special attention during the last few years because of their unique properties in promoting regeneration of adult mammalian central nervous system (CNS) components. However the molecular and cellular characteristics responsible for this capacity remain to be revealed. Such studies are presently hindered by the lack of a plentiful source of homogenous OEG. Thus the availability of immortalized OEG lines maintaining the regenerative characteristics of the primary cultures would represent an unlimited source of OEG for use not only in biochemical analyses of neuroregenerative mechanisms but also to characterize their regenerative properties in models in culture and in vivo. We have immortalized primary rat OEG using the SV40 large T antigen expressed from a constitutive cellular promotor, and report here the isolation and characterization of clonal lines. These OEG clonal lines were comparable to primary OEG and Schwann cells in the promotion of axonal regeneration of mature rat retinal ganglion neurons (RGN) but, significantly, this culture assay system more closely reflects the in vivo reparative properties of OEG on transected nerves than other assays of neuritogenesis in that it revealed OEG cells to promote the growth of a larger number of long axons than Schwann cells. Using this assay we were able to grade our OEG lines for their neuroregenerative capacity, opening the possibility of identifying molecules with correlative expression levels in these cells. Our preliminary characterization revealed that the expression level of a classical OEG marker, the p75-NGF receptor, does not correlate with neuroregenerative capacity.     

Distinct responses of cones and melanopsin-expressing retinal ganglion cells in the human electroretinogram

ABSTRACT: BACKGROUND: The discovery of the novel photoreceptor, melanopsin-expressing retinal ganglion cells (mRGCs), has raised researchers' interest in photoreceptive tasks performed by the mRGC, especially in non-image-forming visual functions. In a prior study, we investigated the mRGC response to light stimuli independent of rods and cones with the four-primary illumination system, which modulates stimulus levels to the mRGC and cones independently, and mRGC baseline responses were recorded in the electroretinogram (ERG). METHODS: In the present study, we used the same illumination system to compare independent responses of the mRGC and cones in five subjects (mean +/- SD age, 23.0 +/- 1.7 years). The ERG waveforms were examined as direct measurements of responses of the mRGCs and cones to stimulation (250 msec). Implicit times (the time taken to peaks) and peak values from 30 stimuli given to each subject were analyzed. RESULTS: Two distinct positive peaks appeared in the mRGC response, approximately 80 msec after the onset of the stimuli and 30 msec after their offset, while no such peaks appeared in the cone response. The response to the mRGC stimulus was significantly higher than that to the cone stimulus at ~80 msec (p < 0.05) and tended to be higher than the cone stimulus at ~280 msec (p = 0.08). CONCLUSIONS: Implicit time of the first peak was much longer than that to the b-wave and this delay might reflect mRGC's sluggish responses. This is the first report of amplitudes and implicit time in the ERG from the response of the mRGC that is independent of rods and cones and obtained using the four-primary illumination system.     

ROCK2 is a major regulator of axonal degeneration,neuronal death and axonal regeneration in the CNS

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.