Chapter One - Neurotrophic Factors and the Regeneration of Adult Retinal Ganglion Cell Axons

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Abstract

The adult central nervous system (CNS) has only a limited capacity to regenerate axons after injury. This is due to a number of factors including the presence of extrinsic inhibitory factors that limit plasticity, lack of effective trophic support, and intrinsic changes in neuronal responsiveness. In this review, we describe the expression and role of neurotrophins in retinal ganglion cells (RGCs) during development and adulthood, and the receptors and miscellaneous signaling systems that influence axonal regeneration after injury. The impact of exogenous neurotrophic factors on adult RGCs injured at different sites in the visual pathway is described for several modes of delivery, including recombinant factors, viral vectors, cell transplantation, as well as combinatorial treatments involving other pharmacotherapeutic agents. Indirect, off-target effects of neurotrophic factors on RGC axonal regeneration are also considered. There remain unresolved issues relating to optimal delivery of neurotrophic factors, and we emphasize the need to develop safe, reliable methods for the regulation of exogenous supply of these factors to the injured CNS.

Introduction

The adult central nervous system (CNS) normally has only a limited ability to regenerate axons after injury. There appear to be many reasons for this, including a lack of sufficient trophic support and presence of extrinsic inhibitory factors that limit plasticity, as well as intrinsic changes in neuronal responsiveness that negatively impact regenerative potential. Although direct injury to the optic nerve (ON) in humans is not common, over the past 30 years or so the mammalian retinal ganglion cell (RGC) has been a consistent target of experimenters attempting to understand how best to promote the viability and regenerative capacity of adult neurons after CNS trauma (Benowitz and Yin, 2008, Berry et al., 2008, Chierzi and Fawcett, 2001, Harvey et al., 2006). The retina and ON are embryologically part of the CNS, and RGCs are useful model neurons because they can be directly targeted by injections into the vitreal chamber and the ON is a white matter tract containing oligodendroglia that is accessible and relatively easy to manipulate, either by crush, stretch, or full or partial transection injury. Further, RGC survival and axonal regrowth can readily be quantified, and the connectivity and topographic order of regenerating RGC axons can be assessed using anatomical, physiological, and behavioral methods. RGC axons can also be targeted more centrally, at sites further away from the eye, by performing intracranial ON injuries or by lesioning the optic tract, usually between the thalamus and superior colliculus (SC). In this way, regenerative potential of RGCs can be compared in vivo after proximal versus distal axonal injuries.

The brief given to us for this review was a focus on neurotrophic factors and axonal regeneration. We will discuss a range of diffusible peptides with trophic actions on RGCs, including the classic neurotrophins such as brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), as well as other factors such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell-derived neurotrophic factor (GDNF), insulin-like growth factor-1 (IGF-1), the fibroblast growth factors (FGFs), and hepatocyte growth factor (HGF). It is, of course, axiomatic that the regrowth of injured axons requires viable parent neurons; thus, the impact of these factors on RGC survival also needs to be considered. In this context, studies aimed at developing therapies to enhance RGC responsiveness after ON trauma have direct ophthalmological relevance because RGC loss is seen in clinical conditions such as glaucoma, retinal ischemia, retinitis pigmentosa, diabetic retinopathy, optic neuritis, multiple sclerosis, and Alzheimer's disease (e.g., Harvey et al., 2006, Johnson et al., 2011, Kern and Barber, 2008 for reviews). Many studies have examined the impact of neurotrophic factors on RGC survival in animal models of these ophthalmic conditions, but while acknowledging their seminal importance, here we focus on how these various factors influence the RGC regenerative process per se. We also briefly comment on signaling pathways recruited by these factors and how they (i) overcome the loss of intrinsic regenerative capacity in adult RGCs and (ii) interact with signals from the extracellular environment to ameliorate growth-inhibitory components of the injured mature CNS.

Section snippets

Trophic Dependence of RGCs During Development

The commonly held view is that RGCs compete for neurotrophic support; those that in some way fail to obtain sufficient support die, resulting in activation of proapoptotic pathways and the loss of over half the RGC population during the developmental period. Thus, early postnatal removal of a central target such as the SC during the period of naturally occurring cell death causes a rapid and massive increase in RGC death, while addition of factors such as BDNF or NT-4/5 results in increased RGC

Axonal Regeneration: Optic Nerve Injuries

The likely sensitivity of maturing RGCs to multiple trophic factors is relevant to adult RGC responses to axotomy because many of these factors are already present within the eye. Even in neonatal rats there is evidence that intraretinally derived neurotrophic factors play a role in maintaining RGC viability (de Araujo and Linden, 1993, Seki et al., 2003, Spalding et al., 2004). In the adult, trophic factors including CNTF, BDNF, basic FGF and GDNF are produced by cells within the retina or ON

Receptors and Miscellaneous Signaling Pathways that Influence Axonal Regeneration

Although this review is focused on neurotrophic factors, the reader should keep in mind that any axogenic effects elicited in injured adult RGCs by these factors should be integrated into what is known about the intracellular signaling pathways that need to be either activated or inactivated to overcome intrinsic (Moore et al., 2011, Sun and He, 2010) or extrinsic (Berry et al., 2008) restrictions on axonal growth. Any impact of endogenous, retinally derived trophic factors is likely to be

Exogenous Neurotrophic Factors and RGC Axonal Regeneration

Neurotrophic factors influence RGC viability and regenerative potential signal via different receptor complexes and signaling cascades, although there is some convergence of these intracellular pathways. These pathways have been reviewed in detail many times, but a brief summary is pertinent here because these cascades relate to other mechanistic studies alluded to in previous paragraphs.

BDNF and NT-4/5 activation of MAPK–ERK and PI3K–AKT pathways results in process outgrowth, cellular

Mode of Delivery: Recombinant Factors

Neurotrophic factors can be delivered to injured RGCs in various ways, either into the vitreal chamber of the eye itself or by application to the injured ON or to central target regions for subsequent retrograde transport by viable projecting neurons. Factors can be injected as a recombinant protein, via nonviral or viral vector-based delivery of an appropriate gene, or by transplantation of cells expressing an appropriate neurotrophic factor.

In general, mature RGCs are less responsive to

Neurotrophic Support and Autologous PN Grafts

As alluded to above, autologous PN grafts containing Schwann cells provide a bridging environment more conducive to the long-distance regeneration of adult RGC axons (Berry et al., 1988, Bray and Aguayo, 1989, Harvey et al., 2006, Heiduschka and Thanos, 2000, Watanabe, 2010). Using this model, it is possible not only to count the number of regrowing RGC axons but also retrogradely label regenerating neurons for morphological characterization and determination of the proportion of viable RGCs

Nonviral Delivery Systems for Neurotrophic Factors

As discussed earlier, bolus injections of relatively high, almost certainly nonphysiological, concentrations of neurotrophic factors into the eye may not necessarily be the optimal approach for eliciting maximal regeneration of RGC axons because of compensatory responses in RGCs and other retinal cells. In addition, it is expensive to give repeat injections of recombinant growth factors, and the injections themselves will initiate inflammatory and other changes in the eye (Cao et al., 2001).

Cellular Delivery of Neurotrophic Factors

An alternate strategy for delivering neurotrophic factors to RGCs over a prolonged period, without the need for repeated intraocular injections, is to implant cells—either as free suspensions or encapsulated in a semipermeable framework—into the vitreal chamber (Zanin et al., 2012). Such an approach has been used in studies targeting photoreceptors (e.g., Emerich and Thanos, 2008, Lawrence et al., 2004, Lund et al., 2003, Sieving et al., 2006). Suspension injections require attention to

Viral Vector Delivery of Neurotrophic Factors

Viral vectors are modified, replication-deficient viruses in which the viral genome is replaced by a therapeutic gene, providing an in vivo method for long-term, targeted supply of a trophic factor to injured neurons, including RGCs (Hellström & Harvey, 2011). Initial studies used a modified adenovirus (AdV) to deliver neurotrophic factors for protective RGC therapy. AdV vectors have been trialed that encode CNTF (van Adel et al., 2005, Weise et al., 2000), BDNF (Di Polo et al., 1998, Isenmann

Combined Gene Therapy and Pharmacotherapy

Previous studies using rCNTF showed that coinjection with the cyclic AMP analogue CPT-cAMP significantly increased the proportion of surviving RGCs capable of regenerating an axon into an autologous PN graft (Cui et al., 2003). This enhancement is mediated by a number of kinase systems including PKA, PI3K/AKT, and MAPK/ERK (Park et al., 2004), and also by moderation of a CNTF-induced increase in SOCS3 expression (Park et al., 2009). Interestingly, CPT-cAMP does not augment RGC regenerative

Administration of Neurotrophic Factors to the Axonal Growth Environment

Provision of neurotrophic support to the somata of injured RGCs clearly enhances their survival and regenerative capacity, the extent of each depending on the factor that is introduced into the eye. Elsewhere in the CNS, some studies have used vectors or pumps to supply neurotrophic factors to the cell bodies of injured neurons, but the majority, especially, for example, those examining therapies for spinal cord injury, apply factors primarily in and around the lesion site. To link this type of

Axonal Regeneration and Optic Tract Injury Studies

Mention was made earlier regarding the location of axotomy relative to the parent cell body, and how this has a profound influence on the ability of an injured adult neuron to regenerate an axon. Most modern-day visual system regeneration studies use the ON, and almost all crush or transect the nerve within 1–1.5 mm of the eye. After injury at greater distances from the eye, including after intracranial ON transection, there may be greater RGC survival but very few if any RGCs regenerate an axon

Indirect Actions of Neurotrophic Factors

Up to this point, we have mostly focused on the actions of exogenously applied neurotrophic factors that are mediated by cognate receptors expressed by the injured RGCs themselves. However, this is an oversimplification: there is considerable evidence that intraocular trophic factor injections activate other cellular constituents in the eye which in turn produce factors that may indirectly contribute to RGC survival and axonal regeneration (e.g., Berry et al., 2008, Cui et al., 2009). For

Conclusions

Exogenous neurotrophic factors have a beneficial effect on the regeneration of adult RGC axons, not only by potentiating the effects of intrinsic growth-promoting programs but also by counteracting at least some of the inhibitory signaling within, as well as extrinsic to, the neuron. Some factors such as CNTF elicit long-distance regeneration and can be applied at the soma or in the vicinity of the regrowing axons themselves, other factors such as BDNF or NT-4/5 elicit mostly terminal sprouting

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