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Review
The intrinsic determinants of optic nerve regeneration
ZHU Rui-lin, CHO Kin-Sang, GUO Chenying, CHEW Justin, CHEN Dong Feng and YANG Liu
Department of Ophthalmology, Peking University First Hospital; Key Laboratory of Vision Loss and Restoration,
Ministry of Education, Beijing 100034, China (Zhu RL and Yang L)
Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School,
Boston, MA 02114, United States (Zhu RL, Cho KS, Guo CY, Chew J, and Chen DF)
Correspondence to: Prof. YANG Liu, Department of Ophthalmology, Peking University First Hospital; Key Laboratory
of Vision Loss and Restoration, Ministry of Education, Beijing 100034, China (Email: [email protected])
This study was supported by grant from National Natural Science Foundation of China (No. 81170837)
Key words: nerve regeneration; retinal ganglion cells; intrinsic determinants; Bcl-2;
cAMP; KLF; mTOR; PTEN; SOCS3
Objective: To review the functions of these intracellular signals in their regulation of
RGC axon regeneration.
Data Sources: Relevant articles published in English or Chinese from year 1970 to
present were selected from PubMed. Searches were made using the terms “intrinsic
determinants, axon regeneration, retinal ganglion cells, optic nerve regeneration, central
nervous system axon regeneration”.
Study selection: Articles studying the mechanisms controlling retinal ganglion cell and
central nervous system (CNS) axonal regeneration were reviewed. Articles focusing on
the intrinsic determinants of axon regeneration were selected.
Results: Like other CNS neurons of mammals, retinal ganglion cells (RGCs) undergo a
developmental loss in their ability to grow axons as they mature, which is a critical
contributing factor to the failure of nerve regeneration and repair after injury. This growth
failure can be attributed, at least in part, by the induction of molecular programs
preventing cellular overgrowth and termination of axonal growth upon maturation. Key
intracellular signals and transcription factors, including Bcl-2, cyclic adenine
monophosphate (cAMP), mammalian target of rapamycin (mTOR), and Krüppel-like
transcription factors (KLFs), have been identified to play central roles in this process.
Conclusions: Intense effort and substantial progress have been made to identify the
various intrinsic growth pathways that regulate RGC axonal regeneration. More work is
needed to elucidate the mechanisms and inter-relationship between the actions of these
factors and to achieve successfully regeneration and repair of the severed RGC axons.
INTRODUCTION
The retina and optic nerve are part of the central nervous system (CNS). Retinal ganglion
cells (RGCs) are a population of neurons located in the innermost layer of the retina that
convey visual signals from the retina along their axons to the brain. As with other
mammalian CNS neurons, RGC axons are unable to regenerate after optic nerve damage
that can, in severe cases, cause complete visual loss with devastating consequences to the
patient’s life1. Causes of axonal damage are varied. To date no therapeutic treatment is
available to substantially stimulate axon regeneration and to functionally repair disrupted
axonal connections in the visual pathway1. Understanding why CNS axons cannot
regenerate after injury in the adult mammals has been a major challenge for both basic
and clinical neuroscientists2.
In the past few decades, many studies have focused on the involvement of the
extraneuronal milieu in the failure of maturing CNS axons to regrow over long distances.
However, in those studies, the regenerative capacity is expressed by a limited population
of neurons3. Simply removing extracellular inhibitory activities is insufficient for
successful axon regeneration in the adult CNS2. Study the development of CNS tracts
suggests that mature neurons have lost substantial capacity for axon regeneration. For
example, embryonic retinal explants can extend axons into embryonic or adult brain
explants, but adult retinal explants cannot extend axons into either, suggesting that the
problem is within the retina or RGCs3. Purified embryonic RGCs extended their axons up
to 10-fold faster than postnatal or adult RGCs, and altering the extrinsic environment did
not change this property of neurons4. Other report studying the spinal cord regeneration
discovered the similar results5. Thus, the failure of older CNS axons to regenerate is not
only due to changes in the extraneuronal environment; moreover, the intrinsic changes
within the neurons also limit their regenerative ability3,5,6. Activating intrinsic growth
programs may be essential for enabling CNS axonal regrowth7.
In recent years, several key intracellular signals and transcription factors that play
important roles to boost the intrinsic growth programs of CNS axons have been identified.
These include cAMP (cyclic adenosine monophosphate), mammalian target of rapamycin
(mTOR), Bcl-2 (B cell lymphoma/leukemia 2), Krüppel-like Transcription Factors (KLFs)
and several other factors. In this review, we introduce the functions of intracellular
signals that have been shown to regulate RGCs axonal regeneration. According to their
different roles in regulating axonal growth, we divided these intrinsic factors into two
groups: enhancers and repressors.
AXON GROWTH ENHANCERS
Bcl-2 (B cell lymphoma/leukemia 2)
The proto-oncogene Bcl-2 is well known for its function as an anti-apoptotic protein. It
was found to play a key role in the neuronal growth8. The Bcl-2 gene codes for a protein
that acts as a powerful inhibitor of cell death9. Bcl-2 expression declines in the
developing CNS as neurons lose their ability to grow axons, but are maintained at a high
level in adult neurons of the peripheral nervous system, where axons regenerate robustly
throughout life10.
In the absence of Bcl-2, the ability of embryonic neurons to elaborate axons is reduced,
even in the presence of potent neuronal growth factors8. By contrast, overexpression of
Bcl-2 enhances neurite outgrowth in several neuronal cell lines and promotes the
regeneration of RGC axons into a permissive brain environment in culture8. Cho et al11
found that before postnatal day 4, when astrocytes are immature, overexpression of Bcl-2
alone supported robust and rapid optic nerve regeneration over long distances, leading to
innervation of brain targets by day 4 in mice12. However, some regenerating fibers were
found to deviate from the optic pathway and grew into the forebrain or form aberrant
projections13. Concurrent induction of Bcl-2 and attenuation of reactive gliosis reversed
the failure of CNS axonal re-elongation in postnatal mice up to 2 weeks of age11.
Therefore, these findings suggest Bcl-2 plays a very important role in optic nerve
regeneration.
Cyclic adenine monophosphate (cAMP)
Cyclic adenine monophosphate (cAMP) is a secondary messenger used in intercellular
signal transduction and affects a number of cellular processes. In the mammalian nerves
system, cAMP functions as one of the intrinsic regulators controlling the regenerative
capacity of axons during development14. The endogenous cAMP level of RGCs in
embryonic neurons is significantly higher than that in older neurons, and it drops to low
level by birth15. The intracellular level of cAMP correlates with the distinct
responsiveness of axonal growth to myelin and a specific myelin component, myelinassociated glycoprotein (MAG), in neurons at different developmental stages: axonal
growth is promoted by myelin and MAG in younger neurons when intracellular cAMP
level is high, while the axonal growth is inhibited by MAG in the same types of neurons
when they grow older15.
A high level of endogenous cAMP in young CNS neurons is required for the promotion
of neurite outgrowth by MAG and myelin. When blocked with PKA inhibitor, KT5720,
or cAMP antagonist, Rp-cAMP, the growth promoting activity of MAG is eliminated14,15.
In goldfish, cAMP levels increase in RGCs at the time when they support extensive axon
outgrowth and navigate within the optic tectum to re-establish topographic connections14.
Similar to cAMP signaling in other tissue systems, cAMP functions through the
PKA/CREB pathway in the CNS to regulate axonal growth during development. Studies
have shown that activation of CREB is required to overcome MAG-mediated axon
growth inhibition16.
Mammalian target of rapamycin (mTOR)
Recently, it has been proposed that coordination of local protein synthesis and
proteosome-mediated protein degradation may play a critical role in robust axon
regeneration in the CNS17. The upstream molecules regulating protein synthesis, such as
target of rapamycin (TOR), were required for growth cone formation and axon
regeneration17. Activation of mTOR leads to protein synthesis and growth, likely
including those that are required for axonal growth18.
Two of the well-studied targets of the mTOR kinase are ribosomal S6 kinase 1 (S6K1)
and the eukaryotic initiation factor 4E (eIF4E)-binding protein 4E-BP118,19. Antibodies to
phospho-S6 (p-S6) are used to monitor the activity of the mTOR pathway in RGCs. Park
et al19 found that strong p-S6 signals can be seen in most embryonic neurons but are
diminished in 90% of adult RGCs, suggesting that mTOR signaling is down-regulated in
the majority of adult RGCs, with only a small subset retaining considerable mTOR
activity. Thus, activation of mTOR seems inversely correlating with the regenerative
ability of RGCs. Following optic nerve injury, mTOR activity is almost abolished in
mature RGCs shown by down regulation of p-S619. Since phosphatase and tensin
homolog (PTEN) and TSC1/2 are negatively regulators of mTOR, suppression or
knockdown of these molecules could enhance mTOR activity and its downstream
pathway such as p-S6. Optic nerve injury was performed in PTEN or TSC1 knockout
mice, and robust optic nerve regeneration was observed. The regenerating RGC was
shown to be p-S6 positive19. Taken together, stimulating mTOR activates an intrinsic
program of axon regeneration.
AXON GROWTH REPRESSORS
Krüppel-like Transcription Factors (KLFs)
Krüppel-like transcription factors (KLFs) are a family of transcriptional regulators found
throughout many tissues20. To date, 17 KLF family members have been identified,
numbered accordingly from KLF-1 through KLF-17. These KLFs were found to play a
major role in the developmental process across the entire organism and are responsible
for cellular differentiation, proliferation, and survival20,21. The mammalian KLFs can be
roughly grouped into six groups from both phylogeny and structural similarity of the
transactivator domain: the “AIN” subfamily, “AHN” subfamily, BTEB-like subfamily,
PVALS/T subfamily, SID/R2-3 subfamily, and the remaining three KLFs which cannot
be grouped as readily21. 15 of 17 KLF family members are expressed in RGCs22.
In a broad screening of 111 candidate molecules that change expression levels during
RGC development, in 2009 Moore et al22 uncovered KLF-4 as one of the most effective
suppressors of neurite outgrowth, decreasing average length by 50%. KLF-4 in vivo is
dramatically upregulated from E20 through P1 to its maximum sustained level,
corresponding to the time frame in which embryonic neurons lose their ability to
regenerate22,23. Overexpression of KLF-4 in these embryonic neurons was found to
reduce average axon length, dendrite length, neurite branching, and the percentage of
neurons that extended neurites. Furthermore, the rate of axon growth was slower in
neurons expressing KLF-4 compared to their control counterparts22. Altogether, these
data indicate that KLF-4 is a strong repressor of neurite growth. In RGCs in which KLF-4
was conditionally knocked out, the regenerative potential increased in response to optic
nerve crush injury, further confirming KLF-4’s role as a neurite growth repressor22. In
RGCs, overexpression of KLF9 significantly decreased growth, similar to KLF422.
Although most KLFs members are axon growth-suppressing KLFs, the “AHN” KLFs
(KLF-6 and -7) have been shown to be potent enhancers of neurite growth and are highly
expressed during development in the mouse21. KLF-7 is critical for the development of
the central nervous system, and KLF-7-/- mice display serious neurological defects,
specifically with regards to RGC axon pathfinding24.
KLFs may interact with each other as well as with their downstream targets. In
overexpression experiments using combinations of growth suppressive (KLF4 and -9)
and growth enhancing (KLF6 and -7) KLFs, Moore et al found that the suppressors
dominated: KLF6 and -7 could never enhance growth in the presence of KLF4 or -9, and
KLF4 could suppress neurite growth even when KLF6 or -7 were co-overexpressed22.
Since KLFs play an important role in RGC axon growth, manipulating multiple KLF
genes may be a useful strategy to add to existing approaches to increase the intrinsic
regenerative capacity of mature CNS neurons damaged by injury or disease.
Phosphatase and tensin homolog (PTEN)
Phosphatase and tensin homolog (PTEN) is a well-known negative regulator of cellular
growth, as evident by its designation as a tumor suppressor gene25,26. PTEN encodes a
lipid and protein phosphatase that negatively regulates phosphoinositide-3-kinase (PI3K)
signaling and controls cell growth and migration. In the nervous system, PTEN mutations
lead to neuronal hypertrophy and defects in cell migration, dendrite arborization and
myelination25,26. Cantrup et al25 identified the PTEN phosphatase as a critical regulator of
retinal tissue morphogenesis. Conditional knockout of PTEN causes RGC, horizontal and
amacrine cell hypertrophy and expansion of the inner plexiform layer25. Drinjakovic et
al27 demonstrated that PTEN, as a key downstream target of Nedd4, played an important
role in regulating RGCs terminal arborization in vivo.
With conditionally PTEN knockout mice, Park et al19 showed that after optic nerve crush,
the RGCs of PTEN knockout mice displayed a significant increase in survival and longdistance axon regeneration. Among the different mouse lines they examined (Rb, P53,
Smad4, Dicer, LKB1, and PTEN), those with a PTEN deletion showed the largest effects
on both neuronal survival and axon regeneration19.
Suppressor of cytokine signaling 3 (SOCS3)
The SOCS family of proteins is comprised of eight family members that function as
intracellular inhibitors of cytokine signaling. SOCS proteins primarily inhibit JAK-STAT
signaling through binding to JAK and/or specific phospho-tyrosine residues on cytokine
receptors. Many physiological functions are regulated by SOCS proteins, including
inflammatory, immune, endocrine, and oncogenic responses28,29.
Smith et al30 showed that SOCS3 deletion in RGCs promotes both neuronal survival and
axon regeneration following optic crush injury. Double knockout of SOCS3 and gp130
revealed a significant loss of the axon regeneration observed in SOCS3-deleted mice,
suggesting that axon regeneration induced by SOCS3 deletion is largely dependent upon
gp130-mediated signaling30. However, neuronal survival in the double mutants is higher
than SOCS3f/f mice or gp130f/f with AAV-Cre mice, suggesting a contribution of gp130independent pathways to the effects of SOCS3 deletion on neuronal survival30.
Hellströmetal et al31 demonstrated that overexpression of SOCS3 resulted in an overall
reduction in axonal regrowth and almost complete regeneration failure of RGCs
transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated
expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal
rCNTF injections, indicating that high levels of SOCS3 could also block cell survival
pathway7,31.
INTERACTIONS BETWEEN THESE INTRINSICAL DETERMINANTS
Evidence suggests that Bcl-2, which usually resides in the endoplasmic reticulum (ER),
regulates ER Ca2+ content by decreasing ER Ca2+ uptake and increasing ER Ca2+ efflux32.
In neurons, this activity of Bcl-2 causes higher intracellular Ca2+ than usual after neural
injury and leads to the activations of mitogen-activated protein kinase and CREB which
promote axonal regrowth32.
On the other hand, SOCS3 deleted RGC upregulates mTOR at 3 and 7 days post crush,
allowing RGCs to respond to injury-triggered factors30. Sun et al33 showed that
simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon
regeneration, suggesting that PTEN and SOCS3 regulate two independent pathways that
act synergistically to promote axon regeneration33.
A recent study demonstrated that administration of Zymosan, cAMP in PTEN knockout
mice following optic nerve crush led to long distance regeneration of RGC axon34.
Unlike the previous experiments using SOCS3 or PTNE knockout mice, this study
examined the mice at later time points. Surprisingly, at 10 week post-injury, most
regenerating axons crossed the midline of the optic chiasm. The injured mice first showed
visual behavior response such as optomotor response and circadian activity34. Thus, the
data suggest that synaptic re-connections may be developed between regenerating RGC
axons and the visual targets.
CONCLUSIONS
Intense effort and substantial progress have been made to induce RGC axonal
regeneration in the last decade. Nevertheless, successful regeneration and repair of the
severed RGC axons or optic nerve pathway remains a challenge. Manipulating the
regulators of axon growth, even when simultaneously overcoming environmental
inhibition, only partially restores regeneration, suggesting that additional intrinsic axon
growth regulators remain to be identified22. The various intrinsic growth pathways and
factors suggests that not one single target will be adequate to increase optic nerve
regeneration35. More work is needed to clarify the interactive effect of these factors and
their mechanisms that regulate the neuronal capacity for axon growth. One promising
strategy is to study neurons during the developmental transition that limits axon growth36.
In order to develop clinically feasible and applicable therapies, studies are needed to
further elucidate the interactive effect of these factors and their mechanisms that regulate
the neuronal capacity for axon growth.
REFERENCES
1. Fischer D, Leibinger M. Promoting optic nerve regeneration. Prog Retin Eye Res
2012;31:688-701. PMID: 22781340
2. Sun F, He Z. Neuronal intrinsic barriers for axon regeneration in the adult CNS. Curr
Opin Neurobiol 2010;20:510-518. PMID: 20418094
3. Chen DF, Jhaveri S, Schneider GE. Intrinsic changes in developing retinal neurons
result in regenerative failure of their axons. Proc Natl Acad Sci U S A 1995;92:72877291. PMID: 7638182
4. Goldberg JL, Klassen MP, Hua Y, Barres BA. Amacrine-signaled loss of intrinsic
axon growth ability by retinal ganglion cells. Science 2002;296:1860-1864. PMID:
12052959
5. Blackmore MG, Wang Z, Lerch JK, Motti D, Zhang YP, Shields CB, et al. Kruppellike Factor 7 engineered for transcriptional activation promotes axon regeneration in
the adult corticospinal tract. Proc Natl Acad Sci U S A 2012;109:7517-7522. PMID:
22529377
6. Moore DL, Apara A, Goldberg JL. Kruppel-like transcription factors in the nervous
system: novel players in neurite outgrowth and axon regeneration. Mol Cell Neurosci
2011;47:233-243. PMID: 21635952
7. Yang P, Yang Z. Enhancing intrinsic growth capacity promotes adult CNS
regeneration. J Neurol Sci 2012;312:1-6. PMID: 21924742
8. Chen DF, Schneider GE, Martinou JC, Tonegawa S. Bcl-2 promotes regeneration of
severed axons in mammalian CNS. Nature 1997;385:434-439. PMID: 9009190
9. Cenni MC, Bonfanti L, Martinou JC, Ratto GM, Strettoi E, Maffei L. Long-term
survival of retinal ganglion cells following optic nerve section in adult bcl-2
transgenic mice. Eur J Neurosci 1996;8:1735-1745. PMID: 8921264
10. Merry DE, Korsmeyer SJ. Bcl-2 gene family in the nervous system. Annu Rev
Neurosci 1997;20:245-267. PMID: 9056714
11. Cho KS, Yang L, Lu B, Feng MH, Huang X, Pekny M, et al. Re-establishing the
regenerative potential of central nervous system axons in postnatal mice. J Cell Sci
2005;118:863-872. PMID: 15731004
12. Yang L, Peng Y, Chen DF. Long time survival of the regenerated optic nerve axons
in Bcl-2 transgenic mice. Chin J Ophthalmol 2009;45:43-49. PMID: 19484930
13. Yang L, Chen DF. Guidance of regenerative axons in optic nerve regeneration in Bcl2 overexpressing mice. Chin J Ophthalmol 2006;42:100-103. PMID: 16643722
14. Rodger J, Goto H, Cui Q, Chen PB, Harvey AR. cAMP regulates axon outgrowth and
guidance during optic nerve regeneration in goldfish. Mol Cell Neurosci 2005;30:452464. PMID: 16169247
15. Cai D, Qiu J, Cao Z, McAtee M, Bregman BS, Filbin MT. Neuronal cyclic AMP
controls the developmental loss in ability of axons to regenerate. J Neurosci
2001;21:4731-4739. PMID: 11425900
16. Gao Y, Deng K, Hou J, Bryson JB, Barco A, Nikulina E, et al. Activated CREB is
sufficient to overcome inhibitors in myelin and promote spinal axon regeneration in
vivo. Neuron 2004;44:609-621. PMID: 15541310
17. Verma P, Chierzi S, Codd AM, Campbell DS, Meyer RL, Holt CE, et al. Axonal
protein synthesis and degradation are necessary for efficient growth cone
regeneration. J Neurosci 2005;25:331-342. PMID: 15647476
18. Chong ZZ, Shang YC, Zhang L, Wang S, Maiese K. Mammalian target of rapamycin:
hitting the bull's-eye for neurological disorders. Oxid Med Cell Longev 2010;3:374391. PMID: 21307646
19. Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, et al. Promoting axon regeneration
in the adult CNS by modulation of the PTEN/mTOR pathway. Science 2008;322:963966. PMID: 18988856
20. Dang DT, Pevsner J, Yang VW. The biology of the mammalian Kruppel-like family
of transcription factors. Int J Biochem Cell Biol 2000;32:1103-1121. PMID:
11137451
21. Moore DL, Apara A, Goldberg JL. Kruppel-like transcription factors in the nervous
system: novel players in neurite outgrowth and axon regeneration. Mol Cell Neurosci
2011;47:233-243. PMID: 21635952
22. Moore DL, Blackmore MG, Hu Y, Kaestner KH, Bixby JL, Lemmon VP, et al. KLF
family members regulate intrinsic axon regeneration ability. Science 2009;326:298301. PMID: 19815778
23. Wang JT, Kunzevitzky NJ, Dugas JC, Cameron M, Barres BA, Goldberg JL. Disease
gene candidates revealed by expression profiling of retinal ganglion cell development.
J Neurosci 2007;27:8593-8603. PMID: 17687037
24. Laub F, Lei L, Sumiyoshi H, Kajimura D, Dragomir C, Smaldone S, et al.
Transcription factor KLF7 is important for neuronal morphogenesis in selected
regions of the nervous system. Mol Cell Biol 2005;25:5699-5711. PMID: 15964824
25. Cantrup R, Dixit R, Palmesino E, Bonfield S, Shaker T, Tachibana N, et al. Cell-type
specific roles for PTEN in establishing a functional retinal architecture. PLoS One
2012;7:e32795. PMID: 22403711
26. Marino S, Krimpenfort P, Leung C, van der Korput HA, Trapman J, Camenisch I, et
al. PTEN is essential for cell migration but not for fate determination and
tumourigenesis in the cerebellum. Development 2002;129:3513-3522. PMID:
12091320
27. Drinjakovic J, Jung H, Campbell DS, Strochlic L, Dwivedy A, Holt CE. E3 ligase
Nedd4 promotes axon branching by downregulating PTEN. Neuron 2010;65:341-357.
PMID: 20159448
28. Newbern JM, Shoemaker SE, Snider WD. Taking off the SOCS: cytokine signaling
spurs regeneration. Neuron 2009;64:591-592. PMID: 20005813
29. Croker BA, Kiu H, Nicholson SE. SOCS regulation of the JAK/STAT signalling
pathway. Semin Cell Dev Biol 2008;19:414-422. PMID: 18708154
30. Smith PD, Sun F, Park KK, Cai B, Wang C, Kuwako K, et al. SOCS3 deletion
promotes optic nerve regeneration in vivo. Neuron 2009;64:617-623. PMID:
20005819
31. Hellstrom M, Muhling J, Ehlert EM, Verhaagen J, Pollett MA, Hu Y, et al. Negative
impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal
ganglion cell axons. Mol Cell Neurosci 2011;46:507-515. PMID: 21145973
32. Jiao J, Huang X, Feit-Leithman RA, Neve RL, Snider W, Dartt DA, et al. Bcl-2
enhances Ca(2+) signaling to support the intrinsic regenerative capacity of CNS axons.
Embo J 2005;24:1068-1078. PMID: 15719013
33. Sun F, Park KK, Belin S, Wang D, Lu T, Chen G, et al. Sustained axon regeneration
induced by co-deletion of PTEN and SOCS3. Nature 2011;480:372-375. PMID:
22056987
34. de Lima S, Koriyama Y, Kurimoto T, Oliveira JT, Yin Y, Li Y, et al. Full-length
axon regeneration in the adult mouse optic nerve and partial recovery of simple visual
behaviors. Proc Natl Acad Sci U S A 2012;109:9149-9154. PMID: 22615390
35. Moore DL, Goldberg JL. Four steps to optic nerve regeneration. J Neuroophthalmol
2010;30:347-360. PMID: 21107123
36. Blackmore M, Letourneau PC. Changes within maturing neurons limit axonal
regeneration in the developing spinal cord. J Neurobiol 2006;66:348-360. PMID:
16408302