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REVIEW Goal” to discover means to repair RGC con- nections with the (https://nei.nih.gov/ audacious). The major questions that drive research on Regenerating optic pathways visual restoration and RGC repair are simple but challenging: What strategies support RGC from the to the brain regeneration after damage? Can regenerating RGC form functional synapses with their Bireswar Laha,1 Ben K. Stafford,1 Andrew D. Huberman1,2,3* targets in the brain?

Humans are highly visual. Retinal ganglion cells (RGCs), the neurons that connect Extrinsic factors unique to the CNS the to the brain, fail to regenerate after damage, eventually leading to blindness. limit optic regeneration Here, we review research on regeneration and repair of the optic system. Intrinsic Various environmental influences limit RGC developmental growth programs can be reactivated in RGCs, neural activity can regeneration. Although neurons with cell bodies enhance RGC regeneration, and functional reformation of eye-to-brain connections is in the peripheral (PNS) avidly possible, even in the adult brain. Transplantation and gene therapy may serve to replace regenerate, neurons such as RGCs, whose cell or resurrect dead or injured retinal neurons. Retinal prosthetics that can restore vision bodies reside in the in animal models may too have practical power in the clinical setting. Functional (CNS), fail to reextend after injury (3, 4). Even restoration of sight in certain forms of blindness is likely to occur in human patients if the rodent is completely tran- in the near future. sected, a peripheral nerve graft allows RGC axons to regenerate and form synapses with their ight is crucial for humans to navigate the ing their correct synaptic relationships. Un- targets in the brain (5, 6). Thus, the damage en- world. Under normal, healthy conditions, fortunately, mammalian RGC axons do not vironment constrains the regeneration of mature our eyes and brain create sight so automat- regenerate after damage and damaged RGCs rodent RGCs. Unfortunately for humans, the PNS ically that only when our visual pathways eventually die, never to be replaced. So prevalent nerve graft approach holds limited therapeutic S are damaged do we fully appreciate the ex- is this cause for blindness that the U.S. National potential because it involves massive neuro- on June 9, 2017 tent to which eyesight defines our experience. For Eye Institute has set forth as their “Audacious surgeries. Nevertheless, these studies underscore the many who suffer visual impairments, it is ur- gent that we discover strategies to regenerate ret- inal neurons and convert those strategies into clinically viable therapeutics. Vision begins in the , the thin trilayered Photoreceptors Light pathway neural tissue at the back of the eye (Fig. 1); there, Horizontal cells Signal output pathway photoreceptors transform light information into Bipolar cells electrical signals that the rest of the can understand. The retinal interneurons—the Amacrine cells horizontal, bipolar, and amacrine cells—then pass Retinal ganglion cells (RGCs) that information to the retinal ganglion cells http://science.sciencemag.org/ (RGCs), the output neurons of the eye. There are ~30 different types of RGCs, each firing action Light response potentials depending on the quality and location Action potentials of visual stimuli in the environment (1). Those Retina action potentials propagate down the optic and into the brain, where they are translated into and light-mediated behaviors. Downloaded from The importance of retinal ganglion cells RGCs are a bottleneck for vision. Even when the rest of the visual system is healthy, if RGCs are dead or dysfunctional, vision is impossible. RGCs are protected by the , the thick, durable tissue that encompasses the back of the eye. How- ever, the path that RGC axons take to reach the Myelinated brain renders them vulnerable to damage in re- optic nerve sponse to impacts to the head or eye. , with its attendant elevated eye pressure, is the most common cause of irreversible blindness (2). Intact optic nerve Optic nerve damage Research on visual repair has therefore focused

SCIENCE on sustaining RGCs after injury, encouraging axon regrowth down the optic nerve, and reestablish-

1Department of Neurobiology, Stanford University School of Fig. 1. Visual information is transmitted from the eye to the visual centers in the brain via the 2 Medicine, Stanford, CA 94305, USA. Department of optic nerve. (Top)Lightreachingtheretinaisconvertedintoelectrical potentials that eventually cause Ophthalmology, Stanford University School of Medicine, action potentials in the ganglion cells (RGCs). (Middle)Themyelinatedopticnervetransmitsaction Stanford, CA 94305, USA. 3BioX, Stanford University School of Medicine, Stanford, CA 94305, USA. potentials (Bottom left)tothevisualprocessingcentersinthebrain.(Bottom right)Afterdamage,

GRAPHIC: ADAPTED BY K.*Corresponding SUTLIFF/ author. Email: [email protected] RGC axons degenerate. In the absence of therapeutic interventions, blindness ensues.

Laha et al., Science 356,1031–1034 (2017) 9 June 2017 1of4 REPAIR AND REGENERATION

the principle that RGCs can regen- Retina erate if given the appropriate milieu. RGCs Injured RGCs Inhibitory effects of made active myelin proteins Normally, myelin insulates axons, increasing conduction velocity of electrical signals (Fig. 1). In the PNS, where regeneration is inher- Injured RGCs ent to the system, Schwann cells provide myelination. In the CNS, are the myelinat- ing glial cells and have an inhib- itory effect on axon regeneration. Oligodendrocytes present a variety of proteins inhibitory to axon re- Site of optic growth, including myelin-associated nerve damage glycoprotein, the neurite-outgrowth inhibitor “Nogo,” - myelin glycoprotein, and semaphor- Fig. 2. Electrical activity can promote RGC axon regeneration. Increasing the activity of RGCs after optic ins (7, 8). Neutralization of these nerve damage can facilitate the repair of degenerating axons in the optic nerve (25, 33), with many extending past proteins has been shown to enhance the site of damage into the brain and partially restoring sight in animal models (25). RGC axon regeneration in vitro (7). However, experiments assessing the consequences mia inhibitory factor, and ciliary neurotrophic considering their clinical applications. First, the of removing these proteins in vivo reveal little factor (CNTF) from and other non-neural increase in phosphor-mTOR has to be in place

or no regeneration (9), challenging whether cells (16, 17). In the adult, CNTF up-regulates a before axon injury in order for regeneration to on June 9, 2017 these proteins actually constitute major brakes transcriptional pathway involving suppression occur (25). Second, mTOR broadly affects cell on regeneration. Neutralizing Nogo can enhance of cytokine signaling factor 3 (SOCS3) in RGCs, growth (26)andthusmaycauseretinaltumor regeneration if RGCs are shifted into a growth thus limiting axon regeneration (18). In the ab- formation (27). Any therapeutic approach that state (10), but overall, the effects of reducing sence of SOCS3, CNTF can, however, enhance relies on enhancing mTOR signaling thus would myelin-associated proteins on RGC regeneration regeneration by activating gp130-dependent ki- have to include safeguards. Third, mTOR en- are subtle. Thus, attention has expanded to con- nase signaling (19). Thus, the pathways that af- hancement alone (or mTOR plus SOCS3 dele- sider other extrinsic influences that might un- fect RGC regeneration depend on the signaling tion) triggers regeneration of RGC axons only as derlie RGC regenerative failure and that might context, which imposes complexity on potential far as the (23–25). This suggests that constitute targets for enhancing regeneration in therapeutic strategies. there are inhibitory cues at the and the clinic. Zinc released from amacrine interneurons af- that more potent stimulators of RGC regeneration ter injury is internalized by RGCs and limits their may be needed to inspire RGC axon growth Reactive scarring and regeneration (20). Other extrinsic factors, how- into the brain. In some mice, axons regenerate http://science.sciencemag.org/ As with any injury, damage to the optic pathway ever, can promote regeneration: Lens injury causes to the optic chiasm but then turn away from the recruits cellular and molecular processes to buf- macrophages to release oncomodulin, which sup- brain and grow into the other optic nerve, toward fer the injury response, some of which affect the ports RGC axon extension via a Ca++/calmodulin the contralateral eye (28). Thus, not all regener- regenerative potential of RGCs (11). Astrocytes— pathway (21). Lesion-reactive cells and proteins ation is productive. Other manipulations such as the glial cells that support synapse development, in both the eye and in the optic nerve can either knockdown of the growth-inhibiting transcrip- transmission, and plasticity (12)—create physi- help or hurt regeneration. The key is to discover tional repressor KLF4 can also encourage RGCs cal and molecular barriers after injury that can when and why. regeneration (29), but again, not the full distance

prevent RGC axons from regrowing. Some of back into the brain. Downloaded from these include chondroitin sulfate proteoglycans Intrinsic factors that limit (CSPGs) and tenasins (11, 13). Lesions of the ro- RGC regeneration Reconnection to targets in the brain dent induced in a context of minimal RGC axons down-regulate their axon growth AfewstudiesshowregenerationofRGCaxons astrocyte reactivity allow for robust axon re- speed more than 1000-fold as they transition beyond the optic chiasm into the brain. En- generation, even through myelin (14), under- from embryonic to postnatal ages, likely because hancement of mTOR combined with augmen- scoring the extent to which astroglial scarring of molecular programs intrinsic to RGCs (22). As tation of adenosine 3′,5′-monophosphate (cAMP) might limit RGC regeneration. Other work shows, RGCs mature, they down-regulate expression of and injections of oncomodulin promoted long- however, that glial scars can actually promote phosphorylated mammalian target of rapamycin range regeneration of RGC axons (30)tobrain regeneration in the rodent spinal cord (15). The (phosphor-mTOR), a growth-promoting molecule. structures, including the dorsal lateral genic- field of visual repair awaits studies that evaluate Deletion of an mTOR inhibitor, phosphatase and ulate nucleus (dLGN), which relays visual in- the role of scar-related factors in the optic nerve tensin homolog (PTEN) in RGCs, greatly enhances formation to the cortex. Mice that received the in vivo. This needs to be addressed directly in their axonal regeneration capacity after injury combined treatment of PTEN-knockdown/cAMP- the visual system because the wiring architec- (23). Some regenerated axons even extend from increase/oncomodulin also recovered some visual

SCIENCE ture of the eye-to-brain pathway differs vastly the lesion site located just behind the eye all the function, although the connections eventually from that of the spinal column. Spinal lesions way to the optic chiasm, a distance of many mil- regressed. typically injure axons at locations that allow limeters. This degree of regeneration represents Therefore, strategies must support both RGC for collateral (side-branch) sprouting around the atriumphforthefield,andwhencombinedwith regeneration and long-term survival of circuits. lesion, something not possible for RGC lesions knockdown of SOCS3, mTOR enhancement causes Some of the same factors that can promote regen- located near the optic nerve head (Fig. 1). even more RGCs to regenerate (24). eration also can promote degeneration of RGCs Lesions can cause local production of inflammation- Combining mTOR activation and SOCS3 inhi- after injury. Removal of dual leucine zipper ki-

GRAPHIC: ADAPTED BY N.related CARY/ cytokines such as interleukin-6, leuke- bition is promising, but caveats pertain when nase (DLK) from RGCs enhances their survival

Laha et al., Science 356,1031–1034 (2017) 9 June 2017 2of4 after optic nerve injury by altering apoptotic Similarly, in the spinal cord the central branch regeneration, may reflect the lack of axon mye- pathways but also limits PTEN-knockdown–based of dorsal root neurons (DRGs) regenerates in a lination or perhaps failure of correct proteins to enhancement of axon regeneration (31). topographic- and laminar-specific manner, unless reaccumulate at retino-dLGN synapses (38). The myelin-associated factors such as Nogo are per- good news is that with modern approaches such Neural activity facilitates turbed (36). Axon-guidance molecules that serve as single-cell RNA-sequencing that enable the RGC axon regeneration in development to direct RGC axons to their targets genetic makeup of specific sets of neurons to be During development, spontaneous and visually may thus get up-regulated after injury, a feature evaluated, one can now compare the molecular driven electrical activity refine RGC connec- known to occur in cold-blooded that attributes of normal and regenerated CNS neu- tions (32)andenhanceRGCaxonoutgrowthby naturally add new RGCs across their life span (37). rons and synapses. increasing responsiveness to trophic factors (22). In the adult animal, increasing RGC firing per- Transplantation of RGCs mits their axons to regenerate through optic Therapeutic intervention with regeneration- nerve lesions (Fig. 2) (25, 33)andbooststheim- inducing stimuli should be successful only dur- pact of molecular stimulants of axon growth such “What strategies support ing a limited period after injury, when RGCs as mTOR (25). Conversely, reducing activity levels are still alive. Although the mammalian retina of RGCs inhibits their survival after damage (25). RGC axon regeneration after may harbor a stem cell population in the ciliary Increasing RGC firing alone was not sufficient to margin (39), there is no evidence they replace support RGC axon regeneration into the brain damage? Can regenerating RGCs damaged by injury or disease. The lack of unless phosphor–mTOR was increased (25). RGC axons form functional endogenous RGC replacement in is RGC axons that regenerate back to their tar- in stark contrast to the scenario in fish and gets apparently fail to undergo myelination and synapses with their targets , which add new RGCs throughout therefore suffer slower conduction of electrical in the brain?” their life span, a feature thought to arise at least potentials (34). RGC firing induces myelinating in part from the presence of a specific proneural oligodendrocytes during development (35)but transcriptional factor, Ascl1, made by retinal apparently not during adulthood. This informs Thresholds for reversing blindness Müller glia in cold blooded vertebrates but not

us that only some of the mechanisms that ini- Regeneration of axons is only part of the story; by mammalian Müller glia (40). on June 9, 2017 tially establish visual circuitry are available for restoration of functional visual capacity is re- Three general approaches to replace RGCs reactivation in adulthood; others may need to quired. The mere presence of regenerated RGC include (i) syngeneic transplantation of adult be replaced. axons at a target in the brain does not predict induced pluripotent stem cells (iPSs) that have visual function (25, 34). For example, combining been programmed to assume RGC-phenotypes, Target and cell-type specificity of RGC activity with mTOR enhancement led to (ii) allogeneic transplantation of RGCs from regenerated connections the recovery of the mice’s visual reflex to avoid healthy eyes into host eyes, and (iii) possible Can regenerating RGC axons rewire appropri- overhead looming stimuli (25), but even when reprogramming of endogenous Müller glia into ately in the brain? One idea is that adult mam- RGC axons regenerated to the dLGN, those RGCs (40). iPS cells with RGC-like characteristics malian CNS circuits avoid regeneration because connections failed to lead to enhanced depth have been created in vitro (41)butnotyetused it is better to have no regeneration than incorrect —awell-establishedpropertyofthe to rebuild functional eye-to-brain circuitry. regeneration. Evidence, however, indicates that retino-dLGN-cortical pathway (25). The fact that Allogeneic transplantation of RGCs led to sub- regenerated RGCs form correct connections and some visual functions are restored whereas oth- stantial integration of RGCs into existing ret- http://science.sciencemag.org/ avoid targets incorrect for their function (25). ers are not, even in the presence of structural inal circuitry in rats (42). The transplanted RGCs

Allogeneic transplantation RGCs from healthy eyes into host eyes may represent a viable strategy for curing irreversible forms of blindness Site of optic nerve damage Downloaded from

Transplanted Intrinsic signals Complete integration donor RGCs drive initial neurite into retina and axon and axonal outgrowth grows into optic nerve SCIENCE

Fig. 3. Transplantation of RGCs to restore vision. (Left and top) Injected donor RGCs (red) differentiate and integrate into the retina after nerve damage, whereas endogenous RGCs (blue) degenerate. (Bottom right)AsubsetofthetransplantedRGCsextendaxons(redcables)downtheoptic

GRAPHIC: ADAPTED BY K.nerve SUTLIFF/ and ultimately reach the brain (41).

Laha et al., Science 356,1031–1034 (2017) 9 June 2017 3of4 REPAIR AND REGENERATION

adopted on-type or off-type or on-off-type photic with light-activated channels are both now ready A responses to light signals and extended axon pro- for testing in humans. It seems likely that a combi- jections from the eye and to central visual targets nation of therapies may be needed to get full re- in the brain (Fig. 3) (42). Thus, the isolation of RGCs covery of visual function. Regardless, the idea of from the of recently deceased humans for regenerating eye-to-brain connections in humans transplantation into recipient humans may ac- is becoming an exciting and realistic possibility. tually represent a clinically viable strategy for curing otherwise irreversible forms of blindness. REFERENCES AND NOTES 1. T. Baden et al., Nature 529,345–350 (2016). Synthetic materials for replacing 2. H. A. Quigley, A. T. Broman, Br. J. Ophthalmol. 90,262–267 (2006). light-driven electrical responses 3. J. L. Goldberg, B. A. Barres, Annu. Rev. Neurosci. 23,579–612 When blindness results from disabled retinal (2000). 4. L. C. Case, M. Tessier-Lavigne, Curr. Biol. 15,R749–R753 light sensing, two approaches are likely to prove (2005). helpful: (i) prosthetic devices that directly stim- 5. P. M. Richardson, U. M. McGuinness, A. J. Aguayo, Nature 284, ulate the RGCs or (ii) gene therapy to activate 264–265 (1980). Light stimulation quiescent remaining photoreceptors. Flexible 6. A. J. Aguayo, M. Vidal-Sanz, M. P. Villegas-Pérez, G. M. Bray, B Ann. N. Y. Acad. Sci. 495,1–9(1987). microarrays could be implanted into the hu- 7. M. E. Schwab, Curr. Opin. Neurobiol. 14,118–124 (2004). man eye to convert light to electrical signals and 8. J. L. Goldberg et al., J. Neurosci. 24,4989–4999 (2004). then passed to RGCs (Fig. 4, A and B). The effici- 9. B. Zheng et al., Proc. Natl. Acad. Sci. U.S.A. 102,1205–1210 Pulse generator ency of some of the modern implantable electrode (2005). 10. D. Fischer, Z. He, L. I. Benowitz, J. Neurosci. 24,1646–1651 arrays is comparable with light stimulation in (2004). terms of generation of action potentials in the 11. M. T. Fitch, J. Silver, Exp. Neurol. 209,294–301 (2008). inner retina of animal models (43). The preci- 12. J. B. Zuchero, B. A. Barres, Development 142,3805–3809 sion with which these devices can stimulate the (2015). Electrical signal 13. G. Yiu, Z. He, Nat. Rev. Neurosci. 7,617–627 (2006). visual pathways is impressive; some are starting 14. S. J. Davies et al., Nature 390,680–683 (1997).

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SCIENCE signals by electrodes, which generate spiking mine which specific molecular pathways are safe 47. B. W. Jones et al., J. Comp. Neurol. 464,1–16 (2003). 48. C. Gall et al., PLOS ONE 11,e0156134(2016). in RGCs, restoring eye-to-brain communication to trigger in humans and how to combine those and, potentially, sight. (C)Adeno-associated with protocols that support RGC survival and ACKNOWLEDGMENTS viruses (AAVs) can be used to deliver light- regrowth. Research will determine whether these Work in the laboratory of these authors was supported by the sensitive ion channels (halorhodopsin) to methods enhance visual function in humans, and National Eye Institute, the Glaucoma Research Foundation, the degenerated photoreceptors, allowing light whether that function derives from RGC axon McKnight Foundation, the Pew Charitable Trusts, and the E. Matilda Ziegler Foundation for the Blind. stimulation at the appropriate wavelength to regeneration, from central plasticity or both (48).

GRAPHIC: ADAPTED BY N.generate CARY/ spiking in the RGCs. Meanwhile, prosthetic implants or gene therapy 10.1126/science.aal5060

Laha et al., Science 356,1031–1034 (2017) 9 June 2017 4of4 Regenerating optic pathways from the eye to the brain Bireswar Laha, Ben K. Stafford and Andrew D. Huberman (June 8, 2017) Science 356 (6342), 1031-1034. [doi: 10.1126/science.aal5060]

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