In the August issue of Nature Neuroscience, Lim and coworkers
reported on their study of axonal regeneration in
the adult optic nerve, revealing that combined activation
of mammalian target of rapamycin (mTOR) signaling and
enhancing neural activity leads to successful restoration
of axonal trajectories to visual brain centers (1). Here, we
shed light on their breakthrough findings and how these
may represent a major leap forward in the search for novel
strategies for central nervous system (CNS) repair.
Along the line of evolution, animals have lost the capacity
to repair their CNS. The presence of axonal outgrowthrepressing
molecules, the lack of neurotrophic factors and
axonal guidance cues, and the repressed intrinsic growth
state of adult neurons, have turned the adult mammalian
CNS into a hostile environment for regenerating neurons/
axons. Because of this limited ability of self-repair, in
conjunction with the lack of preventive treatments, CNS
injury and diseases lead to irreversible, often progressive
debilitation and have a tremendous impact on the patients’
quality of life. Furthermore, given the rising life expectancy
and the progress in other medical fields (e.g., cardiovascular
and cancer treatments), neurodegenerative diseases have
turned into an increasing socio-economical challenge and
research into “healthy aging” has finally gained attention of
policy makers.
In recent years, the focus of neuroregenerative research
has somewhat shifted from manipulating extrinsic inhibitors
of axonal regeneration to reprogramming the intrinsic
growth capacity of neurons. Various experimental paradigms
in animal models of CNS injury were tested with varying
success in order to rebuilt axonal trajectories, and the optic
nerve has proven a particularly valuable research model
in this context. Research into several signaling pathways
for intrinsic growth control (e.g., JAK/STAT, PI3K/Akt/
mTOR and MAPK/ERK) has led to the identification of
multiple molecules that regulate axogenesis. Among them,
the mTOR kinase stands out as one of the most promising
factors to promote CNS regeneration. Upon axonal injury,
mTOR activity is markedly reduced and maintenance
of pre-injury mTOR activity levels, e.g., via inhibition/
downregulation/deletion of phosphatase and tensin
homolog (PTEN)—which at least in part acts by enhancing
mTOR activity—induces axonal regeneration in the adult
mammalian CNS. Nevertheless, most potent regeneration
has been obtained by combining mTOR activation with
other treatments affecting intrinsic or extrinsic factors, such
as SOCS3 deletion, c-myc overexpression, and hyper IL-6
expression (2-4). Although these combinatorial experimental
treatment paradigms can induce robust sprouting of retinal
ganglion cell axons and navigation beyond the optic chiasm,
correct navigation and synapse formation with their proper
brain targets seems still out of reach.
With their study, Lim et al.(1) add a novel strategy to
this growing list of pro-regenerative interventions, i.e., increasing electrical activity of retinal ganglion cells to
enhance regrowth of their axons. This builds upon a large
body of evidence—both from preclinical work and clinical
trials—for its success in peripheral nerve regeneration,
where electrical stimulation is a well-known treatment
to promote neural regeneration and functional recovery(5,6). The molecular mechanism of this stimulated
peripheral nerve regeneration is believed to largely revolve
around an elevation of cyclic AMP (cAMP) in response to
electrical stimulation, which activates a variety of pathways,
including the CREB transcription factor, which leads to
subsequent upregulation of neurotrophic factors and their
receptors, as well as various other growth associated genes (7-9). Interestingly, evidence for a neuroprotective and
regeneration-promoting effect of electrical stimulation
has also been found in the retina, thereby suggesting
similar mechanisms to be at play in the CNS. Indeed, in an
in vitro study, Goldberg et al. (10) demonstrated that retinal
ganglion cell survival and neurite outgrowth greatly increase
upon electrical stimulation. Similarly, stimulating retinal
neurons with a transcorneal electrode proved to increase
in vivo survival and axon preservation following optic nerve
axotomy and crush, respectively (11,12).
In their manuscript “Neural activity promotes longdistance,
target-specific regeneration of adult retinal axons”,
the research group led by prof. Huberman integrated two
experimental paradigms in order to create a synergistic
effect on axonal regeneration in the optic nerve. They
cleverly combined activation of mTOR signaling with
visual stimulation to boost neuronal activity in the
damaged neurons. Whereas mTOR activation appeared a
prerequisite for axonal outgrowth, enhancing neural activity
of the injured neurons proved to be the indispensable
trigger in this study to not only induce axonal outgrowth
but also navigation and target reinnervation. Furthermore,
with an elegantly designed set of experiments in which
neural activity was either abolished or promoted by use
of Designer Receptors Exclusively Activated by Designer
Drugs (DREADD)-based chemogenetic tools, Lim et al.
were able to pinpoint neuronal spiking as the driving force
for this regeneration-promoting effect. Notably, although
their combined treatment did successfully induce axonal
outgrowth beyond the optic nerve lesion site, it appeared
only modestly effective in doing so—e.g., more robust
sprouting is seen upon pten deletion. What is so striking
about this study, however, is that these axons were capable
of long-distance navigation and even reinnervation
of several nuclei of the central and accessory visual system. Besides the work of the Benowitz (13) and Park
laboratories (14), who reported axonal regeneration up to
the superior colliculus and suprachiasmatic nucleus,
respectively, no study ever described a successful
reinnervation of as many visual system nuclei as now
observed by Lim et al. Tracing studies of genetically
labelled retinal ganglion cell subtypes, moreover, confirmed
that their axons travelled to the proper target neurons in
the brain, while partial recovery of some vision-driven
behaviors confirmed that at least part of them successfully
established new synapses. Overall, these data provide the
first indication of an experimental treatment that can
overcome (some aspects of ) optic nerve injury-induced
blindness by reactivating the intrinsic growth potential of
retinal ganglion cells. Notably, whereas the first part of
the study stands out for its exquisite experimental design
and cutting-edge technology, the second half—describing
long-distance regeneration to and functional recovery in
several brain targets—is somewhat more exploratory and
comes with a few limitations. In particular, the possibility
that spared rather than regenerating axons are (at least
in part) responsible for the observed effects has not been
convincingly excluded. The critical reader would therefore
be looking forward to follow-up studies with a deeper focus
on the timing and success of target reinnervation, e.g., with
increased animal numbers, extensive visual testing before,
immediately after and at late time points post lesion, and
additional electrophysiological read-outs in the brain
target areas.
The novel experimental paradigm to enhance visual
system repair introduced by Lim et al. provides an
outlook on what future CNS regenerative therapies may
look like. Visual stimulation—as a proxy for enhancing
neuronal electrical activity—initiates intracellular signaling
mechanisms for cell survival and neurite outgrowth. By
influencing protein synthesis, modification and activation,
with or without altering gene expression, it enhances an
endogenous network of signaling cascades and therefore
elicits a more potent response than any other intervention
targeting—or should one say, outbalancing?—a single
molecule or pathway. Furthermore, its effect is likely to be
multifactorial, potentially not only affecting the intrinsic
growth capacity of a neuron. Electrical stimulation of
neurons has indeed been suggested to also alter their
metabolic regulation, neurotrophin secretion, receptor
profile, responsiveness of growth cones to extrinsic growth/
guidance cues, synapse formation/stabilization, etc. (15).
This fits the idea that a future CNS restorative therapy will highly likely be a combinatorial treatment, tackling
the multiple underlying causes of the adult CNS poor
regenerative capacity. Notably, in line with this multi-target
approach, a prerequisite for successful axonal regeneration
is to make sure that a sufficient population of neurons
survives the initial insult, where after they can regrow
their axons. Besides being a fruitful approach for axonal
regeneration, the treatment paradigm presented by Lim and
co-workers also proved to have a profound neuroprotective
effect (with a 30% increase in retinal ganglion cell survival),
which could have added to the robust axonal regeneration
that they observed.
At the same time, the study also unveils some challenges
that still stand in between the successful development of
therapies for CNS restoration. Mirroring the time course
of axonal regeneration, a therapy would have to induce
neuroprotection followed by axonal sprouting, elongation
and navigation, and target reinnervation. With most of
the research still focusing on robust initiation of axon
outgrowth, the current thinking is that promoting correct
navigation over long distances and anatomical decision
points, is the next big challenge in this research field. The
data presented by Lim et al., however, suggest that axonal
guidance might be less of an issue than expected. Again, the
cellular and molecular mechanisms that have been tied to
enhancing neuronal activity via electrical stimulation, might
explain. These include alignment of astroglial processes—
which serve as guiding scaffolds for growing axon growth
along them—favoring growth cone navigation over stalling/
retraction, enhancing the intrinsic sensitivity of a neuron
to growth-promoting factors and cues. Or, alternatively,
as suggested by the authors, ligands and receptors that
mediate developmental axon navigation in the CNS may
still be present and/or become upregulated upon injury.
This finally leads us the ultimate stages of CNS repair:
synapse formation and refinement, as well as remyelination.
The study of Lim et al. did not explore these in depth, yet
the varying degree of functional recovery for different visual
tasks—corresponding to different brain nuclei in the visual
pathway—that they observed, clearly indicates that more
work is needed. Of note, adding to the holistic approach
of neural activity stimulation, neuron-to-target activity is
important for stabilizing synapses.
To conclude, this report extends the existing literature on
the beneficial effects of neural activity on neuroprotection
and axonal regeneration, which all used electrical
stimulation (15). Over the last decade, new methods to
artificially control neural activity have become available,
such as optogenetics and chemogenetics using DREADDs.
Both methods show great promise by allowing genetic
targeting of specific cell populations, which might prove
to be the next scientific breakthrough—given that activity
patterns will likely need be tailored to neuronal subtypes to
further increase efficacy—and a significant advantage over
non-specific electrical stimulation. Whereas these methods
are well-established tools to dissect neural circuitry, this
report now also highlights their potential in the fields of
CNS regeneration and protection (16,17). The take-home
message of the article by Lim et al. thus is a message of
hope, pointing out that a fairly easy-to-translate approach
such as neural stimulation may force a breakthrough in the
fight for sight and CNS repair in general.
