ArticlePDF AvailableLiterature Review

Can injured adult CNS axons regenerate by recapitulating development?

October 2017
Development 144(19):3417-3429

October 2017
144(19):3417-3429

Authors:

University of British Columbia

In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS.

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REVIEW

Can injured adult CNS axons regenerate by recapitulating

development?

Brett J. Hilton*and Frank Bradke*

ABSTRACT

In the adult mammalian central nervous system (CNS), neurons

typically fail to regenerate their axons after injury. During

development, by contrast, neurons extend axons effectively. A

variety of intracellular mechanisms mediate this difference,

including changes in gene expression, the ability to form a growth

cone, differences in mitochondrial function/axonal transport and the

efficacy of synaptic transmission. In turn, these intracellular

processes are linked to extracellular differences between the

developing and adult CNS. During development, the extracellular

environment directs axon growth and circuit formation. In adulthood,

by contrast, extracellular factors, such as myelin and the extracellular

matrix, restrict axon growth. Here, we discuss whether the

reactivation of developmental processes can elicit axon

regeneration in the injured CNS.

KEY WORDS: Axon regeneration, Central nervous system,

Development, Spinal cord, Spinal cord injury

Introduction

Neurons transmit signals to their targets through specialized cellular

processes called axons. These processes extend to other neurons,

muscles, sensory organs and glands, conducting electrical impulses

across long distances. The axons of most neurons grow towards their

targets during embryonic development. The neuron differentiates

and elaborates processes. It specifies one axon and several

dendrites, which are shorter signal-receiving processes (Witte and

Bradke, 2008). The axon elongates towards its target by sensing

guidance molecules (O’Donnell et al., 2009). After reaching its

target, the axon forms a specialized connection there called a

synapse (Shen and Scheiffele, 2010). The coordinated growth and

connectivity of axons during development is an impressive feat. In

mammals, billions or trillions of synapses are formed between

neurons and targets that are often long distances away from each

other (Kasthuri et al., 2015; Sporns et al., 2005). As such,

developmental axon growth is an intricate process. It relies on an

axonal environment that enables directed elongation (Seiradake

et al., 2016) and on a variety of intracellular mechanisms that allow

the developing neuron to grow its axon long and rapidly (O’Donnell

et al., 2009).

In contrast to the situation in development, and except in a few

unusual cases (Fenrich and Rose, 2009; Jin et al., 2016; Omura

et al., 2015), the axon of an adult mammalian neuron cannot grow

back to its target in the central nervous system (CNS) following

injury; the membrane of the axon reseals to re-establish ionic

homeostasis (Bradke et al., 2012) but axon growth is typically

limited. Most adult neurons fail to express the genes that orchestrate

developmental axon elongation and instead express genes that

restrict growth (He and Jin, 2016). Most adult neurons may also lack

the energy necessary for axon regeneration (Zhou et al., 2016). The

environmental cues that attract the axon to its target during

development are not appropriately expressed in the adult system

(Giger et al., 2010). Instead, the adult environment responds to

injury by expressing factors that impede growth (Cregg et al., 2014;

Schwab and Strittmatter, 2014). In some cases, such as in the

descending corticospinal and rubrospinal systems, and in the

primary sensory neurons that ascend to the brain in the direct dorsal

column pathway, adult neurons survive axonal injury but remain

disconnected from their targets (Kwon et al., 2002; Nielson et al.,

2010; Ylera et al., 2009). In other cases, such as in most subtypes of

retinal ganglion cells (RGCs), injured neurons respond to axon

severance (axotomy) by eventually dying (Duan et al., 2015). Axon

regeneration failure can permanently disrupt CNS connectivity and

can lead to substantial dysfunction in cases of trauma, stroke or

neurodegenerative disease.

A tantalizing strategy for enhancing axon regeneration is to

recapitulate the processes that underlie developmental axon growth

within adult neurons. By examining the processes that mediate axon

outgrowth during development, it may be possible to reactivate the

developmental growth program within the adult neuron and to

unleash robust axon regeneration. Indeed, many experimental

therapeutic strategies to enhance CNS regeneration that

manipulate the extracellular environment or intrinsic growth

capacity of a neuron converge on processes underlying

developmental axon growth (He and Jin, 2016; Schwab and

Strittmatter, 2014). Still, not every process that mediates axon

regeneration is active during embryonic development, and there are

clear differences between these forms of axonal elongation.

In this Review, we highlight the cellular and molecular

mechanisms that orchestrate axon growth during development in

various mammalian species. We then examine how these are

impeded or fail to be activated following adult injury. We describe

divergences between developmental and regenerative axon growth,

and discuss recent work showing that it is possible to promote

regeneration by reactivating developmental growth processes in

adult neurons following CNS injury. Ultimately, a further

understanding of how axons extend and form functional circuits

during development may allow us to recapitulate these processes in

adult neurons and to restore connectivity after disease or injury.

Axon regenerative capacity in the mammalian CNS: a

developmental decline

Although axon regeneration is limited in the adult mammalian CNS,

this is not the case when the CNS is immature. For example,

opossums (such as Monodelphis domestica) are born with an

Laboratory for Axon Growth and Regeneration, German Centre for

Neurodegenerative Diseases (DZNE), Sigmund-Freud-Strasse 27, 53127, Bonn,

Germany.

*Author for correspondence (brett.hilton@dzne.de; frank.bradke@dzne.de)

B.J.H., 0000-0003-2813-2294

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DEVELOPMENT

immature CNS, including a two-layer cortex and a rudimentary

cerebellum (Kraus and Fadem, 1987). Axons injured early after

birth regenerate and form functional circuits (Saunders et al., 1995).

However, the CNS restricts axon growth after a developmental

transition. For example, retinal ganglion cells (RGCs) in the

opossum regenerate if their axons are injured before postnatal day 12

but not afterwards (MacLaren and Taylor, 1997). Although the

timeframe varies among organisms and pathways, this transition is

virtually ubiquitous in mammals (Nicholls and Saunders, 1996). It

has been referred to as a critical period of CNS axon regeneration,

and understanding its mechanistic basis has been a focus of

regeneration research for many decades.

Classically, it has been thought that extracellular molecules

expressed in adulthood after injury but not during development

underlie this transition. Oligodendrocytes start myelinating axons at

around the same time as the transition and their myelin inhibits axon

growth (Schwab and Strittmatter, 2014). Similarly, astrocytes

respond to developmental and adult lesions differently (Cregg

et al., 2014). However, when neural progenitor cells (NPCs) are

transplanted into the injured adult mammalian spinal cord and

provided with trophic factors and a fibrin matrix, they differentiate

into neurons with a virtually limitless capacity for axon growth (Lu

et al., 2012). These results demonstrate that, when properly

stimulated and in the right environment, newly differentiated

neurons can grow axons that are very long after adult injury. There

are many mechanisms that contribute to the growth capacity of

immature neurons. In the first part of this Review, we focus on

intracellular mechanisms underlying this higher growth capacity

(Fig. 1). In the second part, we discuss how the extracellular

environment changes as the CNS matures in a way that contributes

to regeneration failure. Importantly, intracellular and extracellular

factors are not separate: there is a complex interplay between them,

as the intrinsic growth capacity of a neuron is at least partially

instructed by extracellular interactions (Burnside and Bradbury,

2014; Geoffroy and Zheng, 2014; Tedeschi and Bradke, 2017).

The developing and adult CNS: intracellular factors involved

in regeneration

Gene expression

The difference in gene expression between immature and mature

mammalian neurons is a primary factor that underlies their

differential capacity for axon growth. The capacity of a neuron to

regenerate is defined in part by its expression of regeneration-

associated genes (Tetzlaff et al., 1991). The expression of these

genes is activated in developing neurons and is restricted in adult

neurons (Puttagunta et al., 2014; Tedeschi and Bradke, 2017). For

example, developing mammalian neurons express Krüppel-like

factor 7 (KLF7) and Sox11, but downregulate these transcription

factors as they mature (Blackmore et al., 2012; Moore et al., 2009;

Wang et al., 2015). KLF7 or Sox11 activation in the adult neuron

promotes axon regeneration (Blackmore et al., 2012; Wang et al.,

2015). Similarly, the α9 integrin receptor is downregulated as

primary sensory neurons mature, and the overexpression of α9

promotes axon regeneration (Andrews et al., 2009). In most cases,

transcription factors have been tested singularly for roles in

regeneration. However, bioinformatic approaches have recently

determined functional interactions between pathways involved in

axonal outgrowth and may reveal transcription factor combinations

that can optimize regeneration (Belin et al., 2015; Chandran et al.,

2016; Venkatesh and Blackmore, 2016).

Instead of expressing regeneration-associated genes, mammalian

adult neurons express genes that restrict axon growth. In a screen of

genes differentially expressed by RGCs during the developmental

transition to a restricted capacity to regenerate, Moore and

colleagues identified KLF4 as a transcriptional repressor of

regeneration (Moore et al., 2009). RGCs do not express KLF4

during development but upregulate it upon maturation, when it

restricts growth. Why adult neurons express genes that prohibit

regeneration is unclear. One possibility is that the expression of

these genes is an evolutionarily conserved mechanism to prevent

ectopic axon growth and aberrant synapse formation (Tedeschi and

Bradke, 2017). Indeed, synaptogenesis instructs the expression of

genes that prevent axon outgrowth (Tedeschi et al., 2016), as we

discuss later.

At the translation level, developing neurons may be able to more

effectively generate proteins involved in axonal elongation than

adult neurons (Shigeoka et al., 2016), and signalling via the

mechanistic target of rapamycin (mTOR) pathway may be involved

in this difference (Park et al., 2008). Developing neurons have high

levels of mTOR signalling but this decreases as they mature (Liu

et al., 2010; Park et al., 2008). Enhancing mTOR signalling by

knocking out phosphatase and tensin homolog (Pten) boosts axon

regeneration (Geoffroy et al., 2016; Liu et al., 2010; Park et al.,

2008). The mTOR pathway is pivotal for regeneration following

Pten knockout (Park et al., 2008). Although mTOR regulates many

intracellular events (Laplante and Sabatini, 2012), its activation of

protein translation is particularly important for regeneration (Yang

et al., 2014). To stimulate translation, mTOR activates S6 kinase 1

(S6K1) to generate new ribosomes (Chauvin et al., 2014), and S6K1

activation enhances axon regeneration in the mouse optic nerve

(Yang et al., 2014). In addition to S6K1, mTOR activates eukaryotic

initiation factor 4E (eIF4E) to initiate cap-dependent protein

translation (Brunn et al., 1997). Accordingly, it has been shown

that EIF4E is necessary for regeneration following Pten knockout in

adult mice (Yang et al., 2014). Together, these results support a

model in which developing neurons signal through mTOR to

translate proteins more effectively than adult neurons do, and

suggest that enhancing mTOR-mediated protein translation in

adulthood can boost axon regeneration. However, it has been

reported that S6K1 restricts mouse corticospinal axon regeneration

(Al-Ali et al., 2017). These results highlight the complexity of

mTOR signalling in regeneration and suggest that more research is

needed to understand how different effectors of the PI3K/Akt/

mTOR pathway influence axon growth.

Growth cone formation

Developmental axon growth relies on the formation of a subcellular

structure at the distal tip of the axon called the growth cone

(Fig. 2A). The growth cone comprises an actin-rich peripheral

domain and a microtubule-rich central domain, separated by a

transition zone in which dynamic interactions between these

cytoskeletal elements take place (Coles and Bradke, 2015). It

serves vital functions both in the guidance of the axon to its target

and in the process of axonal outgrowth itself (Dent et al., 2011). At

its distal tips, finger-like filopodia and sheet-like lamellipodia

rapidly extend and retract as they sense the axonal

microenvironment. During outgrowth, filamentous actin (F-actin)

polymerizes, driving the extension of new membrane at the distal

edges of the growth cone (Goldberg and Burmeister, 1986). The

proximal growth cone then stabilizes into the axonal shaft,

elongating the axon (Dent et al., 2011; Goldberg and Burmeister,

1986). After synaptogenesis, axon growth continues to occur,

corresponding to the size of the growing organism, but it does so via

stretch-based mechanisms (Smith, 2009).

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After injury, the formation of a growth cone or growth cone-like

structure is vital for axon regeneration (Bradke et al., 2012). Injury

exposes the interior of the axon to calcium, triggering membrane

sealing and growth cone formation. Peripheral nervous system

(PNS) axons form new growth cones after injury in order to

regenerate (Ertürk et al., 2007). However, CNS axons fail to form

growth cones (Bradke et al., 2012; Ertürk et al., 2007). Instead, they

are tipped with dystrophic retraction bulbs; these structures

accumulate anterogradely transported vesicles and mitochondria,

and show microtubule disassembly (Fig. 2A-B). In the human

spinal cord, retraction bulbs have been observed more than four

decades after injury (Ruschel et al., 2015), highlighting their role in

regeneration failure.

Growth cones contain organized microtubules that form tight

bundles parallel to the axonal axis, whereas retraction bulbs have

highly dispersed and disorganized microtubules (Ertürk et al., 2007).

Application of the microtubule-destabilizing agent nocodazole

transforms growth cones into retraction bulb-like structures in vivo,

resulting in halted axon growth (Ertürk et al., 2007). Conversely,

microtubule stabilization through the administration of taxol

interferes with retraction bulb formation and facilitates growth

cone formation in vivo (Fig. 2B,C). When applied in vivo after

CNS injury, taxol administration boosts optic nerve axon regeneration

(Sengottuvel et al., 2011) and serotonergic axon regeneration (Hellal

et al., 2011). Microtubule stabilization also promotes locomotor

recovery in rodent models of traumatic brain injury (Cross et al.,

2015) and spinal cord injury (Hellal et al., 2011). Since microtubule

stabilization drives axon outgrowth during development (Gomis-

Rüth et al., 2008; Witte et al., 2008), these studies highlight how

regeneration can be achieved by activating developmental processes.

Importantly, microtubule stabilization can be stimulated

noninvasively through systemic administration of epothilone B

(Ruschel et al., 2015). This drug binds to the αβ-tubulin heterodimer

subunit of microtubules, decreasing its rate of dissociation and

thus stabilizing the microtubule (Goodin et al., 2004). In vitro,

epothilone B encourages growth cone formation (Ruschel et al.,

2015), while in the adult rat, epothilone B administration

improves skilled locomotion and enhances serotonergic axon

growth following spinal cord injury (Ruschel et al., 2015). These

functional improvements are abrogated by pharmacological ablation

of serotonergic innervation, suggesting that the serotonergic axon

growth is necessary for recovery. In addition to influencing

growth cone formation, microtubule stabilization reduces fibrotic

scarring by mitigating fibroblast polarization and migration

(Hellal et al., 2011; Ruschel et al., 2015) (Fig. 2C,D). Thus,

microtubule stabilization may be a viable strategy for promoting

axon regeneration and functional improvements following CNS

injury.

KLF7

Sox11

KLF4

DNA

Regeneration-associated

gene

Growth-restricting gene

Motor

protein Mitochondrion

Depolarized

mitochondrion

Syntaphilin

Miro/milton complex

ATP

ADP

B Injured adult neuron failing to regenerate axon

A Developing neuron with growing axon

Microtubule

Retraction

bulb

Growth

cone

Nucleus

Key

Fig. 1. Intracellular processes in a developing neuron and its growing axon, and in an adult neuron failing to regenerate. (A) A developing neuron with a

growingaxon. Developing neurons express regeneration-associated genes, such as Klf7 and Sox11, that facilitate axon growth.Mitochondria are highly motile in the

axon, are associatedwith Miro/milton complexes and aretransported by motor proteins (kinesinand dynein) along microtubules. ATPis at a level sufficient to supply

the energy requiredfor axon growth. The axon has a growth cone at its tip (purple) that senses guidance molecules(not shown) and mediates axonal outgrowth. (B)

A schematic of an injured adult neuron failing to regenerate. Adult injured neurons express genes that restrict axon outgrowth, such as Klf4. Mitochondria

are depolarized after injury and are less motile in the adult axon due to the expression of the anchor protein syntaphilin. ATP levels are lower and ADP levels are

higher, such that the axon does not have enough energy to grow. The axon has a retraction bulb with disorganized and disassembled microtubules at its tip.

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DEVELOPMENT

Actin dynamics are also key to growth cone formation. Following

axotomy, F-actin destabilizes at the injured stump, contributing to

retraction bulb formation (Nawabi et al., 2015). Overexpression of

doublecortin like kinase 2 (DCLK2) can prevent this destabilization

and promotes both growth cone formation and axon regeneration

(Nawabi et al., 2015). DCLK2 is enriched in the transition zone of the

growth cone, at the interface between the actin-rich peripheral

domain and microtubule-rich central domain, where it is thought to

help coordinate microtubule/actin dynamics (Bielas et al., 2007).

Interestingly, although overexpression of either the microtubule-

binding domain or actin-regulatory domain of DLK2 promotes axon

regeneration, simultaneous overexpression of both does not (Nawabi

et al., 2015). These results highlight the complexity of molecular

interactions within the distal tip of the axon following injury.

Although DCLK2 encourages growth cone formation by stabilizing

F-actin, some actin destabilization may be beneficial as it permits

microtubules to protrude into distal areas of the growth cone as the

axon shaft is consolidated (Bradke and Dotti, 1999; Flynn et al.,

2012). Understanding the role of actin in different compartments of

the growth cone is thus essential for understanding how it influences

axon regeneration. Moreover, how the neuron coordinates actin/

microtubule dynamics to effectively elongate its axon remains an

important avenue for further research (Coles and Bradke, 2015).

Mitochondrial function and axonal transport

Axon growth is an energetically intensive process that requires

mitochondrial biogenesis and adenosine triphosphate (ATP)

production (Vaarmann et al., 2016). Although ATP diffuses freely

in the cytosol, neurons, like many other cells, traffic mitochondria to

regions of high ATP consumption (Schwarz, 2013). During axon

specification and outgrowth in development, the growth cone

contains a particularly high concentration of mitochondria (Bradke

and Dotti, 1997; Morris and Hollenbeck, 1993). Axons transport

mitochondria both in the anterograde and retrograde direction along

Fibroblasts

Fibrotic

scar

F-actin

bundles

Filopodium

Lamellipodium

Actin

meshwork

Developmental growth cone Retraction bulb

Disorganized and

disassembled

microtubules

Vesicle

Mitochondrion

Accumulation

of vesicles

C Spinal cord injury with microtubule stabilization (epothilone B or taxol treatment)

B Spinal cord injury

Organelle

components

Cytoskeletal

structure

Microtubules

Fig. 2. The growth cone orchestrates

developmental axon growth and is a

target for CNS regeneration. (A)

Cytoskeletal structure and organelle

components of a developmental growth

cone and a retraction bulb. In the growth

cone, microtubules are oriented with the

axonal axis. The distal aspect of the growth

cone is actin rich (purple) and contains

F-actin bundles, filopodia and lamellipodia.

In the retraction bulb, microtubules are

disorganized. Its actin structure is poorly

understood. Mitochondria and vesicles are

found at a higher concentration in the

growth cone than in the axon shaft. In the

retraction bulb, vesicles and mitochondria

accumulate distally. (B) After spinal cord

injury, sensory axons have retraction bulbs

at their tips and axon growth is restricted by

fibroblasts that form scar tissue at the injury

site. (C) Microtubule stabilization (using

epothilone B or taxol treatment) mitigates

fibrotic scarring and enhances the formation

of growth-cone-like structures at the distal

tips of injured sensory axons, enabling

regeneration.

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DEVELOPMENT

microtubules by using a motor/adaptor complex that contains

kinesin and dynein in addition to the proteins Miro (RhoT1/2) and

milton (Trak1/2) (Schwarz, 2013). The motor adaptor Trak1 is

required for axonal mitochondria transport (van Spronsen et al.,

2013) and its depletion inhibits axon outgrowth (van Spronsen et al.,

2013). Thus, mitochondria and their axonal transport are essential

for sustained axon growth during development.

Axotomy depolarizes mitochondria and depletes ATP in injured

axons (Cavallucci et al., 2014; O’Donnell et al., 2013; Zhou et al.,

2016). When axons do not have mitochondria, they rapidly

degenerate (Rawson et al., 2014). Conversely, boosting

mitochondrial ATP production facilitates axon regeneration.

Cytokines stimulate axon regeneration in part by inducing signal

transducer and activator of transcription 3 (STAT3) to translocate

to the inner membrane of the mitochondria (Luo et al., 2016).

There, STAT3 enhances ATP production by optimizing the

function of the electron transport chain (Luo et al., 2016;

Wegrzyn et al., 2009). In cytochrome c oxidase-deficient mice,

which have defective mitochondrial ATP production, cytokine-

stimulated RGC regeneration is reduced (Luo et al., 2016). Thus,

although the cell can produce ATP via glycolysis in the cytoplasm,

ATP generated by the electron transport chain in mitochondria

is important for axon regeneration, and the efficiency of this

process is a target for repair.

Enhancing axonal transport of mitochondria also promotes

regeneration. Adult mammalian neurons transport mitochondria in

their axons less than do developing neurons because they express

syntaphilin (SNPH), a protein that anchors axonal mitochondria to

the axon (Kang et al., 2008). In adult mouse RGCs that have a high

regenerative ability due to dual genetic knockout of Pten and

suppressor of cytokine signalling 3 (Socs3), armadillo repeat

containing X-linked 1 (Armcx1) is highly expressed (Sun et al.,

2011). Armcx1 encodes a mitochondrial protein and belongs to a

family of genes unique to placental mammals (López-Doménech

et al., 2012). The protein contains a putative outer mitochondrial

membrane-targeting sequence, flanking a transmembrane domain

necessary for its mitochondrial localization (Cartoni et al., 2016).

Overexpression of Armcx1 in adult mouse increases mitochondrial

motility and enhances RGC survival and axon regeneration (Cartoni

et al., 2016). In addition, upon simultaneous Pten and Socs3

knockout, which greatly enhances neuronal intrinsic growth

capacity (Sun et al., 2011), Armcx1 mediates neuron survival and

axon regeneration (Cartoni et al., 2016). Similarly, a high

mitochondrial density is crucial for axon regeneration in the

nematode, C. elegans (Han et al., 2016). However, it is unclear how

the axonal transport of mitochondria influences regeneration. While

enhancing ATP production in the axon is likely to be one factor,

mitochondrial density might also influence regeneration by altering

signalling through changes in metabolite production or intracellular

calcium levels (Chandel, 2014; Williams et al., 2013). It is also

known that mitochondria can regulate axon branching by

determining sites of axonal protein synthesis (Spillane et al.,

2013) and that mitochondrial deficiency can activate signalling

cascades that result in dendritic branching (Gioran et al., 2014). The

relationship between the mitochondria in the signal transduction

cascades that mediate axonal outgrowth is an important issue for

future research.

Synaptic transmission

During embryonic mammalian development, axons are connected

to far more targets than those in the adult (Luo and O’Leary, 2005).

This exuberance is mediated in part by neurons extending linearly

and rapidly past their target cells (Stanfield et al., 1982). Following

this, side branches form on the elongated axon and grow slowly into

the target area. The overshooting axon degenerates, and

terminations are refined during a protracted period of

synaptogenesis and synapse elimination (Hua and Smith, 2004;

Low and Cheng, 2006; Purves and Lichtman, 1980). Together,

exuberant outgrowth and pruning during development generate

precise connectivity in the adult (Luo and O’Leary, 2005).

After adult connectivity is established, neurons employ multiple

strategies to avoid ectopic axon growth and to maintain appropriate

synaptic connectivity (Shen and Scheiffele, 2010). These same

strategies might restrict axon regeneration and rely on synaptic

transmission. In an elegant series of experiments, Lorenzana and

colleagues used in vivo two-photon imaging of adult mouse spinal

dorsal column sensory axons to assess whether a surviving intact

branch influences the ability of the neuron to regenerate (Lorenzana

et al., 2015). These axons present a particularly good model for this

because they bifurcate into an ascending branch, which extends

rostrally towards the brain, and a descending branch, which courses

down the spinal cord. Eliminating either of these branches is

followed by a poor regenerative response but eliminating both

increases regeneration. Thus, an intact axonal process suppresses

regeneration. Although the neurons projecting these central axonal

processes do not effectively regenerate them, they can regenerate

their axonal processes in the PNS. One signal underlying this

difference is electrical activity (Enes et al., 2010). After peripheral

nerve injury, electrical activity is lost, which may signal regeneration

to initiate. In contrast, electrical activity is maintained after central

axon injury and may suppress growth by triggering an increase in

intracellular calcium concentration (Enes et al., 2010). Thus, an

intact axonal process can discourage regeneration and may do so by

maintaining electrical activity. This exciting possibility needs to be

directly tested.

The synaptic-based suppression of axon growth is something that

can be targeted therapeutically. Cacna2d2, the gene encoding the

α2δ2 subunit of voltage-gated calcium channels, limits sensory

axon regeneration (Tedeschi et al., 2016). α2δ2 subunits promote

synapse formation and enhance the probability of synaptic

transmission (Eroglu et al., 2009). Thus, they may act as a

molecular switch to synaptically suppress axonal outgrowth.

Gabapentinoids, including pregabalin and gabapentin, are

clinically approved drugs used to treat epilepsy, neuropathic pain

and fibromyalgia, which bind with high affinity and selectivity to

α2δ1/2 subunits (Gee et al., 1996). Systemic gabapentinoid

administration induces adult mouse sensory axon regeneration

following spinal cord injury (Tedeschi et al., 2016). A meta-analysis

found that spinal cord-injured individuals that received these drugs

have enhanced motor recovery if they are administered in the first

month after injury (Warner et al., 2017). Together, these promising

results pave the way for a detailed exploration of gabapentinoid use

to treat spinal cord injury and other neurological disorders

characterized by paralysis.

In summary, intracellular processes define the capacity of

developing neurons, and the inability of adult neurons, to

regenerate. In turn, these processes occur together with changes in

the extracellular environment of the neuron that are also instructive

to axon growth and restraint.

Developing and adult CNS: extracellular factors involved in

regeneration

In addition to intracellular differences between developing and adult

neurons, the extracellular environment of the neuron changes as the

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CNS matures in a way that restricts regeneration. However, it is

becoming clear that extracellular factors that guide developmental

axon growth can facilitate regeneration in the adult.

Axon guidance cues

As an axon extends during development, its growth cone senses an

array of guidance molecules expressed by the environment that

allow it to steer towards the correct target. Several phylogenetically

conserved families of guidance cues have been discovered,

including netrins, slits, semaphorins and ephrins (O’Donnell

et al., 2009). The growth cone integrates these signals and

transmits them into cytoskeletal changes that underlie steering

decisions, such as attraction and repulsion (O’Donnell et al., 2009).

Some guidance molecules that function primarily in an inhibitory

or repulsive role developmentally are expressed following adult

injury and inhibit axon growth (Giger et al., 2010). For example,

during development, Wnt proteins are expressed in spinal cord grey

matter along the rostral/caudal axis in a concentration gradient that

decreases caudally (Liu et al., 2005). Corticospinal axons express

the repulsive Wnt receptor Ryk (related to receptor tyrosine kinase),

which repels these axons to steer them away from the brain and

towards their spinal cord targets (Li et al., 2009; Liu et al., 2005)

(Fig. 3A,B). Wnt proteins are virtually undetectable in the adult

spinal cord (Fig. 3C), but their expression is induced around the

injury site following spinal cord injury, where they inhibit axon

growth (Liu et al., 2008) (Fig. 3D). Reducing the expression or

axonal detection of repulsive guidance cues can thus enhance axon

growth. Blocking Wnt/Ryk signalling enhances corticospinal axon

sprouting in adult mice after cervical spinal cord injury (Hollis et al.,

2016; Liu et al., 2008) (Fig. 3E). The corticospinal tract mediates

skilled forelimb motor control in rodents and primates (Lawrence

and Kuypers, 1968; Starkey et al., 2005). Interestingly, although

blocking Wnt-Ryk signalling can promote corticospinal sprouting,

this is not sufficient to promote forelimb motor recovery after spinal

cord injury (Hollis et al., 2016). Instead, recovery requires the

combination of this blockade with a form of rehabilitative, task-

specific training. If mice are coaxed to perform repeated reaching

movements, Ryk inhibition promotes extensive forelimb motor

recovery. Importantly, this recovery is associated with motor

cortical reorganization. Motor cortical areas that drive hindlimb

motor responses prior to injury acquire the capacity to activate

forelimb muscle groups controlled by motor neurons located rostral

to injury in Ryk-inhibited, rehabilitative trained mice. This provides

strong evidence that releasing corticospinal axons from Wnt

inhibition promotes circuit plasticity when combined with

activity-based rehabilitative approaches (Dietz and Fouad, 2014).

More generally, these results highlight the importance of

developing approaches that direct growing axons into forming

functional circuits and of strengthening connections.

In addition to blocking the action of repulsive or inhibitory

guidance cues, an alternative strategy to promote regeneration is to

provide injured axons with attractive signals that they receive

developmentally (Fig. 4). Grafting NPCs into sites of spinal cord

injury in the adult rat promotes robust corticospinal axon

regeneration via contact-based mechanisms (Kadoya et al., 2016).

However, NPCs derived from the rat spinal cord facilitate greater

regeneration than do NPCs derived from the rat brain. Moreover,

NPCs driven toward a spinal cord-like fate via treatment with

retinoic acid, which caudalizes the cells in a concentration-

dependent manner (Okada et al., 2004), also facilitate more

corticospinal axon regeneration than do brain-derived NPCs.

Interestingly, the maturity of rat NPCs following their engraftment

is not a primary factor in the regenerative response: when NPCs are

matured for 70 days prior to axonal injury, they support regeneration

as well as do immature NPCs grafted the same day as the injury

(Kadoya et al., 2016). These data support a model in which the

regional similarity of transplanted cells to the original substrate of an

axon is an essential factor in determining their capacity to support

regeneration. Understanding the molecular factors underlying this

effect may allow us to provide these signals to axons without the

need for cell transplantation (Assinck et al., 2017).

Astrocytes and the lesion site scar

Cells of the astrocyte lineage play a vital role in axon pathfinding

and circuit formation during development (Clarke and Barres, 2013;

Fitch and Silver, 1997). They secrete extracellular matrix (ECM)

Wnt

Rostral

Caudal

Wnt

Development

(P 1) Adult Adult

injury

Adult

injury

Ryk KO

Wnt

GM WM GM WM GM WM GM WM

ABCD

Ryk

Frizzled

Wnt

Ca2+

Growth

cone

repulsion

TRP

channel

(Injury site) (Injury

site)

Fig. 3. Wnt signalling directs corticospinal axon guidance during development but restricts corticospinal sprouting after injury. (A) A Wnt/Ryk

signalling pathway underlies corticospinal axon repulsion (Li et al., 2009). Wnt (blue) binds to the Ryk (orange) and Frizzled (black) receptors, leading to

activation of TRP channels (green). Calcium influx through TRP channels leads to growth cone repulsion. (B) During development, pioneer corticospinal

axons (red) steer away from the brain towards their spinal targets in white matter (WM) by sensing Wnt, a repulsive guidance cue expressed in grey matter (GM)

that instructs the axons to grow caudally. (C) In adulthood, the corticospinal tract is fully mature, with axonal sprouts extending into the GM, and Wnt is not

expressed. (D) After spinal cord injury, corticospinal axons are inhibited from sprouting by Wnt, which is expressed atthe injury site. (E) Knocking out the repulsive

Wnt receptor Ryk enables corticospinal axon sprouting around a site of spinal cord injury.

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REVIEW Development (2017) 144, 3417-3429 doi:10.1242/dev.148312

DEVELOPMENT

molecules, such as chondroitin sulphate proteoglycans (CSPGs),

that form repulsive barriers to developmental axon growth (Brittis

et al., 1992). After adult injury, astrocytes become reactive and

direct the formation of a scar at the injury site by walling off a core of

cells, including fibroblasts, macrophages, NG2 glia, pericytes and

ependymal cells (Cregg et al., 2014). This response to injury serves

an important purpose: in directing scar formation, astrocytes

mitigate inflammatory damage and are thus neuroprotective

(Faulkner et al., 2004; Hilton et al., 2016b). However, many scar

cells inhibit axon growth, typically by physically interacting with

the distal tips of axons (Filous et al., 2014) or by secreting ECM

molecules such as CSPGs (Burnside and Bradbury, 2014; Cregg

et al., 2014; Tan et al., 2011). When single mouse sensory axons are

axotomized by a laser with minimal scarring, the axons regenerate

robustly a few days after receiving a peripheral conditioning lesion

that boosts their intrinsic growth potential (Ylera et al., 2009). In

contrast, sensory neurons that are growth competent due to a

peripheral conditioning lesion cannot regenerate axons through

mature scar territory (Ylera et al., 2009).

Therapeutic strategies that target these extracellular impediments

to axon regeneration can reactivate developmental growth

processes. For example, during development, visual circuits are

plastic during a well-defined critical period that ends as the ECM

matures (Hubel and Wiesel, 1970; Pizzorusso et al., 2002).

Digesting CSPGs via treatment with chondroitinase-ABC

reactivates this developmental plasticity (Pizzorusso et al., 2002),

while chondroitinase-ABC treatment enhances axon growth in part

by facilitating expression of the same regeneration-associated genes

that mediate developmental outgrowth (Bradbury et al., 2002).

Interestingly, chondroitinase-ABC decreases the amplitude and

charge of excitatory postsynaptic currents in vitro, suggesting that

its influence on axon growth may relate to its modulation of synaptic

activity (Pyka et al., 2011). Of note, treatment with the microtubule-

stabilizing drugs taxol and epothilone B reduce CSPG deposition

and fibrotic scarring after spinal cord injury (Hellal et al., 2011;

Ruschel et al., 2015).

Immature astrocytes can support the regeneration of adult central

axons (Filous et al., 2010; Reier et al., 1986; Smith et al., 1986) and

synthesize less CSPGs than adult astrocytes do (Dow et al., 1994).

This is similar to glial cells in lower vertebrate species, which form

multicellular structures (‘bridges’) that provide a substrate onto

which axons regenerate and form new connections after injury

(Butler and Ward, 1967; Zukor et al., 2011). In zebrafish, this

bridging is directed by connective tissue growth factor a (CTGFa)

signalling (Mokalled et al., 2016). Interestingly, some adult

astrocytes retain a capacity to form bridges that facilitate

regeneration following Pten knockout in adult mouse (Liu et al.,

2010). It is tempting to speculate that axon growth could be

promoted by manipulating astrocytes into forming regenerative

bridges. However, it would be important to promote bridge

formation in a way that does not stop astrocytes from preventing

inflammatory cell loss.

Recent work has suggested that astrocytic scar formation aids

CNS axon regeneration (Anderson et al., 2016). Sensory axons

regenerate after spinal cord injury when they are administered a

peripheral conditioning lesion, trophic factors and hydrogels.

However, axons do not regenerate after this treatment if lesion site

astrocytes are depleted by selectively killing proliferating astrocytes

or preventing astrocytic STAT3 signalling (Anderson et al., 2016).

As reactive astrocytes sequester inflammatory cells to protect

nervous tissue following injury (Faulkner et al., 2004), their ablation

likely enhances inflammatory cell damage (Sofroniew, 2015),

Rostral

Caudal

Telencephalon

NPCs

Hindbrain

NPCs

Spinal cord

NPCs

Caudalized

NPCs

Human

iPSCs

Rostralized

NPCs

Neural

induction

medium

Neural

induction

medium

+ RA

CST

SCI and

NPC graft

CST

SCI (no graft)

Rostral Caudal

A E14 rat CNS

B Adult rat CNS

C NPC culture

Telencephalon

Hindbrain

Spinal cord

Fig. 4. The capability of neural progenitor cells to support regeneration is influenced by their site of origin. (A) A schematic of an embryonic day

(E) 14 rat embryo showing itsdeveloping CNS. During embryonic development, the rostrocaudal axis of the CNS is specified by morphogens such as retinoic acid

(RA), which caudalizes tissue in a concentration-dependent manner (green). This process distinguishes the spinal cord (green) from the more rostral

hindbrain (khaki) and telencephalon (orange). (B) In the adult, axons in the corticospinal tract (CST) project from the cortex to the spinal cord and fail to regenerate

after spinal cord injury (SCI). (C) Neural progenitor cells (NPCs) can be dissociated and cultured from an E14 rat CNS, embedded in a fibrin matrix with a cocktail

of trophic factors, and grafted into sites of spinal cord injury. NPCs derived from the telencephalon do not support CST regeneration into the graft (orange),

whereas spinal cord graft NPCs do (green). Hindbrain-derived NPCs (khaki) support some CST regeneration (but less than that produced byspinal-cord NPCs).

In addition, human induced-pluripotent stem cells (iPSCs) driven towards a rostralized NPC fate (orange) do not support CST regeneration but iPSCs caudalized

by treatment with RA (green) do.

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DEVELOPMENT

precluding the possibility of regeneration (Silver, 2016). Indeed,

astrocytic scar formation is driven by type 1 collagen, which

instructs astrocytes to adhere to one another by activating the

integrin/N-Cadherin signalling pathway (Hara et al., 2017).

Blockade of collagen 1 signalling in reactive astrocytes, which

prevents scar formation without depleting reactive astrocytes from

the lesion site, enhances axon regeneration (Hara et al., 2017). Thus,

although astrocyte depletion does not promote regeneration, some

astrocyte phenotypes produced in response to spinal cord injury

inhibit axon growth.

Intriguingly, recent work in mouse and rat has highlighted the

heterogeneity of reactive astrocytes following CNS disease and

injury by providing evidence that classically activated microglia

induce astrocytes to become neurotoxic (Liddelow et al., 2017).

Microglia secrete interleukin 1α(IL1α), tumour necrosis factor

(TNF) and complement component 1, subcomponent q (C1q),

inducing the formation of neurotoxic ‘A1’astrocytes that kill RGCs

after axotomy (Liddelow et al., 2017). In contrast, trophic-factor

producing ‘A2’astrocytes form in response to ischemia and may be

neuroprotective. The relationship of this type of astrocyte

heterogeneity to the bridge-forming or scar-forming

characteristics of some astrocytes is unknown. Nonetheless,

blocking the neurotoxic activity of A1 reactive astrocytes or

directing A2 astrocyte formation may promote neuron survival and

regeneration following disease or injury.

Oligodendrocytes and myelin

Myelin insulates axons and accelerates the conduction of electrical

impulses (Fancy et al., 2011). Most myelin-forming

oligodendrocytes are born in the early postnatal period at

approximately the same time as the developmental transition

occurs that restricts axon regeneration (Fancy et al., 2011;

Keirstead et al., 1992). As such, they have long been considered

potential mediators of this transition (Schwab et al., 1993).

Suppressing the onset of myelination can extend the permissive

period of functional regeneration in the chick (Keirstead et al.,

1992). Various myelin-associated molecules inhibit axon

outgrowth, including Nogo, myelin-associated glycoprotein

(MAG) and oligodendrocyte-myelin glycoprotein (OMgp)

(Schwab and Strittmatter, 2014). Nogo blockade boosts central

axon growth and circuit plasticity following a stroke in an adult rat if

followed by rehabilitation training to consolidate newly grown

axons into mature circuits (Wahl et al., 2014).

Although oligodendrocytes restrict axon growth, they may play a

vital role after injury by myelinating newly regenerated axons

(Fig. 5). If the axon has regenerated, it may require new myelin for

efficient conduction and to form a functional circuit, particularly if

that axon was well myelinated prior to injury. In adult mice,

knockout of Pten and Socs3 or overexpression of osteopontin

(OPN), insulin-like growth factor 1 (IGF1) and ciliary neurotrophic

factor (CNTF) induces RGC regeneration (Bei et al., 2016). After

regenerating, some axons form functional synapses with their

presumptive targets in the superior colliculus. However, these axons

lack myelin, fail to conduct action potentials and do not restore

visual function. Administration of the voltage-gated potassium

channel blocker 4-aminopyridine (4-AP) improves visual function

by promoting conduction in regenerated axons (Bei et al., 2016).

Neurotrophin 3 expression guides sensory axon regeneration and

synapse formation but the axons remain unmyelinated (Alto et al.,

2009). In contrast, after Pten knockout, cAMP administration and

inflammatory stimulation, regenerating RGC axons are myelinated

and assemble nodes of Ranvier (Marin et al., 2016). After NPCs are

transplanted into the injured spinal cord and differentiate into

neurons that extend axons long distances, about a quarter of their

axons are myelinated by host oligodendrocytes (Hunt et al., 2017).

As such, myelination of newly regenerated axons is not an automatic

process and varies based on the treatment that induces regeneration to

begin with. Similar to analyses of axon regeneration, assessment of

myelination in regeneration studies must be carefully interpreted

because these results can be confounded by the presence of myelin on

uninjured axons (Tuszynski and Steward, 2012).

The signals that direct myelination of regenerating axons are

unclear. Following focal chemical demyelination, oligodendrocytes

effectively regenerate myelin on denuded axons in a process that

recapitulates aspects of developmental myelination (Fancy et al.,

2011). Both oligodendrocytes and Schwann cells are capable of

regenerating myelin (Plemel et al., 2017), with oligodendrocyte

remyelination relying on myelin regulatory factor signalling

(Duncan et al., 2017) and Schwann cell remyelination requiring

neuregulin 1 signalling (Bartus et al., 2016). One possibility is that

denuded axons contain signals necessary for myelination that

regenerated axons may lack. In this regard, one potential signal is

electrical activity (Fields, 2015). Another possibility is that

myelination of regenerated axons requires inflammatory

stimulation, given that macrophages drive oligodendrocyte

differentiation during remyelination of denuded axons (Miron

et al., 2013). Understanding the signals that regulate the myelination

of regenerated axons is a new area of research that will yield

valuable insight into developing effective repair strategies.

Do adult axons continue to diminish in their regenerative

capacity with age?

Although the CNS diminishes in regenerative capacity early after

birth, one issue of substantial therapeutic relevance is whether it

continues to decline as it ages. In other words, do mid-adult-aged

neurons have even less capacity to regenerate relative to their young

adult counterparts? Age is an important risk factor in cases of stroke

and glaucoma (Leske et al., 1995; Sacco et al., 1997), both of which

would benefit from axon regeneration therapies. Additionally, the

average age of incidence of spinal cord injury has increased since

the 1970s and now occurs in the late 30s or early 40s (DeVivo and

Chen, 2011). However, assessing the role of aging in axon

regeneration has been a challenge because young adult mammals

already have an extremely low capacity to regenerate to begin with.

A number of key insights into neuronal aging have come from

studies of C. elegans. After C. elegans reach adulthood, aging

diminishes their ability to generate growth cones and to extend axons

after injury (Byrne et al., 2014). This restricted axon regeneration is

not merely a consequence of organismal aging. Instead, it is an active

intracellular process: in aged C. elegans, insulin signalling via the

insulin growth factor 1 (IGF1) receptor inhibits forkhead

transcription factor daf-16/FOXO activity, suppressing

regeneration (Byrne et al., 2014). In the mammalian peripheral

nervous system, axon regenerative capacity declines with age

because Schwann cells are impaired in their ability to dedifferentiate

and clear myelin debris (Painter et al., 2014). Mammalian central

neurons also decline in their regenerative capacity as they age from

young adulthood to mid-adulthood (Geoffroy et al., 2016). After

Pten knockout in 4-week-old mice, corticospinal and rubrospinal

neurons regenerate axons past sites of spinal cord injury (Geoffroy

et al., 2016; Liu et al., 2010). However, when Pten is knocked out at

older ages (10 weeks or older), axons fail to navigate past the injury

site by 8 weeks post-injury (Geoffroy et al., 2016). Importantly, Pten

knockout boosts axon sprouting above the lesion in older animals,

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DEVELOPMENT

demonstrating that it remains effective in enhancing axon growth.

Pten restricts axon regeneration in C. elegans independently of age

(Byrne et al., 2014). Hence, Pten is likely an evolutionarily

conserved inhibitor of regeneration. After knockout of the myelin

associated inhibitor Nogo, the regeneration of corticospinal and

serotonergic axons is much less after spinal cord injury in 14-week-

old mice relative to 8-week-old mice (Cafferty et al., 2007).

How mammalian neurons continue to decline in their

regenerative capacity as they age is unknown. As in C. elegans,

specific signalling pathways might be activated in older mammalian

neurons that further restrict their regeneration, but this is unclear.

Another possibility is that older neurons have the capacity to

regenerate following treatment but their growth through scar

territory is much slower. When Pten is knocked out in

corticospinal neurons a full year after spinal cord injury, axon

regeneration is observed 7, but not 4, months later (Du et al., 2015).

This is a much longer timeframe for regeneration than in studies in

young adult mice, where knockout is induced prior to injury

(Geoffroy et al., 2016; Liu et al., 2010). In those cases, axon

regeneration is observed 6-12 weeks post-injury. One major

difference between these scenarios is that the scar at the injury

site is significantly denser and more mature at 1 year post-injury

(Cregg et al., 2014). When the mid-adult mouse spinal cord is

injured, it has enhanced macrophage density and astrogliosis

relative to an injured spinal cord from a young adult (Geoffroy

et al., 2016). Moreover, in the adult rat, aging increases the density

of CSPGs sulphated at position 6, which cause more inhibition of

axon growth, and decreases CSPGs sulphated at position 4, which

are more growth permissive (Foscarin et al., 2017). As such, aging

might accelerate the formation or maturity of inhibitory scar

territory, making it harder for axons to regenerate. Understanding

the mechanistic basis of this decline in central axon regeneration

from young adulthood to mid-adulthood is an exciting avenue of

research with important implications for CNS repair.

Divergences between developmental and regenerative axon

growth

Although recapitulating aspects of developmental growth enhances

axon regeneration, it is instructive to understand how these two

processes differ. In zebrafish, axon regeneration is mediated by

activating regeneration-associated gene expression using promoter

elements that are not active during embryonic development

(Udvadia et al., 2001). Similarly, mouse DRG neurons that

regenerate because of a conditioning lesion rely on signalling

pathways that are partially distinct from those mediating their

outgrowth during development (Liu and Snider, 2001). Indeed, the

global patterns of gene expression that are active during peripheral

axon regeneration only partially overlap with those that are active

during developmental axon growth (Chandran et al., 2016; Tedeschi

et al., 2016). Thus, the gene expression programs underlying

regeneration in non-mammalian vertebrates and in the mammalian

PNS are at least partially distinct from those underlying

developmental axon growth.

Some intracellular processes key to axon outgrowth are either

unique to or particularly important for regeneration. For example, in

adulthood, the distance between the axon tip and cell body of most

neurons is far longer than at any stage of embryonic development. In

Axotomy

*Cell death

A No treatment

Axotomy

B PTEN and SOCS3 knockout or

overexpression of OPN/IGF1/CNTF

Regeneration

and

synapse

formation

C PTEN knockout, cAMP

and inflammatory stimulation

Axotomy

Regeneration,

synapse

formation

and

myelination

RGC

Dying

RGC

Target

cell *Injury site

Degenerating

distal axon

Regenerated

axon and

synapse

Original

myelin

New

myelin

Key

Fig. 5. Regenerated axon myelination is not an automatic process and

may involve inflammatory signalling. (A) A mouse retinal ganglion cell

(RGC) projects its axon via the optic nerve to a target cell in the superior

colliculus. The axon is myelinated by oligodendrocytes. After axotomy, the

RGC axon distal to the injury site degenerates and the RGC eventually dies.

(B) The combined knockout of PTEN and SOCS3 or the overexpression of

OPN, IGF1 and CNTF stimulates an axotomized RGC to survive and to

regenerate its axon back to, and form a synapse with, its putative target.

However, the regenerated axon lacks new myelin, leading to poor action

potential conduction, which prevents functional recovery. (C) PTEN knockout,

cAMP and inflammatory (zymosan) stimulation also prompts an axotomized

RGC to regenerate its axon back to its target, where it forms a synapse. With

this treatment, the regenerated axon is myelinated by oligodendrocytes and

forms new nodes of Ranvier.

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DEVELOPMENT

line with this, axonal transport is crucial for regeneration (He and

Jin, 2016). In the retrograde direction, initiation of the regenerative

program requires signalling from the injured axon to the cell body

(Plunet et al., 2002). The upregulation of regeneration-associated

genes in response to injury is less pronounced in the neuron when it

is axotomized at a longer distance from its cell body compared with

when it is axotomized closer to its cell body (Fernandes et al., 1999).

STAT3 and dual leucine zipper kinase (DLK) are retrograde

messengers key to injury signalling (Lee et al., 2004; Shin et al.,

2012). In the anterograde direction, the building blocks of the axon,

including membrane, proteins and organelles are transported

towards the tip from the cell body to generate new axon. We

surmise that the inability of long descending and ascending axons in

the adult mammalian spinal cord to regenerate is due in part to the

failure of these systems to adequately transport new materials.

Conclusions

Recent studies have demonstrated that recapitulating aspects of

developmental axon growth can promote regeneration in the adult.

However, many challenges remain. For example, it is unclear how

regenerating axons form functional circuits. For many

manipulations, whether regenerated axons find their targets, form

synapses, are myelinated or become functional circuits following

injury or disease is unknown. The development of circuit-specific

genetic technologies to activate or silence neurons will allow

researchers to assess functional connectivity following CNS injury

(Hilton et al., 2016a; Kadoya et al., 2016). These technologies allow

transient activation or silencing of neuronal activity in response to

light (Deisseroth, 2011) or to designer drugs (Roth, 2016). For

example, a dual-virus approach was recently used to express an

engineered G-protein-coupled receptor, the designer receptor

exclusively activated by designer drug hM4Di (DREADD),

within dorsolaterally projecting mouse corticospinal neurons

(Hilton et al., 2016a). hM4Di activation transiently hyperpolarizes

the neuron and suppresses presynaptic glutamate release (Roth,

2016), thus allowing the role of the dorsolateral corticospinal

pathway in mediating spontaneous recovery following cervical

spinal cord injury to be determined (Hilton et al., 2016a). In

principle, such strategies will allow researchers to assess whether a

manipulation promotes synaptic transmission or to probe the

necessity of specific neuron populations in function after injury

(Hilton et al., 2016a; Jayaprakash et al., 2016; Kadoya et al., 2016).

The employment of novel rabies and adeno-associated viral

strategies to dissect the anatomy and functionality of genetically

defined circuits will also help the field of neuroregeneration (Azim

et al., 2014; Esposito et al., 2014; Zampieri et al., 2014). Bioengineered

rabies viruses permit the illumination and interrogation of synaptic

partners of genetically and/or anatomically defined subpopulations

of neurons, and were recently used to analyse synaptic connectivity

between transplanted cells and host axons after spinal cord injury

(Adler et al., 2017). With the recent development of self-

inactivating rabies virus (Ciabatti et al., 2017), which expands the

temporal window of the study of neural circuitry, it is possible to

precisely determine the synaptic connectivity of regenerating axons.

Ultimately, our understanding of the relationship between axon

regeneration and functional recovery remains rudimentary. In some

cases, axon regeneration is associated with worse functional

outcome (Takeoka et al., 2011; Wang et al., 2015), signifying the

importance of understanding the relationship between these

variables. In this regard, investigating synaptic connectivity

during embryonic development may provide clues as to how

regenerating axons can be directed into forming functional circuits.

For example, classical studies in the visual system have

demonstrated the influence of neuronal activity in synapse

formation, elimination and re-arrangement (Katz and Shatz,

1996). Whether neuronal activity can influence synaptic

connectivity of regenerating axons in a similar manner is unclear.

At the same time, axotomized neurons can form new circuits,

underlying recovery following injury in the absence of regeneration-

promoting treatment (Bareyre et al., 2004). Thus, exploring how

therapies that promote axon regeneration can build on, and not

antagonize, endogenous repair mechanisms will be essential for

their translation to the clinic.

Acknowledgements

We thank Dr Daniele Bano and Dr Andreas Husch for discussions on the review.

Competing interests

The authors declare no competing or financial interests.

Funding

B.J.H. is supported by a Wings for Life (WfL) Aguayo-Tator Mentoring Fellowship

and a non-stipendiary European Molecular Biology Organization (EMBO) long-term

fellowship (ALTF 28-2017). F.B. is supported by the Deutsches Zentrum fu

̈r

Neurodegenerative Erkrankungen, the International Foundation for Research in

Paraplegia, WfL and the Deutsche Forschungsgemeinschaft.

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REVIEW Development (2017) 144, 3417-3429 doi:10.1242/dev.148312

DEVELOPMENT

Osteopontin enhances the effect of treadmill training and promotes functional recovery after spinal cord injury

Article

Full-text available

Nov 2023

In this study, we examined the combined impact of osteopontin (OPN) and treadmill training on mice with spinal cord injury (SCI). OPN was overexpressed by injecting AAV9-SPP1-GFP into the sensorimotor cortex, followed by a left incomplete C5 crush injury two weeks later. Mice (Ex or Ex + OPN group) were trained at 50% maximum running speed for 8 weeks. To analyze the effects, we used biotinylated dextran amine (BDA) for tracing the corticospinal tract (CST) and performed Western blotting and immunohistochemical methods to assess the activation of the mammalian target of rapamycin (mTOR). We also examined axonal regeneration and conducted behavioral tests to measure functional recovery. The results demonstrated that treadmill training promoted the expression of neurotrophic factors such as brain-derived neurotrophic factor (BNDF) and insulin-like growth factor I (IGF-1) and activated mTOR signaling. OPN amplified the effect of treadmill training on activating mTOR signaling indicated by upregulated phosphorylation of ribosomal protein S6 kinase (S6). The combination of OPN and exercise further promoted functional recovery and facilitated limited CST axonal regeneration which did not occur with treadmill training and OPN treatment alone. These findings indicate that OPN enhances the effects of treadmill training in the treatment of SCI and offer new therapeutic insights for spinal cord injury.

Anatomical Diversity of the Adult Corticospinal Tract Revealed by Single-Cell Transcriptional Profiling

Article

Full-text available

Sep 2023

The corticospinal tract (CST) forms a central part of the voluntary motor apparatus in all mammals. Thus, injury, disease, and subsequent degeneration within this pathway result in chronic irreversible functional deficits. Current strategies to repair the damaged CST are sub-optimal in part due to underexplored molecular heterogeneity within the adult tract. Here we combine spinal retrograde CST tracing with single-cell RNA sequencing in adult male and female mice to index corticospinal neuron (CSN) subtypes that differentially innervate the forelimb and hindlimb. We exploit publicly available datasets to confer anatomical specialization among CSNs and show that CSNs segregate not only along the forelimb and hindlimb axis but also by supraspinal axon collateralization. These anatomically defined transcriptional data allow us to use machine learning tools to build classifiers that discriminate between CSNs and cortical layer 2/3 and non-spinally terminating layer 5 neurons in M1, and separately identify limb specific CSNs. Utilizing these tools, CSN subtypes can be differentially identified to study postnatal patterning of the CST in vivo , leveraged to screen for novel limb-specific axon growth survival and growth activators in vitro , and ultimately exploited to repair the damaged CST after injury and disease. Significance Statement Therapeutic interventions designed to repair the damaged corticospinal tract (CST) after spinal cord injury have remained functionally sub-optimal in part due to an incomplete understanding of the molecular heterogeneity among subclasses of corticospinal tract neurons (CSNs). Here, we combine spinal retrograde labeling with scRNAseq and annotate a CSN index by the termination pattern of their primary axon in the cervical or lumbar spinal cord and supraspinal collateral terminal fields. Using machine learning we have confirmed the veracity of our CSN gene lists to train classifiers to identify CSNs among all classes of neurons in M1 to study the development, patterning, homeostasis, and response to injury and disease, and ultimately target streamlined repair strategies to this critical motor pathway.

Prenatal PM2.5 exposure impairs spatial learning and memory in male mice offspring: from transcriptional regulation to neuronal morphogenesis

Article

Full-text available

Apr 2023

Background As one of the environmental risk factors for human health, atmospheric fine particulate matter (PM2.5) contributes to cognitive deterioration in addition to respiratory and cardiovascular injuries. Recently, increasing evidence implicates that PM2.5 inhalation can affect neurological functions in offspring, but the sex-specific outcomes and the underlying biological processes are largely unknown. Objectives To observe the influence of prenatal PM2.5 exposure on cognitive performance in offspring, to elucidate the neuronal morphological alterations and possible transcriptional regulation based on mRNA-sequencing (mRNA-Seq) data after birth, and to determine the key components of PM2.5 contributing to the adverse effects. Methods Pregnant C57BL/6J mice were exposed to sterile saline or PM2.5 suspension. Morris water maze test was used to assess the cognitive function in weanling offspring. Microscopic observation was applied to detect neuronal morphogenesis in vivo and in vitro. The cortex tissues from male offspring were collected on postnatal days (PNDs) 1, 7, and 21 for mRNA-Seq analysis. The organic and inorganic components of PM2.5 were separated to assess their contributions using primary cultured neurons. Results Prenatal PM2.5 exposure impaired spatial learning and memory in weanling male mice, but not female mice. The sex-specific outcomes were associated with mRNA expression profiles of the cortex during postnatal critical windows, and the annotations in Gene Ontology (GO) of differentially expressed genes (DEGs) revealed that the exposure persistently disrupted the expression of genes involved in neuronal features in male offspring. Consistently, axonal growth impairment and dendritic complexity reduction were observed. Importantly, Homeobox A5 (Hoxa5), a critical transcription factor regulating all of the neuronal morphogenesis-associated hub genes on PNDs 1, 7, and 21, significantly decreased in the cortex of male offspring following PM2.5 exposure. In addition, both inorganic and organic components were harmful to axonal and dendritic growth, with organic components exhibiting stronger inhibition than inorganic ones. Conclusion Prenatal PM2.5 exposure affected spatial learning and memory in male mice by disrupting Hoxa5-mediated neuronal morphogenesis, and the organic components, including polycyclic aromatic hydrocarbons (PAHs), posed more adverse effects than the inorganic components.

PTEN inhibition dedifferentiates long-distance axon-regenerating intrinsically photosensitive retinal ganglion cells and upregulates mitochondria-associated DYNLT1A and LARS2

Article

Full-text available

Apr 2023
DEVELOPMENT

Central nervous system (CNS) projection neurons fail to spontaneously regenerate injured axons. Targeting developmentally regulated genes in order to reactivate embryonic intrinsic axon growth capacity, or targeting pro-growth tumor suppressor genes such as Pten, promotes long-distance axon regeneration in only a small subset of injured retinal ganglion cells (RGCs), despite many RGCs regenerating short-distance axons. A recent study identified αRGCs as the primary type that regenerates short-distance axons in response to Pten inhibition, but the rare types which regenerate long-distance axons, and cellular features that enabled such response, remained unknown. Here, we used a new method for capturing specifically the rare long-distance axon-regenerating RGCs, and also compared their transcriptomes to embryonic RGCs, in order to answer these questions. We found the existence of adult non-α intrinsically-photosensitive (ip) M1 RGC subtypes that retained features of embryonic cell state, and showed that these subtypes partially dedifferentiated towards an embryonic state and regenerated long-distance axons in response to Pten inhibition. We also identified Pten inhibition-upregulated mitochondria-associated genes, Dynlt1a and Lars2, which promote axon regeneration on their own, and thus present novel therapeutic targets.

Advances in electroactive bioscaffolds for repairing spinal cord injury

Article

Full-text available

Apr 2024
BIOMED MATER

Spinal cord injury (SCI) is a devastating neurological disorder, leading to loss of motor or somatosensory function, which is the most challenging worldwide medical problem. Re-establishment of intact neural circuits is the basis of spinal cord regeneration. Considering the crucial role of electrical signals in the nervous system, electroactive bioscaffolds have been widely developed for SCI repair. They can produce conductive pathways and a pro-regenerative microenvironment at the lesion site similar to that of the natural spinal cord, leading to neuronal regeneration and axonal growth, and functionally reactivating the damaged neural circuits. In this review, we first demonstrate the pathophysiological characteristics induced by SCI. Then, the crucial role of electrical signals in SCI repair is introduced. Based on a comprehensive analysis of these characteristics, recent advances in the electroactive bioscaffolds for SCI repair are summarized, focusing on both the conductive bioscaffolds and piezoelectric bioscaffolds, used independently or in combination with external electronic stimulation. Finally, thoughts on challenges and opportunities that may shape the future of bioscaffolds in SCI repair are concluded.

Axonal Regeneration in the Central Nervous System

Chapter

Apr 2024

Lars Klimaschewski

The second chapter focuses on spinal cord injuries (SCIs). In addition to the biological and clinical basics, the differences to peripheral nerve regeneration are discussed, including the causes of axonal growth inhibition in the adult CNS. The current treatment approaches in the acute and chronic phase following SCIs are presented, and novel therapies using biopolymers, stem cell transplants, electrical stimulation, bioprostheses, and rehabilitation measures are introduced.

Glia in Recovery Processes and Repair

Chapter

Jan 2024

Wolfgang Walz

Axonal regrowth is not possible in the adult mammalian brain due to unsupportive gene expression and microenvironment. Remyelination is different. Recovery of neuronal functions is aided by the secretion of neurotrophic factors from reactive astrocytes. Uninjured and intact axons can sprout and establish new synapses. Most prominent are reparative axonal sprouting and long-distance sprouting. It occurs over long distances and connects functionally related areas. It is therefore most important for functional recovery and compensation. Reactive astrocytes are involved as they repulse and promote the sprouting. In other words, they serve as guides that direct the sprouting axons to their targets. Blocking this astrocytic activity does not lead to functional recovery. This important role of astrocytes pertains only to moderate reactive subtypes but not to scar-forming astrocytes. In addition, reactive astrocytes are known to regulate spine stabilization, plasticity, and maturation of the newly sprouted axons with target dendrites by releasing synaptogenic factors in the appropriate areas. Macrophages can induce a proinflammatory shift in microglia after an injury. This can lead to chronic inflammation which is completely detrimental to the healing process. If this macrophage influence is absent microglia shift after a short pro-inflammatory phase into an anti-inflammatory phase and assist the astrocytes in restoring function by degrading fibrotic tissue and releasing of factors that promote sprouting and synaptogenesis.

Chronic Adaptations in the Dorsal Horn Following a Cervical Spinal Cord Injury in Primates

Article

Dec 2023

Spinal cord injury (SCI) is devastating, with limited treatment options and variable outcomes. Most in vivo SCI research has focused on the acute and early post-injury periods, and the promotion of axonal growth, so little is understood about the clinically stable chronic state, axonal growth over time, and what plasticity endures. Here, we followed animals into the chronic phase following SCI, to address this gap. Male macaques received targeted deafferentation, affecting three digits of one hand, and were divided into short (4–6 months) or long-term (11–12 months) groups, based on post-injury survival times. Monkeys were assessed behaviorally, where possible, and all exhibited an initial post-injury deficit in manual dexterity, with gradual functional recovery over 2 months. We previously reported extensive sprouting of somatosensory corticospinal (S1 CST) fibers in the dorsal horn in the first five post-injury months. Here, we show that by 1 year, the S1 CST sprouting is pruned, with the terminal territory resembling control animals. This was reflected in the number of putatively “functional” synapses observed, which increased over the first 4–5 months, and then returned to baseline by 1 year. Microglia density also increased in the affected dorsal horn at 4–6 months and then decreased, but did not return to baseline by 1 year, suggesting refinement continues beyond this time. Overall, there is a long period of reorganization and consolidation of adaptive circuitry in the dorsal horn, extending well beyond the initial behavioral recovery. This provides a potential window to target therapeutic opportunities during the chronic phase.

Axonale Regeneration im zentralen Nervensystem

Chapter

Sep 2023

Lars Klimaschewski

Targeting pioneer transcription factor Ascl1 to promote optic nerve regeneration

Preprint

Jul 2023

In adult mammalian central nervous system (CNS) neurons, axon regeneration after injury remains limited due to the preset unfavorable gene regulatory programs. Factors enabling comprehensive epigenetic and transcriptional shifts, for example, the pivotal transcription factors which mediate the neurogenesis and morphogenesis may be essential for driving CNS axon regeneration. Based on the analysis of multiple public whole-genome mRNA and chromatin accessibility sequencing datasets of CNS neuronal development, as well as previous functional studies on the regeneration-capable dorsal root ganglion peripheral neurons, we hypothesize that overexpression of the pioneer transcription factor Achaete-Scute homolog 1 (Ascl1) will promote axon regeneration in the adult mammalian CNS neurons. We employed the optic nerve crush (ONC) in mice, a common model for studying CNS axon regeneration, neuron survival and glaucoma, to investigate the effect of Ascl1 overexpression. We found that Ascl1 could sufficiently promote axon regeneration past the crush site and significantly preserve the survival of retinal ganglion cells. Mechanistically, we revealed that the effect of Ascl1 was mediated by known pro-regeneration factor Sox11 but not others. Together, our study established an effective pipeline for discovering functionally important novel target for promoting CNS neuron axon regeneration and survival.

Life-Long Genetic and Functional Access to Neural Circuits Using Self-Inactivating Rabies Virus

Article

Full-text available

Jul 2017
CELL

Neural networks are emerging as the fundamental computational unit of the brain and it is becoming progressively clearer that network dysfunction is at the core of a number of psychiatric and neurodegenerative disorders. Yet, our ability to target specific networks for functional or genetic manipulations remains limited. Monosynaptically restricted rabies virus facilitates the anatomical investigation of neural circuits. However, the inherent cytotoxicity of the rabies largely prevents its implementation in long-term functional studies and the genetic manipulation of neural networks. To overcome this limitation, we developed a self-inactivating ΔG-rabies virus (SiR) that transcriptionally disappears from the infected neurons while leaving permanent genetic access to the traced network. SiR provides a virtually unlimited temporal window for the study of network dynamics and for the genetic and functional manipulation of neural circuits in vivo without adverse effects on neuronal physiology and circuit function.

Brain ageing changes proteoglycan sulfation, rendering perineuronal nets more inhibitory

Article

Full-text available

Jun 2017

Chondroitin sulfate (CS) proteoglycans in perineuronal nets (PNNs) from the central nervous system (CNS) are involved in the control of plasticity and memory. Removing PNNs reactivates plasticity and restores memory in models of Alzheimer's disease and ageing. Their actions depend on the glycosaminoglycan (GAG) chains of CS proteoglycans, which are mainly sulfated in the 4 (C4S) or 6 (C6S) positions. While C4S is inhibitory, C6S is more permissive to axon growth, regeneration and plasticity. C6S decreases during critical period closure. We asked whether there is a late change in CS-GAG sulfation associated with memory loss in aged rats. Immunohistochemistry revealed a progressive increase in C4S and decrease in C6S from 3 to 18 months. GAGs extracted from brain PNNs showed a large reduction in C6S at 12 and 18 months, increasing the C4S/C6S ratio. There was no significant change in mRNA levels of the chondroitin sulfotransferases. PNN GAGs were more inhibitory to axon growth than those from the diffuse extracellular matrix. The 18-month PNN GAGs were more inhibitory than 3-month PNN GAGs. We suggest that the change in PNN GAG sulfation in aged brains renders the PNNs more inhibitory, which lead to a decrease in plasticity and adversely affect memory.

Myelin regulatory factor drives remyelination in multiple sclerosis

Article

Full-text available

Sep 2017
ACTA NEUROPATHOL

Remyelination is limited in the majority of multiple sclerosis (MS) lesions despite the presence of oligodendrocyte precursor cells (OPCs) in most lesions. This observation has led to the view that a failure of OPCs to fully differentiate underlies remyelination failure. OPC differentiation requires intricate transcriptional regulation, which may be disrupted in chronic MS lesions. The expression of few transcription factors has been differentially compared between remyelinating lesions and lesions refractory to remyelination. In particular, the oligodendrocyte transcription factor myelin regulatory factor (MYRF) is essential for myelination during development, but its role during remyelination and expression in MS lesions is unknown. To understand the role of MYRF during remyelination, we genetically fate mapped OPCs following lysolecithin-induced demyelination of the corpus callosum in mice and determined that MYRF is expressed in new oligodendrocytes. OPC-specific Myrf deletion did not alter recruitment or proliferation of these cells after demyelination, but decreased the density of new glutathione S-transferase π positive oligodendrocytes. Subsequent remyelination in both the spinal cord and corpus callosum is highly impaired following Myrf deletion from OPCs. Individual OPC-derived oligodendrocytes, produced in response to demyelination, showed little capacity to express myelin proteins following Myrf deletion. Collectively, these data demonstrate a crucial role of MYRF in the transition of oligodendrocytes from a premyelinating to a myelinating phenotype during remyelination. In the human brain, we find that MYRF is expressed in NogoA and CNP-positive oligodendrocytes. In MS, there was both a lower density and proportion of oligodendrocyte lineage cells and NogoA+ oligodendrocytes expressing MYRF in chronically demyelinated lesions compared to remyelinated shadow plaques. The relative scarcity of oligodendrocyte lineage cells expressing MYRF in demyelinated MS lesions demonstrates, for the first time, that chronic lesions lack oligodendrocytes that express this necessary transcription factor for remyelination and supports the notion that a failure to fully differentiate underlies remyelination failure.

The mTOR Substrate S6 Kinase 1 (S6K1) Is a Negative Regulator of Axon Regeneration and a Potential Drug Target for Central Nervous System Injury

Article

Full-text available

Jun 2017

Themammaliantarget of rapamycin(mTOR)positively regulatesaxongrowth in themammaliancentral nervous system (CNS). Althoughaxon regeneration and functional recovery from CNS injuries are typically limited, knockdown or deletion of PTEN, a negative regulator of mTOR, increases mTOR activity and induces robust axon growth and regeneration. It has been suggested that inhibition of S6 kinase 1 (S6K1, gene symbol: RPS6KB1), a prominent mTOR target, would blunt mTOR’s positive effect on axon growth. In contrast to this expectation, we demonstrate that inhibition of S6K1 inCNSneurons promotes neurite outgrowth in vitro by twofold to threefold. Biochemical analysis revealed that an mTOR-dependent induction of PI3K signaling is involved in mediating this effect of S6K1 inhibition. Importantly, treating female mice in vivo with PF-4708671, a selective S6K1 inhibitor, stimulated corticospinal tract regeneration across a dorsal spinal hemisection between the cervical 5 and 6 cord segments (C5/C6), increasing axon counts for at least 3mmbeyond the injury site at 8 weeks after injury. Concomitantly, treatment withPF-4708671producedsignificantlocomotorrecovery. Pharmacological targeting ofS6K1maytherefore constituteanattractive strategy for promoting axon regeneration following CNS injury, especially given that S6K1 inhibitors are being assessed in clinical trials for nononcological indications.

Comprehensive Monosynaptic Rabies Virus Mapping of Host Connectivity with Neural Progenitor Grafts after Spinal Cord Injury

Article

Full-text available

May 2017

Neural progenitor cells grafted to sites of spinal cord injury have supported electrophysiological and functional recovery in several studies. Mechanisms associated with graft-related improvements in outcome appear dependent on functional synaptic integration of graft and host systems, although the extent and diversity of synaptic integration of grafts with hosts are unknown. Using transgenic mouse spinal neural progenitor cell grafts expressing the TVA and G-protein components of the modified rabies virus system, we initiated monosynaptic tracing strictly from graft neurons placed in sites of cervical spinal cord injury. We find that graft neurons receive synaptic inputs from virtually every known host system that normally innervates the spinal cord, including numerous cortical, brainstem, spinal cord, and dorsal root ganglia inputs. Thus, implanted neural progenitor cells receive an extensive range of host neural inputs to the injury site, potentially enabling functional restoration across multiple systems.

Early Administration of Gabapentinoids Improves Motor Recovery after Human Spinal Cord Injury

Article

Full-text available

Feb 2017

The anticonvulsant pregabalin promotes neural regeneration in a mouse model of spinal cord injury (SCI). We have also previously observed that anticonvulsants improve motor outcomes following human SCI. The present study examined the optimal timing and type of anticonvulsants administered in a large, prospective, multi-center, cohort study in acute SCI. Mixed-effects regression techniques were used to model total motor scores at 1, 3, 6, and 12 months post injury. We found that early (not late) administration of anticonvulsants significantly improved motor recovery (6.25 points over 1 year). The beneficial effect of anticonvulsants remained significant after adjustment for differences in 1-month motor scores and injury characteristics. A review of a subset of patients revealed that gabapentinoids were the most frequently administrated anticonvulsant. Together with preclinical findings, intervention with anticonvulsants represents a potential pharmacological strategy to improve motor function after SCI.

Myelination of axons emerging from neural progenitor grafts after spinal cord injury

Article

Jul 2017
EXP NEUROL

Neural progenitor cells (NPCs) grafted to sites of spinal cord injury (SCI) extend numerous axons over long distances and form new synaptic connections with host neurons. In the present study we examined the myelination of axons emerging from NPC grafts. Rat E14 multipotent NPCs constitutively expressing GFP were grafted into adult C5 spinal cord hemisection lesions; 3 months later we examined graft-derived axonal diameter and myelination using transmission electron microscopy. 104 graft-derived axons were characterized. Axon diameter ranged from 0.15 to 1.70 μm, and 24% of graft-derived axons were myelinated by host oligodendrocytes caudal to the lesion. The average diameter of myelinated axons (0.72 ± 0.06 μm) was significantly larger than that of non-myelinated axons (0.61 ± 0.03 μm, p < 0.05). Notably, the G-ratio of myelinated graft-derived axons (0.77 ± 0.01) was virtually identical to that of the normal, intact spinal cord described in published reports. These findings indicate that axons emerging from early stage neural grafts into the injured spinal cord recapitulate both the small/medium size range and myelin thickness of intact spinal cord axons.

Remyelination therapies: A new direction and challenge in multiple sclerosis

Article

Jul 2017

Multiple sclerosis is characterized by inflammatory activity that results in destruction of the myelin sheaths that enwrap axons. The currently available medications for multiple sclerosis are predominantly immune-modulating and do not directly promote repair. White matter regeneration, or remyelination, is a new and exciting potential approach to treating multiple sclerosis, as remyelination repairs the damaged regions of the central nervous system. A wealth of new strategies in animal models that promote remyelination, including the repopulation of oligodendrocytes that produce myelin, has led to several clinical trials to test new reparative therapies. In this Review, we highlight the biology of, and obstacles to, remyelination. We address new strategies to improve remyelination in preclinical models, highlight the therapies that are currently undergoing clinical trials and discuss the challenges of objectively measuring remyelination in trials of repair in multiple sclerosis.

Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury

Article

Jun 2017
NAT MED

Central nervous system (CNS) injury transforms naive astrocytes into reactive astrocytes, which eventually become scar-forming astrocytes that can impair axonal regeneration and functional recovery. This sequential phenotypic change, known as reactive astrogliosis, has long been considered unidirectional and irreversible. However, we report here that reactive astrocytes isolated from injured spinal cord reverted in retrograde to naive astrocytes when transplanted into a naive spinal cord, whereas they formed astrocytic scars when transplanted into injured spinal cord, indicating the environment-dependent plasticity of reactive astrogliosis. We also found that type I collagen was highly expressed in the spinal cord during the scar-forming phase and induced astrocytic scar formation via the integrin-N-cadherin pathway. In a mouse model of spinal cord injury, pharmacological blockade of reactive astrocyte-type I collagen interaction prevented astrocytic scar formation, thereby leading to improved axonal regrowth and better functional outcomes. Our findings reveal environmental cues regulating astrocytic fate decisions, thereby providing a potential therapeutic target for CNS injury.

Cell transplantation therapy for spinal cord injury

Article

Apr 2017

Spinal cord injury can lead to severe motor, sensory and autonomic dysfunction. Currently, there is no effective treatment for the injured spinal cord. The transplantation of Schwann cells, neural stem cells or progenitor cells, olfactory ensheathing cells, oligodendrocyte precursor cells and mesenchymal stem cells has been investigated as potential therapies for spinal cord injury. However, little is known about the mechanisms through which these individual cell types promote repair and functional improvements. The five most commonly proposed mechanisms include neuroprotection, immunomodulation, axon regeneration, neuronal relay formation and myelin regeneration. A better understanding of the mechanisms whereby these cells promote functional improvements, as well as an appreciation of the obstacles in implementing these therapies and effectively modeling spinal cord injury, will be important to make cell transplantation a viable clinical option and may lead to the development of more targeted therapies.

Can injured adult CNS axons regenerate by recapitulating development?

Abstract

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